Neurodegeneration (1617611190)

NEUROSCIENCE RESEARCH PROGRESS
NEURODEGENERATION: THEORY,
DISORDERS AND TREATMENTS

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NEURODEGENERATION: THEORY,
DISORDERS AND TREATMENTS

ALEXANDER S. MCNEILL
EDITOR

Nova Science Publishers, Inc.
New York

Copyright 2011 by Nova Science Publishers, Inc.

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Library of Congress Cataloging-in-Publication Data
Neurodegeneration : theory, disorders, and treatments / editors, Alexander
S. McNeill.
p. ; cm.
Includes bibliographical references and index.
ISBN 978-1-61324-178-3 (eBook)
1. Nervous system--Degeneration. I. McNeill, Alexander S.
[DNLM: 1. Neurodegenerative Diseases. WL 359]
RC365.N453 2010
616.99'48--dc22
2010026969

Published by Nova Science Publishers, Inc. New York

Contents

Preface vii
Chapter 1 Oxidative Stress-Mediated Neurodegeneration:
A Tale of Two Models 1
Paul K. Y. Wong, Jeesun Kim, Soo Jin Kim, Xianghong Kuang
and William S. Lynn
Chapter 2 Mechanisms of the Motoneuron Stress Response
and Its Relevance in Neurodegeneration 45
Mac B. Robinson, David J. Gifondorwa and Carol Milligan
Chapter 3 Methylene Blue Induces Mitochondrial Complex IV and Improves
Cognitive Function and Grip Strength in old Mice 63
Afshin Gharib

and Hani Atamna
Chapter 4 Receptor Specific Features of Excitotoxicity
Induced Neurodegeneration 87
Sergei M. Antonov and Dmitrii A. Sibarov
Chapter 5 Mitochondrial Uncoupling Proteins Therapeutic
Targets in Neurodegeneration? 107
Susana Cardoso, Cristina Carvalho, Snia Correia,
Renato X. Santos, Maria S. Santos

and Paula I. Moreira
Chapter 6 - Targeting Caspases in Neonatal Hypoxic Ischemic
Brain Injury and Traumatic Brain Injury 125
Xin Wang, Rachna Pandya, Jiemin Yao, He Ma
and Jianmin Li
Chapter 7 Alterations in N-Methyl-D-Aspartate (NMDA) Receptor Function
and Potential Involvement in Anesthetic-
Induced Neurodegeneration 155
Cheng Wang, Xuan Zhang, Fang Liu, Merle G. Paule
and William Slikker Jr.

Contents vi
Chapter 8 Genetics and Molecular Biology of Alzheimer's Disease and
Frontotemporal Lobar Degeneration: Analogies and Differences 173
Daniela Galimberti, Chiara Fenoglio and Elio Scarpini
Chapter 9 The Cholinergic Neuron in Alzheimers Disease 189
Christian Humpel and Celine Ullrich
Chapter 10 Retinal Neurodegeneration Is an Early Event in the Pathogenesis of
Diabetic Retinopathy: Therapeutic Implications 203
Rafael Sim and Cristina Hernndez
Chapter 11 Molecular Imaging and Parkinsons Disease 215
Valentina Berti, Cristina Polito, Maria T. R. De Cristofaro
and Alberto Pupi.
Index 221

Preface

Neurodegeneration is the umbrella term for the progressive loss of structure or function
of neurons, including the death of neurons. Many neurodegenerative diseases including
Parkinsons, Alzheimers, and Huntingtons occur as a result of neurodegenerative processes.
This book presents current research in the study of neurodegeneration, including oxidative
stress-mediated neurodegeneration; preserving motoneuron viability and function during
disease or after traumatic injury; research in aspects of excitotoxicity mechanisms;
uncoupling proteins as therapeutic targets in stroke and neurodegenerative diseases; the
genetics and molecular biology of Alzheimer's Disease; and retinal neurodegeneration in
diabetic retinopathy.
Chapter 1 - Maintenance of a balanced redox status within cells provides a healthy
environment for cellular functions and is critical to the fate of the cell. Alterations in cellular
redox status affect many redox sensitive activities including signal transduction, DNA and
protein synthesis, and protein folding. Significant or prolonged deviations in the intracellular
redox status disrupt cellular processes leading to numerous disease conditions.
This chapter focuses on two mouse models of two human diseases that display disruption
of the redox status of the cells leading to oxidative stress-mediated neurodegeneration (ND).
One model represents the human childhood genetic disorder ataxia telangiectasia (A-T)
lacking a functional ATM (A-T mutated) protein kinase. A-T is primarily a
neurodegenerative disorder that also affects other systems in the human body. Since one of
the key functions of the ATM protein is to maintain normal cellular redox status, the absence
of a functional ATM in cells of the central nervous system (CNS) results in chronic oxidative
stress leading to ND. A second model represents the human HIV-associated dementia (HAD)
and other neurological diseases associated with the accumulation of misfolded proteins. This
model uses a murine retrovirus called ts1 (a mutant of Moloney murine leukemia virus) that
causes oxidative stress, endoplasmic reticulum (ER) stress, and mitochondrial impairment as
a result of virus infection and accumulation of misfolded viral envelope protein in the ER of
astrocytes. Neurons are not productively infected by retroviruses thus neuronal loss induced
by these retroviruses is not directly due to productive infection of neurons, but rather due to
the infection of other cells in the CNS, including astrocytes, oligodendrocytes, microglia and
endothelial cells.
The goals are to understand the pathogenic mechanisms for both of these diseases thereby
helping to develop drugs to prevent neuronal cell loss. Recently the authors have found that
neurological symptoms of both disease models can be prevented by treatment with redox-
Alexander S. McNeill viii
active drugs, notably phthalazine dione, without repairing the initial causes. This finding
suggests that these two animal models share oxidative stress in the CNS as a common
mechanism of neuropathogenesis, although they have different initial causes, one from
genetic mutation and the other from viral infection.
This chapter brings together two important translational topics: elucidation of
neurodegenerative mechanisms and development of therapeutic treatment. These two models
will provide insight into the pathology of oxidative stress-mediated cell death and
demonstrate how mouse models can help in understanding human diseases. Insights from this
understanding may enable us to progress toward improved treatments in humans, not only for
neurodegeneration (ND) but also other related debilitating diseases resulting from oxidative
stress.
Chapter 2 - Preserving motoneuron viability and function during disease or after
traumatic injury is an intense area of research focusing on both the molecular mechanisms of
degeneration and therapeutic interventions to prevent it. Understanding how motoneurons
sense and respond to injury or pathology may help us identify potential targets for therapeutic
intervention. The motoneuron stress response or heat stress response (HSR) has been an area
of investigation spanning now well over a decade and has explored the role of heat shock
protein (HSP) expression during physiological stress and in animal models of
neurodegenerative disease. What we have found from these studies is that, in the midst of a
physiological stress, motoneurons rarely activate a classical stress response as characterized
by increased expression of Hsp70. It has been proposed that this lack of stress response
activation could contribute to pathological motoneuron dysfunction and degeneration.
Understanding the molecular mechanisms responsible for this phenomenon may provide
insights as to why motoneurons are the pathological hallmark in amyotrophic lateral sclerosis
(ALS) and other neurodegenerative conditions.
Chapter 3 - Methylene blue (MB) is very effective in delaying cellular senescence and
enhancing mitochondrial activity of primary human embryonic fibroblasts. At nanomolar
concentrations, MB increased the activity of mitochondrial cytochrome c oxidase (complex
IV), heme synthesis, cell resistance to oxidants, and oxygen consumption. MB is the most
effective among the many agents that has been are reported to delay cellular senescence. The
authors extended these in vitro findings to the investigation of the effect of long-term intake
of MB in old mice. The authors administered MB, in the drinking water (250 M), to old
mice for 90 days. In vivo, MB prevented the age-related decline in cognitive function and
spatial memory. MB also prevented the age-related decline in grip strength. Interestingly, MB
resulted in 100 % and 50 % increases in complex IV activities in the brains and hearts of old
mice, respectively. The age-related decline in protein content of the brain was prevented by
MB. We also found a 39 % decrease in brain monoamine oxidase (MAO) activity in old mice
treated with MB while aging or MB did not affect the activity of brain NQO1. The findings
suggest that the in vitro model for cell senescence may be used for fast and reliable screening
for mitochondria-protecting candidate agents before testing in animal models. The study also
demonstrates simultaneous enhancement of mitochondrial function, improvement of the
cognitive function, and improvement of grip strength in old mice by a drug. Since these are
three major concerns in human aging, MB may be a useful agent for delaying
neurodegeneration and physical impairments associated with aging.
Chapter 4 - Excitotoxicity is a term that describes the neuronal death caused by
neurotoxic effects of glutamate, which is the most abundant excitatory neurotransmitter in the
Preface ix
vertebrate central nervous system. Glutamate is well known to be involved in cognitive
functions like learning and memory, but its excessive accumulation in extracellular space can
lead to neuronal damages and eventual cell death via necrosis and apoptosis. As a result
excitotoxicity contributes to pathogenesis of numerous neurodegenerative diseases. Both
normal function and pathological action imply an activation of the same glutamate receptors
particularly of NMDA- (N-methyl-D-aspartate), AMPA- (-anino-3-hydroxyl-5-methyl-4-
isoxazole-propionate) and KA- (kainate) subtypes.
Many achievements in the mechanisms of neurodegeneration were obtained using
different experimental approaches on primary neuronal cultures. Double successive acridine
orange and ethidium bromide staining combined with confocal microscopy offers fast, easy,
sensitive and reproducible method by which necrosis and apoptosis can be recognized and
quantified in a population of living neurons. Together with immunostaining they provide
many research advantages and allow analysis of protein expression patterns.
The growing quantity of evidence reveals the diversity of apoptosis cascades. Whereas
our data show the same profiles of excitotoxicity for NMDA and KA, we found receptor
subtype specific differences in neuronal death mechanisms. For example, apoptosis caused by
prolonged NMDA receptors activation develops through the caspase-independent cascades
via release of apoptosis inducing factor (AIF) from mitochondria and its direct action on
nuclear chromatin. In contrast AMPA and KA receptors mediated apoptosis includes caspase-
dependent pathway.
On the basis of our data and literature the chapter will review the contemporary state of
research concerning the aspects of excitotoxicity mechanisms discussed above.
Chapter 5 - Uncoupling proteins (UCPs) are mitochondrial inner membrane proteins that
uncouple electron transport from ATP production by dissipating protons across the inner
membrane. UCP1 was the first uncoupling protein described and is present in brown adipose
tissue being involved in the non-shivering thermogenesis. Subsequent studies demonstrated
that neurons express at least three UCPs isoforms including the widely expressed UCP2 and
the neuron-specific UCP4 and UCP5. UCPs control the mitochondrial membrane potential,
free radicals production and calcium homeostasis and thereby influence neuronal function.
Given that mitochondrial energy impairment and free radicals production are thought to be
central players in neurodegeneration, recent data suggest that UCPs may have an important
role in neuroprotection and neuromodulation. The function of neuronal UCPs and their
impact on the central nervous system are attracting an increased interest as potential
therapeutic targets in several disorders including neurodegenerative diseases. Here the authors
will discuss the uncoupling process as an intrinsic mechanism of mitochondria physiology.
The role of UCPs in healthy and pathological brain conditions will be also considered.
Finally, they will discuss UCPs as potential therapeutic targets in stroke and
neurodegenerative diseases.
Chapter 6 - Mounting evidence implicates apoptosis in the pathogenesis of both acute and
chronic neurological disorders. The caspase family of cysteine proteases plays a central role
in the initiation and execution of neuronal apoptosis. So far the caspase family has been
expanded to 18 cysteine protease members. About two decades of investigation involving the
caspase family has produced a wealth of information. Studies indicate that targeting the
caspase family can prevent neuronal cell death in neurological disorders. This chapter will
discuss the role of the caspase family in experimental models of neonatal hypoxia-ischemia
brain injury and traumatic brain injury in vivo and in vitro, as well as in human neonatal
Alexander S. McNeill x
hypoxic-ischemic encephalopathy and traumatic brain injury. Given that elucidation of the
roles of individual caspases could yield multiple points of possible therapeutic intervention,
from the drug discovery and treatment perspective, the review will summarize what is
currently known about the beneficial effects of targeting caspases using a variety of
treatments against neonatal hypoxia-ischemia brain injury and traumatic brain injury. It will
focus on commonalities in the inhibition of caspase in the cell death receptor pathway, the
mitochondrial death pathway and the endoplasmic reticulum death pathway.
Chapter 7 - Advances in pediatric and obstetric surgery have resulted in an increase in the
duration and complexity of anesthetic procedures. It is known that the most frequently used
general anesthetics have either NMDA receptor blocking or -aminobutyric acid (GABA)
receptor activating properties. It is also known that anesthetic agents can cause widespread
and dose-dependent apoptotic neurodegeneration in the developing brain.
Exposure of developing mammals to NMDA-type glutamate receptor antagonists affects
the endogenous NMDA receptor system and enhances neuronal cell death. The NMDA
receptor regulates a calcium channel and calcium influx that overwhelms the mitochondrial
buffering capacity can result in increased production of reactive oxygen species (ROS) and
cell death. Meanwhile, stimulation of immature GABA receptors is thought to be excitatory
early in development but inhibitory in mature neurons. Stimulation of immature neurons by
GABA agonists is thus thought to increase overall nervous system excitability and may
contribute to NMDA receptor-associated increased excitability during early development.
This increased excitability may contribute to abnormal neuronal cell death during
development.
The type of excitotoxic insults that lead to neuronal apoptosis or necrosis are not
adequately understood but surely depend upon animal species, the concentration of stressors,
durations of exposures, the receptor subtypes activated and the stage of development or
maturity of a particular cell type at the time of exposure. It has been proposed that prolonged
blockade of the NMDA receptor in the developing brain by NMDA receptor antagonists such
as the dissociative anesthetics ketamine or phencyclidine (PCP) causes a compensatory up-
regulation of NMDA receptors. Neurons bearing these up-regulated receptors are
subsequently more vulnerable to the excitotoxic effects of endogenous glutamate, because
this up-regulation of NMDA receptors allows for the influx of toxic levels of intracellular
Ca
2+
under normal physiological conditions.
Although many more studies will be necessary in order to develop adequate quantitative
models to explain the relationships between altered NMDA receptor function and anesthetic-
induced neurodegeneration, a general hypothesis has been constructed and tested in an
interactive manner using carefully selected agents as defined by their pharmacological and
physiological properties. The integrative and iterative evaluation of these kinds of models will
lead to a better understanding of the potential neurotoxicity of NMDA antagonists and GABA
agonists in the developing human.
Chapter 8 - Alzheimers disease (AD) is the most common cause of dementia in the
elderly, whereas Frontotemporal Lobar Degeneration (FTLD) is the most frequent
neurodegenerative disorder with a presenile onset. The two major neuropathologic hallmarks
of AD are extracellular Amyloid beta (A|) plaques and intracellular neurofibrillary tangles
(NFTs). Conversely, in FTLD the deposition of tau has been observed in a number of cases,
but in several brains there is no deposition of tau but instead a positivity for ubiquitin.
Preface xi
In some families these diseases are inherited in an autosomal dominant fashion. Genes
responsible for familial AD include the Amyloid Precursor Protein (APP), Presenilin 1 (PS1)
and Presenilin 2 (PS2). The majority of mutations in these genes are often associated with a
very early onset (40-50 years of age).
Regarding FTLD, the first mutations described are located in the Microtubule Associated
Protein Tau gene (MAPT). Tau is a component of microtubules, which represent the internal
support structures for the transport of nutrients, vesicles, mitochondria and chromosomes
within the cell. Mutations in MAPT are associated with an early onset of the disease (40-50
years), and the clinical phenotype is consistent with Frontotemporal Dementia (FTD).
Recently, mutations in a second gene, named progranulin (GRN), have been identified in
some families with FTLD. Progranulin is expressed in neurons and microglia and displays
anti-inflammatory properties. Nevertheless, it can be cleaved into granulins which,
conversely, show inflammatory properties. The pathology associated with these mutations is
most frequently characterized by the immunostaining of TAR DNA Binding Protein 43
(TDP-43), which is a transcription factor. The clinical phenotype associated with GRN
mutations is highly heterogeneous, including FTD, Progressive Aphasia, Corticobasal
Syndrome, and AD. Age at disease onset is variable, ranging from 45 to 85 years of age.
The majority of cases of AD and FTLD are however sporadic, and likely several genetic
and environmental factors contribute to their development. Concerning AD, it is known that
the presence of the c4 allele of the Apolipoprotein E gene is a susceptibility factor, increasing
the risk of about 4 fold. A number of additional genetic factors, including cytokines,
chemokines, Nitric Oxide Synthases, contribute to the susceptibility for the disease. Some of
them also influence the risk to develop FTLD.
In this chapter, current knowledge on molecular mechanisms at the basis of AD and
FTLD, as well as the role of genetics, will be presented and discussed.
Chapter 9 - Alzheimers disease (AD) is a chronic brain disorder characterized by
cognitive decline, neuronal and synaptic loss, beta-amyloid-containing plaques,
neurofibrillary tangles, inflammation and cerebrovascular damage. Numerous studies
revealed that cholinergic neurons in the basal forebrain (septum, diagonal band of Broca,
basal nucleus of Meynert) are affected in AD and a loss of acetylcholine directly correlates
with memory dysfunction. (1) We will give an overview on the cholinergic neurons in the
basal forebrain and discuss the role of the key enzyme choline acetyltransferase (ChAT). (2)
We review the protective role of nerve growth factor (NGF) to support the cholinergic
phenotype. (3) We demonstrate different in vitro and in vivo models, which are used to study
cholinergic CNS neurons. (4) We reconsider if cholinergic neurons degenerate in AD or if
cholinergic neurons only downregulate the key enzyme ChAT. (5) Finally, our review will
summarize recent therapeutic strategies on augmenting cholinergic neurotransmission to
improve or reverse cognitive deficits in AD. In summary our review focuses on the
cholinergic CNS neurons and their role in AD.
Alzheimers disease is a severe and chronic degenerative disorder characterized by a
progressive neurodegeneration, amyloid-containing plaques, neurofibrillary tangles, as well
as cognitive dysfunction. Cholinergic neurons in the basal forebrain are located in six main
central nuclei (Ch1-Ch6). The key enzymes for the cholinergic system, choline
acetyltransferase (ChAT) and acetylcholinesterase (AChE) can be used for
immunohistochemical staining and characterization of the system. Essential for the
development and survival of cholinergic neurons in the basal forebrain is the nerve growth
Alexander S. McNeill xii
factor (NGF). The cholinergic neurotransmitter system in the basal forebrain is severely
affected in AD and loss of the neurotransmitter acetylcholine directly correlates with
cognitive dysfunction (Perry et al., 1981; Francis et al., 1985). Basic research of the
neuropathologic hallmarks and treatment strategies in AD is a fundamental goal, due to
immense costs of caring for patients with AD. The current review will highlight present
knowledge of the cholinergic dysfunction in AD and will demonstrate different models,
which are used to study AD, as well as possible therapeutic approaches.
Chapter 10 - Diabetic retinopathy (DR) remains the leading cause of blindness among
working-age individuals in developed countries. Although tight control of both blood glucose
levels and hypertension are essential to prevent or arrest progression of the disease, the
recommended goals are difficult to achieve in many patients and, consequently, DR develops
during the evolution of the disease. Therefore, new therapeutic strategies based on the
understanding of the pathophysiological mechanisms of DR are needed.
DR has been classically considered to be a microcirculatory disease of the retina due to
the deleterious metabolic effects of hyperglycemia per se, and the metabolic pathways
triggered by hyperglycemia. However, before any microcirculatory abnormalities can be
detected in ophthalmolscopic examination, retinal neurodegeneration is already present. The
two main features of retinal neurodegeneration are apoptosis and glial activation. Most of the
information regarding retinal neurodegeneration has been obtained from rats with
streptozotocin-induced diabetes (STZ-DM). Streptozotocin (STZ) is a potent neurotoxic agent
and is able to produce neural degeneration. Therefore, neurodegeneration observed in rats
with STZ-DM could be due to STZ itself rather than the metabolic pathways related to
diabetes. However, the recent observation that both apoptosis and glial activation also occur
in the retina of diabetic patients, even before any microvascular abnormality could be
detected in ophthalmologic examination, reinforces the concept that neurodegeneration is a
crucial pathogenic factor of DR.
Neuroretinal damage produces functional abnormalities such as the loss of both
chromatic discrimination and contrast sensitivity. These alterations can be detected by means
of electrophysiological studies in diabetic patients with less than two years of diabetes
duration, that is before microvacular lesions can be detected in ophthalmologic examination.
In addition, neuroretinal degeneration subsequently initiates and/or activates several
metabolic and signaling pathways which participate in the microangiopathic process, as well
as in the disruption of the blood-retinal barrier (a crucial element in the pathogenesis of DR).
Therefore, the study of the mechanisms that lead to neurodegeneration will be essential for
identifying new therapeutic targets in the early stages of DR.
Chapter 11 - Parkinsons disease (PD) is a neurodegenerative disorder characterized by
the loss of dopaminergic (DA) terminals in the striatum, resulting in functional changes in
frontostriatal circuits.
DA transporter imaging ([
123I
]FP-CIT SPECT imaging) and brain metabolic imaging
([
18F
]FDG PET imaging) have been broadly employed to explore the biological substrate of
PD, and together they could highlight the pathological processes occurring in early stages of
PD.
To evaluate the functional association between DA degeneration and cortical metabolism
we performed both [
123I
]FP-CIT SPECT and [
18F
]FDG PET in the same PD sample; through a
multiple regression analysis with SPM we explored the correlation between putaminal DA
degeneration and cortical metabolic rate of glucose.
Preface xiii
In the putamen, which is the first and most affected striatal region in PD, the severity of
dopaminergic impairment is directly related to cortical hypometabolism in premotor,
dorsolateral prefrontal, anterior prefrontal and orbitofrontal cortices.
[
123I
]FP-CIT SPECT and [
18F
]FDG PET allow to identify the early functional alterations
in the frontostriatal circuits involved in PD.

In: Neurodegeneration: Theory, Disorders and Treatments ISBN: 978-1-61761-119-3
Editor: Alexander S. McNeill 2011 Nova Science Publishers, Inc.
Chapter 1
Oxidative Stress-Mediated
Neurodegeneration: A Tale
of Two Models
Paul K. Y. Wong, Jeesun Kim, Soo Jin Kim,
Xianghong Kuang and William S. Lynn
Department of Carcinogenesis, The University of Texas, MD Anderson
Cancer Center, Science Park-Research Division, Smithville, Texas, USA
Abstract
Maintenance of a balanced redox status within cells provides a healthy environment
for cellular functions and is critical to the fate of the cell. Alterations in cellular redox
status affect many redox sensitive activities including signal transduction, DNA and
protein synthesis, and protein folding. Significant or prolonged deviations in the
intracellular redox status disrupt cellular processes leading to numerous disease
conditions.
This chapter focuses on two mouse models of two human diseases that display
disruption of the redox status of the cells leading to oxidative stress-mediated
neurodegeneration (ND). One model represents the human childhood genetic disorder
ataxia telangiectasia (A-T) lacking a functional ATM (A-T mutated) protein kinase. A-T
is primarily a neurodegenerative disorder that also affects other systems in the human
body. Since one of the key functions of the ATM protein is to maintain normal cellular
redox status, the absence of a functional ATM in cells of the central nervous system
(CNS) results in chronic oxidative stress leading to ND. A second model represents the
human HIV-associated dementia (HAD) and other neurological diseases associated with
the accumulation of misfolded proteins. This model uses a murine retrovirus called ts1 (a
mutant of Moloney murine leukemia virus) that causes oxidative stress, endoplasmic
reticulum (ER) stress, and mitochondrial impairment as a result of virus infection and
accumulation of misfolded viral envelope protein in the ER of astrocytes. Neurons are not
productively infected by retroviruses thus neuronal loss induced by these retroviruses is
Paul K. Y. Wong, Jeesun Kim, Soo Jin Kim et al. 2
not directly due to productive infection of neurons, but rather due to the infection of other
cells in the CNS, including astrocytes, oligodendrocytes, microglia and endothelial cells.
Our goals are to understand the pathogenic mechanisms for both of these diseases
thereby helping to develop drugs to prevent neuronal cell loss. Recently we have found
that neurological symptoms of both disease models can be prevented by treatment with
redox-active drugs, notably phthalazine dione, without repairing the initial causes. This
finding suggests that these two animal models share oxidative stress in the CNS as a
common mechanism of neuropathogenesis, although they have different initial causes,
one from genetic mutation and the other from viral infection.
This chapter brings together two important translational topics: elucidation of
neurodegenerative mechanisms and development of therapeutic treatment. These two
models will provide insight into the pathology of oxidative stress-mediated cell death and
demonstrate how mouse models can help in understanding human diseases. Insights from
this understanding may enable us to progress toward improved treatments in humans, not
only for neurodegeneration (ND) but also other related debilitating diseases resulting
from oxidative stress.
Introduction
Oxidative stress is a destructive consequence of many disease states, particularly those
involving the CNS. Oxidative stress in the nervous system has multiple causes, including
genetic mutations, viral infection, energy or thiol deprivation, aging, and extreme
environmental conditions. These disease conditions all exhibit oxidative stress, especially in
ER and mitochondria and these events are tightly linked (Figure 1). Different organelles in
the cells could be the sources for reactive oxygen species (ROS) production. Mitochondrial
dysfunction is believed to be the major cause of increased ROS. Another source of ROS could
be the result of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) in
plasma and ER membrane.Extensive accumulation of misfolded protein in the ER lumen can
also lead to production of ROS. If redox balance is not restored in the ER, Ca
2+
is rapidly
released from ER stores and picked up by mitochondria. This could disrupt the mitochondrial
electron transport chain resulting in increased production of ROS. On the other hand,
accumulation of ROS disrupts redox homeostasis in the ER, which impairs protein folding
that facilitates the accumulation of misfolded proteins, leading back into a vicious cycle with
further accumulation of misfolded protein in ER and severe ER stress. Collectively, these
events eventually activate the cellular apoptotic cascade.
This chapter focuses on two animal models for neurodegenerative diseases. One is a
genetic disease with mutated Atm gene as a mouse model for human A-T. The other is a
murine retrovirus mouse model for human HIV-associated dementia and other
neurodegenerative syndromes. In both models, intracellular oxidative stress and oxidative
stress-initiated pathways cause cell dysfunction and death in the CNS. The common
involvement of oxidative stress in these two different diseases contributes to our
understanding of shared mechanisms in human degenerative diseases. Our goals are to
understand the pathogenic mechanisms for both of these diseases, to develop drugs to protect
the viability of CNS cells and to prevent neuronal cell loss, thereby ameliorating NDs.
Potential treatments with a unique antioxidant and anti-inflammatory phthalazine dione drug,
Oxidative Stress-Mediated Neurodegeneration: A Tale of Two Models 3
monosodium luminol (MSL or GVT) and combination drug treatment with short-chain fatty
acid chemical chaperones for these diseases will be addressed.
Oxidative Stress, ER Stress
and Mitochondria Impairment
ROS, or free radicals, are highly reactive molecules due to the existence of unpaired
electrons. In addition, in the presence of Fe
++
, hydrogen peroxide (H
2
O
2
) can be converted
into highly toxic hydroxyl radicals and lipid hydroperoxides. At low physiological levels
H
2
O
2
modulates cell signaling events, including those responsible for cell proliferation and
cell death [1-7]. At high concentrations, however, H
2
O
2
and free radicals can damage cells by
oxidizing proteins, DNA, and lipids, ultimately leading to cell death. Oxidative stress occurs
in cells when the production of ROS exceeds intracellular antioxidant defenses [8, 9].
Oxidative stress is generally accompanied by thiol depletion in cells, because thiol-mediated
antioxidants such as glutathione (GSH) are consumed when antioxidant defenses are
mobilized [10, 11].
Oxidative stress contributes to many human neurodegenerative diseases [12], including
Alzheimers disease (AD) [13, 14], Parkinsons disease (PD) [15], multiple sclerosis (MS)
[16], amyotrophic lateral sclerosis (ALS) [17], Charcot-Marie-Tooth disease (CMT) [16],
Vanishing White matter Disease [16], HIV-associated dementia (HAD) [18-22], neuropathy
associated with endogenous human retroviruses [23] and other viral infections.

Figure 1.
Sources of ROS
Sources of ROS are variable for tissue, cell type, cell organelle and for each stressful
situation. Stress on the redox-sensitive translation system in ER generates hydrogen peroxide.
Stress on the mitochondrial electron transport system liberates oxygen free radical, primarily
superoxide and hydroxyl radicals if free iron is around. Stress in plasma membrane activates
NOX to generate H
2
O
2
(Figure 2). Thus, cells are abundantly equipped to produce ROS in
different organelles. The different sources of ROS are presented below.
The endoplasmic reticulum: protein folding (disulfide-bond formation) in the
ER can lead to formation of H
2
O
2
and ER-generated oxidative stress
It has been estimated that up to 25% of the total ROS generated by eukaryotic cells is a
consequence of oxidative protein folding in the ER (Tu BP and Weissman JS), Oxidative
protein folding in eukaryotes: mechanisms and consequences, JBC, 2004). Protein folding in
the ER is an energy-consuming process, and oxidizing conditions are required for the
formation of intramolecular and intermolecular disulfide bonds [24-26], a process catalyzed
by protein disulfide isomerase (PDI) and ER oxidoreductase (ERO1) [27]. PDI accepts and
donates electrons from protein-folding substrates, resulting in formation of the disulfide bond.
To maintain proper protein thiol redox potentials, the flavoenzyme ERO1 uses a flavin-
dependent electron bypass reaction FAD (flavin adenine dinucleotide) to transfer electrons
from flavin to molecular oxygen (O
2
), resulting in the production of H
2
O
2
[28]. In healthy
cells H
2
O
2
levels are normally low, but with excessive turnover of this thiol redox system,
and increased H
2
O
2
levels, both proliferation (at low levels) or senescence (at high levels)
occur [reviewed in 29, 30].
As nascent proteins fold to their proper conformations, disulfide bonds are broken and re-
formed several times, to achieve proper folding. These isomerization reactions are catalyzed
by the PDI [29]. Reduced and oxidized glutathione (GSH/GSSG ratio) in the ER lumen serve
as the redox catalysis for PDI [30]. Redox stress, caused by an excess of misfolded proteins
with inappropriate disulfide bond formation and/or breakage, disturbs the thiol redox
potentials. As a result, GSH levels in the ER are reduced by ERO1 and transfer of the electron
to O
2
causes the production of H
2
O
2
. These conditions, with the production of H
2
O
2
during
protein oxidation, together with GSH depletion by reduction of abnormal disulfides, can
exacerbate oxidative stress in the cell, leading to the release of Ca
2+
from ER stores, and
activation of mitochondrial apoptotic pathways (Figure 2). These conditions can also affect
the ER environment, such as disruption of ER redox status, leading to further accumulation of
proteins in the ER, causing ER stress. As an adaptive measure response to ER stress, the ER
possesses a signaling network that senses and responds to the presence of accumulated
misfolded proteins and targets them to be degraded by proteolytic systems such as the
proteasome [31]. This signaling network is collectively termed the unfolded protein response
(UPR), or the ER stress response. Irreversibly misfolded proteins are either retained within
the ER lumen, in complexes with molecular chaperones, or they are disposed of by the
ubiquitin-proteasome system, in a process called ER-associated degradation (ERAD).
The activities of the ER surveillance components are highly dependent on the redox
environment of the ER [32, 33]. GSH, the principal thiol compound of the ER, has been
shown to play a critical role in maintaining the ER thiol redox environment [34]. GSH can
also assist in disulfide-bond reduction, when there is an accumulation of misfolded proteins
due to inappropriate disulfide bonds [35].

Figure 2. Cellular components involved in production of H
2
O
2
and ROS
Protein folding within the ER is carried out by a family of protein disulfide isomerases (PDI) and ER
oxidoreductases (ERO1) that catalyze disulfide bond formation and isomerization. Accumulation of
misfolded proteins in the ER lumen can cause ER stress. ER stress, in turn, causes an increase in the
formation of incorrect intra and/or intermolecular disulfide bonds that require breakage (unfolding) and
reformation (refolding) for proteins to attain the appropriate folded conformation. PDI catalyzes
disulfide bond formation and isomerization, whereas GSH reduces improperly paired disulfide bonds.
Reoxidation is mediated by ERO1 with ROS production in the process. Thus, accumulation of
misfolded protein in the ER lumen is sufficient to produce ROS.
If redox balance is not restored, Ca
2+
stored in the ER is released. The excess Ca
2+
is taken up into the
inner membranes of the mitochondria, thereby disrupting the electron transport chain. This diverts the
electrons off-course and allows their release from the mitochondria, to react with molecular oxygen in
the cytoplasm, producing ROS. The ROS produced during these events can cause further Ca
2+
release
from the ER, resulting in amplified accumulation of ROS. Excess Ca
2+
can also activate NOX with
production of ROS, which damages mtDNA, followed by activation of poly (ADP-ribose) polymerase
1 (PARP) that depletes ATP. Together, these events result in permeability transition pore (PTP)
opening, leading to activation of apoptotic pathways. Nox at the plasma membrane can also be
activated by ligand-receptor interaction, resulting in generation of H
2
O
2
. H
2
O
2
, at appropriate (low)
levels functions as a signaling molecule.
Mitochondria: The major source of ROS production
Mitochondria not only play a central role in cellular energy and metabolisms, but are also
the major source of free radical production, particularly when the cell is subjected to redox
stress conditions. Critical for their function is the oxidation-reduction (redox) reactions,
which is essential for cellular respiration and ATP production. This process produces free
radical intermediates. Defects in mitochondrial functions result in many diseases, especially
those involved in metabolism and the nervous system [reviewed in 36, 37]. These and other
disease syndromes are likely to stem from the nature of the electron transfer processes that
underlie the oxidative phosphorylation complex mechanism. These may include the increased
production of toxic levels of ROS by the electron transport chains and altered ion homeostasis
[reviewed in 38].
Mitochondria and ER are physically and physiologically interconnected. A subset of
mitochondria is found in close proximity to the ER, at the opening of the inositol 1,4,5-
triphosphate (IP3)-sensitive Ca
2+
channel. The Ca
2+
released from ER is rapidly sequestered
by mitochondria. In healthy cells, large amounts of Ca
2+
are stored in the ER. Under steady-
state conditions, the ER releases small amounts of free Ca
2+
during signal transduction events
that occurs in many cellular activation processes. This free Ca
2+
is returned to ER stores by
ATP-dependent pumps and remains in the ER in a bound state. Thus, cytosolic free Ca
2+
is
only present at very low levels. However, when certain triggering events occur such as the
initiation of the UPR in response to ER stress, there is a large net release of Ca
2+
from these
ER stores. Much of this Ca
2+
is taken up into the inner membranes of the mitochondria and
disrupts the electron transport chain. During normal energy production, electrons in the
mitochondrial inner membrane flow down the mitochondrial electron transport chain, until
they are joined by two single oxygen ions to form water. When the electron transport is
disrupted, electrons in the transport chain are diverted off-course, and are released from the
mitochondria to react with molecular oxygen in the cytoplasm, producing ROS. The ROS
produced during these events can cause further Ca
2+
release from the ER, resulting in
amplified accumulation of toxic levels of ROS (Figure 2).
Increased Ca
2+
levels could also stimulate NADPH oxidase (NOX) activation to produce
ROS. ROS damages mitochondrial DNA (mtDNA), activating poly(ADP-ribose) polymerase
1 (PARP), which depletes ATP [36]. ROS at toxic levels also activates mitochondrial
apoptotic programs causing mitochondrial transmembrane potential (A
m
) dissipation. This
together with ATP decline is followed by activation of mitochondrial collapse and apoptosis
via opening of the permeability transition pore (PTP) [36, 37, 39-43]. Thus, it is clear that ER
stress and mitochondrial stress are intricately linked [44]. Ultimately, the consequence of
these stresses is amplification of apoptotic signals leading to cell death. As noted above,
numerous studies have linked ER stress and mitochondrial dysfunction to almost all NDs
[reviewed in [14, 36, 37].
NADPH oxidase (NOX Complex): Source of H
2
O
2

Another source of ROS could be the result of NOX action at the cellular membrane.
Originally discovered in neutrophils and phagocytic cells, NOX complex provides host
cellular defense against bacteria via a rapid respiratory burst of ROS. This involves
reduction of molecular oxygen to produce the superoxide anion. Superoxide then is converted
to H
2
O
2
. Use of cell-free systems for subunits of NOX complex, including p47
phox
, p67
phox
,
Rac and p40
phox
have been identified. Phosphorylation of p47
phox
leads to a conformational
change allowing its interaction with p22
phox
at the membrane. This in turn allows p47
phox
to
bring p67
phox
into contact with Nox2. When this occurs, the GTP bound Rac interacts with the
Nox2 and subsequently interacts with p67
phox
. The assembled complex can generate
superoxide by transferring electrons from NADPH to oxygen [reviewed in 45] . Recent
studies have shown that in addition to neutrophils NOX subunits are present in many cell
types, including endothelial cells, neurons and astrocytes. There is growing evidence
supporting the idea that ROS produced by NOX are causative factors in several
neurodegenerative diseases. For example, deletion of Nox2 in mouse models for AD [46], PD
[47], and ALS [48, 49] slows down disease progression and improves cell survival.
Recently, Brennan et al [50] found that neurons exposed to glutamate modulate ROS
levels through NOX rather than mitochondria. Unlike mitochondria, NOX enzymes do not
generate energy when they produce ROS. One electron is released in the cytoplasm when
each electron is transported. The translocation of negatively charged electrons may change
ion fluxes. NOX resides not only at the plasma membrane but also the ER membrane. NOX
enzyme can produce superoxide into the lumen of ER and extracellular environment [51]. In
particular, the ER is highly permeable to protons and could sustain NOX activity suggesting
that this may be another mechanism for the ER to generate ROS.
NOX activation is also interrelated to mitochondria. Recent studies investigating PINK1-
associated Parkinsons disease, suggests that an initial defect in calcium mishandling by
mitochondria leads to activation of NOX resulting in increased ROS in the cytoplasm, which
damages the glucose transporter leading to respiratory impairment [52].
Cellular defense against oxidative stress
Cellular defense responses to oxidative stress occur in a controlled sequence. The first
level of cellular defense involves upregulation of superoxide dismutases (SODs) and catalase
to counteract ROS buildup. If ROS overload causes significant cysteine and GSH depletion,
the second line of cellular defense is deployed via activation and nuclear translocation of the
transcription factor NF-E2 related factor 2 (Nrf2) [53-55]. Nrf2 transcriptional activity is
regulated by several mechanisms, including protein interactions, protein stability, nuclear
cytoplasmic shuffling, and phosphorylation [56]. Under normal conditions, Nrf2 is
sequestered in the cytoplasm by the actin-bound regulatory protein Kelch-like Ech-associated
protein 1 (Keap1) [57]. Multiple cysteine residues on the redox-sensitive Keap1 molecule
allow it to respond to intracellular accumulation of ROS, with release of Nrf2 from its
complex with Keap-1. This change allows Nrf2 phosphorylation and activation, which is
followed by Nrf2 nuclear translocation. In the nucleus, Nrf2 activates the expression of genes
via the antioxidant response element (ARE) promoter sequences. The genes that are activated
include many detoxification enzymes, antioxidant enzymes, and reducing molecules, such as
GSH [11, 58-60]. These products protect the cell from oxidative damage.
In the cytoplasm of resting cells, Nrf2 remains complexed with Keap-1, where it is
cyclically ubiquitinated and degraded through proteasome pathways [61]. Thus, Keap1 serves
as both an adaptor protein docking Nrf2 for ubiquitination and as a sensor for oxidative stress.
Another protein that binds to Nrf2 to keep it in the cytoplasm ready for phosphorylation but
not yet translocated into the nucleus is DJ-1 [62]. Interestingly oxidation of a critical residue
of DJ-1 causes relocation of the protein to the mitochondria sensor of oxidative stress.
Mutations in this protein result in impaired response to oxidative damage and increased cell
death in a PD cell model [63]. Under basal conditions some Nrf2 is also present in the
nucleus, mediating constitutive expression of Nrf2 target genes, such as NADPH quinone
oxidoreductase 1 (Nqo1) and hemoxygenase 1 (HO-1). Activation of Nrf2 in the brain has
been shown to mitigate the effects of oxidative stress in many NDs, including AD and PD
[62, 64, 65]. A recent report shows that Nrf2 activation in astrocytes with resultant increase
secretion of GSH prevents neuronal cell death and ND in mouse models of amyotrophic
lateral sclerosis (ALS) [17]. Thus, Nrf2/ARE pathway has been implicated to be a potential
therapeutic target against NDs.
A key product of the Nrf2 pathway is GSH. In addition to its role in detoxification to
protect cells from oxidative stress, GSH provides the main redox buffer for cells and as such
functions as a net reductant in the ER, either by maintaining ER oxidoreductase in a reduced
state or by directly reducing disulfide bonds in substrate folding proteins [30]. Thus, GSH has
a major role not only in the protein folding process but also in balancing redox reactions
thereby protecting cells from oxidative stress.
Mouse Genetic Model of A-T Associated
Neurodegeneration
A-T is an autosomal recessive genetic disease in which the Atm gene is mutated. Humans
with A-T display pleiotropic phenotypes, including cancer predisposition, immunodeficiency,
and progressive ND, with development of ataxia, telangiectasia, and premature aging.
However, A-T is primarily a neurological disorder. Symptoms of A-T are usually manifested
in the first few years of life, when children exhibit ataxia or wobbly gait. Loss of
neuromuscular control or coordination is relentless and, by ten years of age, children are
usually confined to a wheelchair. A-T patients are also more susceptible to infection,
radiation, pulmonary failure, and /or lymphoid cancer in the second decade of life [66, 67].
Despite increasing interest in recent years, the mechanisms underlying ND in human A-T are
still poorly understood. For this reason, treatments for A-T ND are not available. Gaining
knowledge of how ATM works is key to understanding the molecular basis of A-T associated
NDs and in development of therapeutic treatment for A-T ND.
ATM Regulates Cellular Redox Status and Maintains Redox Balance
The ATM protein is a large kinase that plays critical roles in regulation of cell cycling,
DNA repair, and control of cellular redox status. In unstressed cells, ATM exists as an
inactive form, but ATM becomes enzymatically active by autophosphorylation [68-70]. Until
recently, the molecular mechanisms that trigger ATM activation remain unclear. The cellular
activity of many kinases is known to be redox-sensitive, and this redox sensitivity is primarily
dependent on reactive cysteine residues in the proteins [4, 71]. For this reason, it has been
postulated that ATM might be activated in response to increased levels of H
2
O
2
in cells, e.g.,
as a result of cell metabolism and oxidants generated during postnatal development. At
moderate levels H
2
O
2
acts as a protein-modifying signaling molecule [4]. H
2
O
2
is readily
diffusible intracellularly, and induces protein activation rapidly. This could be one way in
which oxidative stress is sensed by redox-sensitive kinases, including ATM (Figure 3). It
has been shown that H
2
O
2
is directly and specifically responsible for oxidation-induced
disulfide bond formation that leads to conformational alteration and autophosphorylation of
ATM [72].
ATM might regulate cellular ROS levels by (a) increasing production of reductant
precursors, such as NAD+ and NADP+ [73], (b) decreasing energy consumption and ROS
production by cell cycling arrest [74], and (c) slowing electron transport in mitochondria, thus
decreasing ROS production [75]. Cells lacking ATM have increased rates of mitochondrial
respiration, particularly in the brain [73]. This suggests that the absence of ATM leads to
upregulation of intracellular ROS from mitochondria. The brain consumes about 20% of
inhaled oxygen, and energy production by neurons is heavily dependent upon mitochondrial
respiration. This situation makes the brain highly susceptible to oxidative stress [74, 76-83].
This could explain why loss of ATM results in oxidative stress. Persistent oxidative stress in
ATM-deficient cells probably overcomes cellular antioxidant defense systems, resulting in
dysregulation of signaling pathways and a neurological phenotype associated with oxidative
stress-induced damage.
Until now, the most convincing evidence linking oxidative stress to neurological
phenotypes in A-T has been obtained with Atm knockout (Atm
-/-
) mice. ATM-deficient mice
exhibit genomic instability and hypersensitivity to ionizing radiation and other treatments that
generate ROS [77]. Overexpression of SODs that generate H
2
O
2
in these mice exacerbated
certain features of A-T phenotypes [84].

Figure 3. ATM is a redox-sensitive protein and this redox sensitivity is primarily dependant on reactive
cysteine residues in the protein. ATM might be activated in response to increases in H
2
O
2
levels in the
cell. Oxidation of the two SH groups in cysteine results in disulfide bond formation, thereby leading to
conformational alteration and activation of the protein. The activated ATM then performs multiple
functions including DNA repair, cell cycle checkpoint maintenance, redox homeostasis and initiation of
signal transduction.
ROS levels are intrinsically upregulated in Atm
-/-
neural stem cells (NSCs), astrocytes
[81, 82, 85] and thymocytes [85], as well as hematopoietic stem cells [3, 86] suggesting that
ATM-deficient cells are constitutively under oxidative stress. In cultured normal NSCs and
astrocytes, addition of H
2
O
2
results in a rapid and dose-dependent phosphorylation of ATM
[81, 82] that can be suppressed by antioxidants (unpublished data from our lab). Together,
these findings strongly support the notion that activation of ATM can be linked to increased
ROS levels in cells. When ATM is activated, it exerts a systemic influence in the cell,
involving a large number of substrates that control cell functions [87]. Of these, the two best-
known functions have been cell cycle checkpoint control and DNA repair [78]. In recent
years, however, mounting evidence suggests that ATM can be activated by conditions that
increase intracellular ROS independent of its respond to DNA damage [72, 79, 88, 89].
ATM Deficiency and ER Stress
Although ATM deficiency has been shown to induce ER stress through oxidative stress
[77, 86], no conclusive evidence has been documented showing that ATM suppresses ER
stress. We have previously shown that ATM deficiency also induces ER stress in astrocytes
with increased levels of ER stress markers, glucose-regulated protein 78 (GRP78), or BiP,
and activation of caspase-12 cleavage [80]. These markers are also upregulated in cerebella of
Atm
-/-
mice. In keeping with this we also observe that BiP and phosphorylated alpha subunit
of eukaryotic translation initiation factor 2 (p-eIF2alpha) are upregulated in Atm
-/-
thymocytes
relative to Atm
+/+
thymocytes [90]. In another study, using ATM deficient cells or cells treated
with Atm siRNA, He and coworkers [91] show that ATM blocks ER stress induced by the ER
stress inducer tunicamycin. Together, these findings showing ATM regulation of ER stress
substantiates the notion that ATM plays a crucial role in controlling stress-mediated disease
conditions. These data also suggest that oxidative stress is linked to ER stress since ROS
levels are constitutively upregulated in ATM deficient cells. As mentioned above, prolonged
ER stress makes the ER membrane more permeable to Ca
2+
and this in turn results in
perturbation of intracellular Ca
2+
concentration. In view of the close apposition of ER and
mitochondria, perturbed Ca
2+
signaling could lead to mitochondrial collapse and apoptosis via
mitochondrial membrane permeabilization and opening of the permeability transition pore
[39, 40].
Lack of ATM Expression Causes Mitochondrial Dysfunction
A newly uncovered function for ATM is that it may regulate mitochondrial homeostasis
[38]. Shadel and coworkers show that fibroblasts from A-T patients exhibit conditional
mitochondria DNA (mtDNA) depletion independent of DNA damage. These cells also fail to
promote increases in mtDNA when DNA damage was induced by ionizing radiation [92].
Tissue-specific alterations in mtDNA copy number were also observed in Atm
-/-
mouse tissue.
In addition, the structural organization of mitochondria in A-T cells is abnormal compared to
wild type [73]. Moreover, ATM-deficient cells harbor a much larger population of
mitochondria with decreased membrane potential than control cells [93]. Thus, ATM
apparently plays a key role in mitochondrial function under normal growth conditions. Since
ATM also regulates p53 function [94], and since p53 has also been shown to regulate
mitochondrial respiration [95, 96], it is likely that some of the effects of ATM deficiency-
mediated mitochondrial dysfunction may be due to disruption of p53 in the absence of ATM.
This newly uncovered ATM function suggests that mitochondrial impairment may be at least
in part involved in the pathogenic mechanism of ATM deficiency. In addition to the hallmark
characteristics of ND in A-T, some A-T patients are associated with metabolic syndrome and
premature aging, both of which are linked to mitochondrial dysfunction. Mitochondrial
dysfunction also leads to the release of ROS resulting in oxidative stress and apoptosis. Thus,
further investigation of the effects of ATM on mitochondrial function is warranted. This also
may open the avenue for novel therapeutic treatment targeted to mitochondria for A-T
patients.
ATM Deficiency Results in Defective Self-Renewal and Proliferation
of NSCs through an Oxidative Stress-Mediated Neurodegenerative
Signaling Pathway
In the normal brain, the number of NSCs is the result

of a tightly controlled balance
among self-renewal, differentiation,

and death [97]. NSCs undergo asymmetric self-renewing
division to produce neuronal and glial progenitor cells, which differentiate to neurons,
astrocytes, and oligodendrocytes. Thus, proper control of these events is critical in
maintaining the normal numbers of neurons, astrocytes, and oligodendrocytes in the brain
[98]. ATM expression is abundant in NSCs in the normal brain, but is gradually
downregulated as the cells differentiate [99], suggesting that ATM may play an essential role
in NSC survival and proliferation during development. In the absence of ATM, abnormal
neuronal and astrocytic development occurs [80, 99, 100]. This could be the result of
abnormal differentiation of NSCs.
ROS levels are constitutively high in NSCs of Atm
-/-
mice and elevated ROS levels are
associated with defective self-renewal and proliferation of these cells. Treatment with the
antioxidant antioxidant N-acetyl cysteine (NAC) restores normal renewal and proliferation for
these cells. The elevated ROS in Atm
-/-
NSCs results in phosphorylation of p38MAPK (here
after called p38), which is correlated with decreased levels of p-Akt and Bmi-1 [82]. Bmi-1 is
a component of the polycomb repressor complex 1 (PRC1) that represses p21
CIP1
(hereafter
called p21) by chromatin modification. Thus, downregulation of Bmi-1 function results in
p21 upregulation. Furthermore, treatment of the Atm
-/-
NSCs with the p38 inhibitor
SB203580, or with NAC, restores normal levels of p21 and normal proliferation of Atm
-/-

NSCs [82]. These results suggest that ATM is required in NSCs to maintain normal
intracellular redox homeostasis. In the absence of ATM, chronic oxidative stress results in
activation of the p38-Akt-Bmi-1-p21 pathway in NSCs.
Bmi-1 can also separately regulate mitochondrial function and redox homeostasis [101]
by reducing the intracellular levels of ROS [102]. Thus, downregulation of Bmi-1 and ATM
in cells both result in oxidative stress. Whether downregulation of ATM results in
downregulation of Bmi-1 is unclear at the present time, although Bmi-1 is downregulated in
Atm
-/-
NSCs, or when normal NSCs are treated with H
2
O
2
[82]. Interestingly, Bmi-1 deficient
mice exhibit a progressive postnatal depletion of NSCs, leading to neurological abnormalities
and ataxia [103]. Conversely, NSCs over expressing Bmi-1 have increase self-renewal and
proliferation capacities [104] (Kim and Wong unpublished data). Collectively, these
observations strongly support the notion that downregulation of Bmi-1, like ATM deficiency,
contributes to decreased proliferation and self-renewal of NSCs.
Until now, the identity of upstream effectors that might control the levels or function of
Bmi-1 has been unclear. It has been shown that Bmi-1 could be phosphorylated by 3pk
(MAPKAP kinase 3), which is a downstream effector of p38 [105]. Phosphorylation of Bmi-1
by 3pk or by p38 reduces Bmi-1s ability to bind to chromatin, thus reducing its suppressive
effect on p21. Another way in which p38 may regulate Bmi-1 levels is via downregulation of
Akt since oxidative stress-induced activation of p38 attenuates insulin-like growth factor
stimulation of Akt [106]. On the other hand, Bmi-1 may be a substrate of Akt, and
upregulation of p-Akt coincides with upregulation of p-Bmi-1 (Kim and Wong, unpublished
data) probably by stabilizing Bmi-1. However, the question whether Akt can phosphorylate
and upregulate Bmi-1 remains to be addressed. Finally, a recent report has shown that
inhibition of ATM by an ATM-specific inhibitor KU-60019 reduces phosphorylation of Akt
and cell proliferation [107]. Together, these data and the findings described above indicate
that Bmi-1 also plays a critical role in NSC self-renewal and downregulation of Bmi-1 greatly
affects cerebellum development [103], supporting the notion that in the absence of ATM,
chronic oxidative stress results in activation of the p38-Akt-Bmi-1-p21 pathway leading to
defective proliferation and self-renewal of NSCs (Figure 4). This may at least in part
contribute to the defective cerebellum development and neurodegenerative phenotype in
Atm
-/-
mice.

Figure 4. One of the major functions of ATM is to regulate cellular redox status. In the absence of
ATM, reactive oxygen species (ROS) levels are intrinsically high in different cell types. This could lead
to oxidative/ER/mitochondrial stress with activation of cell death pathways. In Atm
-/-
NSCs the elevated
ROS results in upregulation of p-p38, which is correlated with decreased levels of p-Akt and Bmi-1.
Bmi-1 is a component of the polycomb repressor complex 1 (PRC1) that represses p21 by chromatin
modification. Thus, downregulation of Bmi-1 function results in p21 upregulation, which suppresses
cell proliferation. In Atm
-/-
astrocytes increased ROS activates the MEK-ERK pathways also resulting in
downregulation of Bmi-1 with repression of p16, leading to suppression of cell proliferation
Astrocytes Lacking ATM Results in Oxidative Stress-Mediated ERK1/2
Activation, Downregulation of Bmi-1 and Upregulation of P16 Leading to
Retardation of Growth
Interestingly, like NSC from Atm
-/-
mice, primary astrocytes isolated from Atm
-/-
mice
proliferate more slowly than do those from Atm
+/+
mice. Atm
-/-
astrocytes can be passed only
a few times before they become flat and enlarge, with increased intracellular vacuolation,
proliferative arrest and death. Atm
-/-
astrocytes, like Atm
-/-
NSCs, not only proliferated much
slower than Atm
+/+
astrocytes, but their growth was severely arrested [80]. When normal
(Atm
+/+
) astrocytes were treated with H
2
O
2
, with the ATM inhibitor KU55933, or with
siRNA-ATM, they grew significantly more slowly than do untreated C1 astrocytes (Kim J,
2009, unpublished data). This retardation of growth could be corrected, to a large extent, by
the MEK inhibitor or by NAC [81].
Cultured astrocytes from Atm
-/-
mouse brains show signs of increased levels of an
oxidative stress-regulating kinase ERK1/2 or extracellular signal-regulating kinases1/2 [81].
Phosphorylated extracellular signal-regulating kinases1/2, and their downstream mediators,
are now known to be major contributors to CNS pathology in a number of neurodegenerative
diseases, including AD [108-110]. In mice carrying a human tau gene (an animal model of
AD), a specific inhibitor of ERK2 reduces tau phosphorylation, and corrects the animals
motor impairments [109]. Interestingly, ATM downregulates p-ERK1/2 for cell survival after
IR [111]. Thus, in the absence of ATM, oxidative stress-induced upregulation of p-ERK1/2
may have detrimental effects in cells. Consistent with this notion, astrocytes from Atm
-/-
mice
show signs of oxidative stress and increased levels of p-ERK1/2 in vivo [80]. In Atm
-/-
mice
the specialized cerebellar Bergmann astrocytes have increased oxidative stress markers and
increased p-ERK1/2 [80]. These astrocytes are primary supporting cells for the Purkinje
neurons that control movement, and their dysfunction is likely to compromise their support
function to these neurons. One of the downstream events that follow ERK1/2 activation is
upregulation of p16 expression [112], which like p21, is a suppressor of cell cycling and a
major marker of aging [113, 114]. Since p16 expression is regulated at the transcriptional
level, p-ERK1/2 is unlikely to act directly on p16 expression. Instead, as in Atm
-/-
NSCs, Bmi-
1 acts as the bridge for this gap. Phosphorylation of BMI-1, by p-ERK1/2, may lift the normal
repression of the p16 gene by Bmi-1, allowing p16 expression to inhibit astrocyte cell cycling
and proliferation.
Phosphorylation of Bmi-1 could also be inhibited with the MEK inhibitor. Not
surprisingly, p16 levels are constitutively increased in Atm
-/-
astrocytes, but when Atm
-/-
astrocytes are exposed to H
2
O
2
in culture, p16 is further elevated and the elevated levels
persist for up to 16h. In Atm
+/+
astrocytes, by contrast, addition of H
2
O
2
caused a brief
upregulation of p16 at 4 h, but then this was followed by a return to normal levels at 16 h.
This means that oxidative stress due to increased H
2
O
2
is reversible when ATM is present. In
the Atm
+/+
cells, it seems likely that the brief expression of p16 shuts down cell cycling,
allowing time for the cells to repair any damage. Once this task is complete, p16 levels
returned to normal, as a result of ATMs redox balancing action. However, if oxidative stress
was prolonged, as it is in cells lacking ATM, upregulation of p-ERK1/2 persistently increased
p16 expression, resulting in prolonged cell cycle arrest and retardation of cell proliferation
inhibition of p-ERK1/2 activation with MEK inhibitor reduces p16 levels and 4 h after H
2
O
2

treatment [81]. Together, the above findings suggest that chronic oxidative stress results in
the activation of MEK-ERK-Bmi-1 and p16 pathways leading to defective proliferation of
astrocytes (Figure 4).
Whether the two signaling pathways presented above in ATM-deficient astrocytes and
NSCs are linked is at present unclear. Notably, in both Atm
-/-
and NSCs and astrocytes,the
MAPK p38 and ERK pathways converge to downregulate Bmi-1 with resultant defective
proliferation of these cell types. Furthermore, whether these signaling pathways are also
involved in neurons lacking ATM is also unclear. What is clear, however, is that in the
absence of ATM, ROS levels are intrinsically high in all these cell types, which substantiates
the notion that the defects in these cell types are associated with chronic oxidative stress in
the absence of ATM. Interestingly, convergence of the ERK and p38 pathways is also
observed in AD [115].
Mouse Model of Retrovirus Induced
Neurodegenerations
Retrovirus-Mediated ND Is Caused by Oxidative Stress, ER Stress
and Mitochondria Impairment
Oxidative stress appears to also play a critical role in the neurovirulence caused by many
viruses. These include Epstein-Barr Virus [116], respiratory syncytial virus [117], Japanese
encephalitis virus [118-120]. Three groups of retroviruses that cause ND are HIV in humans
(see below), simian immunodeficiency virus (SIV) in nonhuman primates [121], feline
immunodeficiency virus (FIV) [122] and certain murine retroviruses (see below). This
chapter focuses on NDs caused by murine retroviruses and HIV. This approach may yield a
number of important insights into the pathogenesis of NDs induced by these viruses via
oxidative/ER stress and mitochondrial impairment. In addition, using mouse models for
human diseases can bridge the gap between them, and provide better understanding of the
mechanism of pathogenesis and insights into improved treatment not only of HAD but also
other deliberating NDs associated with oxidative stress, ER stress and mitochondrial
impairment.
Retroviruses
Retroviruses are major health hazards because of their ability to cause persistent infection
of the nervous, immune and other systems in our body. A number of retroviruses, including
the murine leukemia viruses (MuLV), feline immunodeficiency virus, simian
immunodeficiency virus and human HIV-1 [123, 124], as well as human endogenous
retroviruses [125], are associated with neurological and systemic disorders. A recently
identified retrovirus, called xenotropic MuLV-related virus, is linked to chronic fatigue
syndrome in humans [126][Lo SC et. al, Detection of MLV-related virus gene sequences in
blood of patients with chronic fatigue syndrome and healthy blood donors, 2010 PNAS, epub
ahead of print]. Despite many years of study however, the mechanisms that underlie the
pathogenesis of retrovirus-induced NDs are not completely understood.
Since retroviruses were discovered, a considerable amount of information about their
biology has become available from studies on the murine retroviruses. In fact, before HIV-
AIDS appeared on the world landscape, retrovirus-associated ND was discovered as a
pathological manifestation of infection by some MuLV strains [reviewed in 127]. Therefore,
goals for HIV-AIDS research should be broadened toward a more basic understanding of
pathogenic mechanisms and disease progression [128] including neuropathogenic MuLVs in
mice.
Neurovirulent Murine Retrovirus
A group of neurovirulent murine retroviruses, represented by the ts1-Moloney murine
leukemia virus, a mutant derived from MoMuLV [127] and FrCas
NC
, derived from the
CasBrE retrovirus [129, 130] cause spongiform encephalopathy in infected mice. The
determinant of neurovirulence of both viruses results from genetic variation in the viral env
gene [130-133]. For both retroviruses, there is now strong evidence for oxidative stress, ER
stress and mitochondrial impairment in the neuropathology that they cause. One puzzling
aspect of these retrovirus-mediated ND is the cellular specificity of the cytotoxicity. These
viruses infected all the cell types, except neurons, in the CNS. However, in all cell types,
except microglia, signs of apoptosis are detected [Wong PK and Yuen PH, 1994, Histol
Histopathol 9:845].
ts1 MoMuLV Model
In ts1 MoMuLV, a single point mutation (Val to Ile) in the env gene results in misfolding
of the envelope precursor protein gPr80
env
. The misfolded gPr80
env
accumulates in the ER,
because it cannot be transported from the ER to the plasma membrane. ts1 infects many cell
types, but gPr80
env
accumulation occurs mainly in infected T lineage lymphocytes in the
immune system and astrocytes (and perhaps oligodendrocytes) in the CNS [123, 134, 135].
This results in a disease that resembles HIV infection in several important ways [123, 124,
136, 137]. The characteristic features of both HIV- and ts1-induced disease include T cell
depletion [138-140] and cell death in astrocytes and neurons in the CNS [124, 136, 141-149]
(see Table 1). T cells and macrophages are primary peripheral targets for both HIV and ts1
[138, 139, 150]. In the CNS, both HIV and ts1 infect microglia, astrocytes, oligodendrocytes,
and endothelial cells, but not neurons [123, 139, 141, 151-153]. Thus, neuronal loss induced
by these retroviruses is not directly due to productive infection of neurons, but rather to
astroglial neuronal support impairment, or the secretion of neurotoxic factors by infected or
activated glia [141, 142, 153-156].
In the CNS, accumulation of the misfolded precursor envelope protein of ts1 in the ER of
astrocytes results in UPR, leading to oxidative stress and ER stress, which in turn results in
mitochondria-mediated cell death [42, 157]. As noted above, since neurons are not directly
infected by ts1 but they die alongside infected astrocytes, neuron death is most likely due to
reduced thiol support from dysfunctional astrocytes (causing oxidative stress and ER stress
and thiol deficiency in neurons) and/or to neurotoxic factors produced by ts1 infected
astrocytes and microglia or due to loss of support from ER- stressed oligodendrocytes.
Table 1. Retrovirus-mediated cell death and NDs are associated with oxidative stress,
ER stress and mitochondrial cell death pathways in glial and neurons
HIV ts1
Oxidative stress| [18, 21, 147, 172-176] [11, 161, 167, 177]
Antioxidants defense+ [146, 178, 179] [11, 161]
ER stress [144, 180] [42, 181, 182]
Dysregulation of calcium homeostasis [175, 183-185] Unpublished data
Mitochondria impairment [180, 184, 186] [42, 181]
Upregulation of xCT
-
[187] [11]
Oxidative stress with accumulation
protein other than viral protein
[188-192] [142]
NOX activation [193] Unpublished data
Elevation of iNOS [194] [157]
Elevated COX-2 levels [195] [196]
Increase in FGF [197] [198]
Protective effect of Nrf2 [199, 200] [11, 167]
Protective effect of Bcl2 [158] [159, 161]
Protective effect of minocycline and
other antioxidants
[201] [167, 177]
Astrocytes and neuronal death [144-149, 202-204] [42, 141, 142, 177, 181]
Role of microglia [123] [123, 141, 153, 205]
In ts1-Infected Astrocyte Cultures and Brainstem Tissues of ts1-Infected
Mice, Oxidative Stress Is Associated with ER and Mitochondrial Stress,
with Initiation of Apoptotic Pathways
In ts1-infected astrocytes increased expression of ER and mitochondrial stress biomarkers
including upregulation of the chaperone proteins GRP78 (BiP), and mitochondrial
degeneration [42] are observed. The UPR is initiated, as shown by activation of the ER-
resident transmembrane protein kinase PERK, which upregulates both the initiation factor-2o
(eIF2o) and CHOP. Interestingly, studies by others have shown that CHOP downregulates
Bcl2 [158], a protein that has protective capability against ts1-induced [159] and HIV-
induced [158] ND. The ER stress-specific enzyme caspase-12 is also activated, cleavage of
procaspase-9 occurs, and caspase-3 is activated leading to apoptosis.
Evidence for mitochondrial involvement in ts1 neuronal death comes from a recent
reports showing p53 accumulation in neurons in the ts1-infected CNS [160] and that
overexpression of Bcl2 in astrocytes confers resistance to ts1-induced cell death [161] and to
ts1-mediated ND in infected mice [159]. In the brainstems of ts1-infected mice, activated
caspase-3 and damaged mitochondria are present in astrocytes. Therefore, it appears that
oxidative, ER, and mitochondrial stress-related apoptotic pathways are involved in ts1-
induced astrocytic death [11, 42].
Nrf2 Mediates Antioxidant Defenses in ts1 Infection,
and It Promotes Cell Survival
As noted above, ts1 infection of astrocytes induces thiol (i.e., GSH and cysteine)
depletion and ROS accumulation, in parallel with viral envelope precursor gPr80
env

accumulation [11]. ts1-infected cultured astrocytes apparently mobilize their antioxidative
defenses by upregulating their levels of Nrf2, and levels of its target genes, including the
xCT
-
cystine/glutamate antiporter, -glutamylcysteine ligase, and glutathione peroxidase.
Thiol depletion appears to accelerate astrocyte cell death, while thiol supplementation
promotes survival of ts1-infected astrocytes. Together, these data substantiate the notion that
ts1 infection may damage astrocytes by oxidative and ER stress, which can be alleviated by
Nrf-2-mediated thiol antioxidant defenses [11].
A Subpopulation of ts1-Infected Cultured Astrocytes Survives by
Upregulating Antioxidant Defenses
As previously mentioned, ts1-infected astrocyte cultures show increased levels of ROS.
Despite this, however, only about half of infected astrocytes die in these cultures. The
surviving cells continue to proliferate and produce virus. To determine how these resistant
cells survive ts1 infection in culture, we established and characterized a subline of these cells
(C1-ts1-S). C1-ts1-S cells proliferate more slowly than do C1 cells, and produce fewer
viruses than do infected C1 cells. They also show reduced H
2
O
2
levels, increased uptake of
cystine, and higher levels of both GSH and cysteine, compared to acutely infected (non-
surviving) cells or to uninfected C1 cells [161]. C1-ts1-S cells also upregulate their thiol
antioxidant defenses by activation of Nrf2 and its target genes and Bcl2, a mitochondrial
protector. We conclude that some astrocytes can survive ts1 infection by successfully
mobilizing their antioxidant defenses via Nrf2 activation and by upregulation of Bcl2 [161].
ts1-Associated Caspase 8 Activation in Astrocytes Is Caused by
Intracellular Events
In ts1-infected astrocytes, caspase 8 is activated by an intrinsic pathway, which starts
with elevation of the death receptor DR5 and the C/EBP homologous protein
(GADD153/CHOP), an ER stress-initiated transcription factor, rather than through TNF2 and
TNF-R1 interaction on the cell surface. Upregulation of CHOP that inhibits Bcl2 may explain
why Bcl2 is downregulated in ts1 infected primary astrocyte cultures. Activated caspase 8
cleaves Bid into tBid, initiating mitochondria-driven apoptosis via tBid translocation. This in
turn amplifies ER stress, contributing to oxidative stress-induced apoptosis. Treatment of ts1-
infected astrocytes with a specific caspase 8 inhibitor reduces ER stress responses. This
occurs because the inhibitor reduces both caspase 8 activation and cleavage of the ER-
associated membrane protein BAP31 into BAP20, of which overexpression exacerbates the
ER stress response. These findings suggest for the first time that the caspase 8- and ER and
mitochondria stress-associated apoptotic pathways are linked [44]. These findings suggest
that caspase 8 could be a new target for treatment of stress-related diseases in humans.
Retrovirus Infection Promotes ROS Production through NOX
Following ts1 infection of astrocytes, levels of NADPH are decreased and levels of
superoxide are increased when compared with uninfected astrocytes, which suggests NOX
activation (Kim and Wong, unpublished data). Moreover, NOX inhibition induces the anti-
apoptotic Bcl-2 and BclxL proteins, and suppresses activation of the pro-apoptotic enzyme
caspase 3 (Kim and Wong, unpublished data).
As noted above, ER stress may be related to intracellular ROS induction in ts1-infected
cells. Due to mutation, the precursor viral envelope protein of ts1 gPr80
env
is unable to fold
properly and unable to proceed to Golgi. The UPR may activate NOX, which might be
localized in the ER membrane. Although Nox2 distribution in ER membranes of astrocytes
has not been well studied, Nox4 in endothelial cells is predominantly expressed in the ER
membrane. Chen et al reported that Nox4-dependent ROS inactivates protein tyrosine
phosphatase (PTP)1B in the ER. PTP1B then serves as a regulatory switch for epidermal
growth factor (EGF) receptor trafficking [51].
Enveloped retroviruses such as HIV and MLV may use ROS induction of cells as a tool
to invade host cells. The viral envelope makes contact with its receptor on the plasma
membrane, and this is followed by viral entry. Notably, retroviral entry could be modified by
the local redox climate [162, 163]. Ryser et al [164] proposed that receptor-bound envelope
glycoprotein gp120 of HIV is reduced by host surface-associated PDI which is colocalized
with CD4 [165]. Inhibition of PDI activity prevents the entry of HIV [166]. Reducing
disulfide bonds causes conformational changes in gp120 and these changes may enhance viral
fusion to the host membrane.
In summary, the key findings in these studies have been that ts1 infection causes
oxidative stress, ER stress, and mitochondrial impairment, all of which play critical roles both
in ts1-induced ND [167-169]. Interestingly, overwhelming evidence now shows that oxidative
stress, ER stress and mitochondrial dysfunction also play a key role in HIV
neuropathogenesis (see Table 1).
Advantages of the ts1 Model for HAD
Despite extensive research on HAD, the mechanisms by which HIV causes death in
neurons of AIDS patients remain unclear. The main problems have been the expense and
ethical costs of human studies and of primate models of HIV infection. These limitations and
a lack of suitable alternative animal models have hampered full understanding of the
mechanisms involved in HIV-induced CNS cell death.
ts1 is a well-characterized murine retrovirus that causes a rapidly developing disease
syndrome. Disease severity and latency are well defined and can be manipulated by the virus
strains used, the dosage of virus, the age of the host when infected, and the mouse strains
employed [123, 127, 132, 136, 137, 170]. ts1-mediated disease is dramatic, easily induced,
and reproducible [127, 132]. With the ts1 model, we can perform both cell biology and
systemic studies of retroviral-induced disease. Because of the availability of gene knockout
and transgenic mouse models, such as mice overexpressing Nrf2 [17] or Bcl-2 [159], this
model can be used to focus on specific proteins involved in ts1-induced ND. These
technologies have the distinct advantage of allowing analysis of disease states within the
context of the whole animal, which more accurately mimic the human disease [171].
Although ts1 does not completely reproduce all pathologic features of HIV infection, and
although the sources of ROS overproduction in ts1 and HIV infection may not be identical,
important similarities exist between the ts1-induced ND model and HAD, as described above
(Table 1). Since oxidative stress, ER stress, mitochondria impairment and downstream events
are involved in both ts1-mediated ND and HAD, similar cell death mechanisms may
contribute to these diseases. Knowledge gained from the studies with animal models should
therefore shed light on the pathogenic mechanisms and on potential therapeutic treatment for
HAD in humans. Furthermore, a better understanding of the cellular and molecular
mechanisms of oxidative stress, ER stress and mitochondrial dysfunction induced by various
agents, including protease inhibitors used in HAART for HIV, may provide useful
information for the development of new therapeutic strategies against these stressful
conditions.
Oxidative Stress in HAD
HIV induces chronic systemic oxidative stress in AIDS patients, which occurs even in
early stages of the disease [206, 207]. Later in the disease course, signs of oxidative damage
have been observed in dying neurons and astrocytes in the brains of HIV associated dementia
HAD patients [18-21, 149, 208]. HIV-induced oxidative stress causes apoptosis in cultured
astrocytes and neurons [146] (see Table 1). In addition, ROS generation by glial cells is
linked to neural cell death induced by HIV in vitro [22]. In general, individuals infected by
HIV-1 show decreased systemic antioxidant defenses suggesting that the nutrient substrates
for oxidant defense are being depleted by ongoing oxidative stress [178]. The exact
mechanisms by which HIV induces oxidative stress, however, remain unclear. Several HIV-1
proteins, in particular gp120 and Tat, have been associated with ROS production and
oxidative stress in cultured infected astrocytes [146, 147, 202]. Although HIV does not infect
neurons [175, 183, 185], both HIV gp120 and Tat disrupt neuronal calcium homeostasis by
perturbing the Ca
2+
regulating system in the plasma membrane and ER, leading to oxidative
stress and mitochondrial dysfunction, which together cause neuronal death. When the HIV-1
protein Tat is added to HIV-1 target cells in culture, levels of ceramide, an oxidative stress
marker, are increased. Ceramide levels are also increased in the brains of HAD patients [209].
Upregulation of xCT
-
after Tat exposure has been documented for human retinal pigment
epithelial cells and retinas of Tat-transgenic mice [187]. In addition, Tat itself activates
oxidative and inflammatory pathways in the brain vascular endothelium [210]. It has been
reported that human astrocytic cell line exposed to HIV-1 underwent apoptosis as a result of
HIV-1 mediated oxidative stress, and that antioxidant compound NAC prevents these HIV-
induced cellular damages [146]. Furthermore, it has been shown that HIV gp 120 is toxic to
astroglial cells, by lipid peroxidation as a result of oxidative stress. This effect of gp120 on
astroglial cells can also be counteracted by NAC [202]. Together, these studies suggest that
oxidative stress may play a critical role in the pathogenesis of HAD.
ER Stress in HAD
Chronic ER stress is a primary component in the neuropathology of a wide variety of
NDs [211, 212]. As noted above, in the oxidative environment of the ER, elevated oxidative
stress can directly cause alterations in protein folding [213, 214]. In addition, accumulation of
misfolded protein may itself alter the redox status of the ER, causing other proteins to misfold
[215, 216]. These conditions can also generate disulfide bond-mediated intermolecular
protein aggregation [217]. Generally, protein misfolding leads to protein accumulation, ER
stress and proteinopathy (accumulation of misfolded proteins), which contribute to many
human NDs, including -amylold in AD [13], -synuclein in PD [15], MS [16], ALS [218],
Prion disease [219] and neuropathy associated with retrovirus infection and endogenous
human retroviruses [23]. Thus, although NDs can have multiple causes, they all seem to share
common mechanisms involving oxidative stress, ER stress, mitochondrial impairment and
proteinopathy (Figure 1). Since both ts1-ND and HAD are associated with oxidative stress, it
is possible that ER stress-initiated oxidative stress and cell damage is a common culprit in cell
death in retrovirus-mediated NDs. As shown above, ER stress and cell death occur in both
astrocytes and neurons in the CNS of patients with HAD [144, 149, 180].
Although the precursor envelope protein of HIV gp160 accumulation has not been
reported for HIV-infected cells in the CNS, retention of gp160 in the ER of T cells [220, 221]
suggests a likely relationship between retention of gp160 in ER and cytopathic effects in HIV
infection [222, 223]. The gp160 molecule requires complex and time-consuming folding in
the ER, and is prone to misfolding and accumulation [220]. Because of this, there is a high
incidence of unusual cysteine variants in HIV envelope proteins in individual patients and this
results in aberrant disulfide bond formation and gp160 accumulation [224]. Thus, it is
possible that accumulation of gp160 in HIV-infected cells results in oxidative and ER stress.
Even though gp160 has not been shown to accumulate in HIV-infected astrocytes, ER stress
may still be a possible cause of astrocyte damage and neuronal death, if oxidative stress
occurs in the cells and ER redox state is disturbed by HIV infection.
The fact that accumulation of -amyloid occurs in HAD brain [189, 190, 192] and inside
neurons [188] provides strong evidence that redox imbalance leading to global protein
misfolding in HIV-infected CNS cells. Recent studies also demonstrate that exposure of
endothelial cells to HIV results in acute and significant increases in their intracellular -
amyloid levels. Although the mechanisms underlying this phenomenon are unclear, a primary
factor is likely to be HIV-mediated oxidative stress and inflammation [191]. Thus, gp160 and
other redox sensitive proteins could misfold as a result of HIV-mediated oxidative
stress/inflammation.
It is important to note that at therapeutic concentrations, HIV protease inhibitors (PIs),
used for the highly activated antiretroviral therapy (HAART) for HIV-1 infection, are now
known to activate the UPR in macrophages. This results in ER stress, depletion of ER
calcium stores and activation of apoptosis in these cells [225]. Although the mechanisms
underlying this effect of HAART are unclear, a recent report has demonstrated enhanced -
amyloid levels in the brains of HAART-treated HAD patients. Thus, a better understanding of
the cellular and molecular mechanisms of UPR activation and ER stress and the events that
follow may provide useful insights for development of new therapeutic strategies for HAD in
HIV-AIDS.
Role of Astrocytes in HAD CNS Cell Death
Astrocytes comprise more than 50% of total cells in the CNS and they have crucial
homeostatic and redox regulatory functions that maintain neuron integrity and essential brain
function [226, 227]. Increasing evidence suggests that astrocytes play a prominent role in
HIV neuropathogenesis. In the CNS, HIV affects astrocyte functions by distinct pathways
[228, 229]. Astrocytes are natural host cells for HIV-1, particularly in advanced HAD [230].
Using state of the art technology, Churchill and coworkers demonstrated that astrocyte
infection is extensive in human patients with HAD [203], occurring up to 19% of astrocytes.
Moreover, astrocytes infection is frequently correlated with the severity of neuropathological
changes, emphasizes the important role of astrocyte infection in HAD. Since the majority of
cells in the CNS are astrocytes, they are a significant reservoir of latent HIV infection. Latent
HIV infection results in global changes in astrocyte gene expression [228]. In addition, a
subpopulation of latently infected astrocytes undergo apoptosis that correlates with severity of
HAD [149, 203].
In the brain, astrocytes are exposed constantly to whole HIV particles, gp120 alone, Tat
alone and other substances produced by HIV-1-infected microglia. It has been shown that
HIV gp120 is toxic to astroglial cells by lipid peroxidation as a result of oxidative stress. In
addition, HIV-1 efficiently binds to astrocytes and induces neuroinflammatory responses
[231]. Since neurons are not infected by retrovirus, neuronal death is most likely due to
reduced thiol support from dysfunctional astrocytes (causing oxidative stress and thiol
deficiency in neurons). It has been reported that productive infection of astrocytes with HIV-1
leads to oxidative stress and cell death and neuropathology in a mouse model for HIV
infection [232-234]. Upregulation of BiP has been documented in astrocytes in the CNS of
HIV-positive individuals [144]. Additionally, Tat released from HIV-infected astrocytes
induces mitochondrial dysfunction and neuronal death [208, 235-237]. Tat also impairs
glutamate uptake in astrocytes, exposing neurons to glutamate excitotoxicity.
Microarray analysis reveals HIV effects on gene expression in both human and mouse
astrocytes [204]. It was demonstrated that similar changes were found in HIV-1-exposed
mouse and human astrocytes in vitro, underscoring the usefulness of the mouse model for
studying HIV-1 pathogenesis. These findings strongly suggest that changes in gene
expression of astrocytes are a major component of the overall molecular profile of disease in
the brains of HIV-1-infected patients, and also suggest that exposure of human or mouse
astrocytes to HIV-1 in culture can be a useful tool for investigating the molecular and
functional changes, involved in the development of HIV-associated dementia.
Proteomic modeling of HIV-1 infected cells, in a study of astrocyte-microglia
interactions, has shown that astrocytes have a profound effect on protein expression in HIV-
infected microglia. This finding provides novel insights into the influence of astrocytes on the
onset and progression of HAD [229]. This and similar studies also provide a new perspective
on previously undisclosed pathogenic mechanisms for retrovirus-induced NDs and on the
importance of CNS cell-cell interactions.
Together the above observations underscore potential implications for therapeutic
approaches toward treating HAD. While HAART reduces HIV-1 viral load and raises CD4+
T cell counts in the peripheral lymphoid system, neurologic damage is not significantly
reduced in treated patients [238-240]. As noted above, this may be due to the fact that these
drugs themselves activate the UPR and induce apoptosis [225] and thus could have adverse
effects. It is possible that these drugs may not have therapeutic effects on HAD mechanisms,
including oxidative stress [18, 21, 172, 174, 176]. Another possibility, as mention above, is
that HAART targets replicating HIV in the CNS produced by macrophage-lineage cells but
does not target HIV latent infection in astrocytes, thereby only providing partial clinical
benefit to HAD patients.
Therapeutic Intervention
Several important criteria must be satisfied before a candidate drug against NDs mediated
by oxidative stress can be deemed suitable. 1) The drug must be able to attenuate, rather than
completely shut-off ROS production, because low (appropriate) levels of ROS are beneficial
to cells. 2) The drug should not only lower oxidation reactions but also increase the cells
capacity to cope with oxidative stress, by attenuating stress signals. 3) The drug must be non-
toxic, stable, and suitable for long-term use because of the slow and progressive courses of
most neurodegenerative diseases. 4) The drug should have a global effect in the cell. 5) The
drug should also be orally bioavailable, and be able to cross the blood-brain barrier (BBB) as
well as penetrate cell membranes. 6) The drug should be able to upregulate or stabilize Nrf2
and restore GSH levels in the cells by supplying cysteine, the precursor of GSH. This is
because cells can develop thiol deficits even during antioxidant treatment, since
neurodegenerative syndromes typically are diagnosed after they are well underway, at a time
when CNS cells are already thiol-depleted, and unable to refill their stores with their own
reducing equivalents. We have been working with two drugs that in combination may meet
these requirements. An antioxidant and redox buffer, MSL, and the antioxidant/thiol
replenishing agent N-acetylcysteine-amide (AD4) may act together to restore the redox state
and to replenish the deleted GSH.
1. Using the Antioxidant Drug MSL Alone, or in Combination with other
drugs such as AD4, for Therapeutic Intervention
Earlier work from our laboratory has shown that ts1-induced damage to the CNS and
thymus of infected mice is suppressed by treatment of infected animals with N-acetyl cysteine
(NAC) [170]. While NAC inhibits ts1-associated thymic atrophy, it has relatively limited
effects against ts1-induced ND. This could be due to its limited ability to cross the BBB [241,
242].

Figure 5.
Recently, we have reported that MSL is much more effective than NAC, or other
antioxidant that we have test in preventing oxidative stress mediated ND in ts1-infected mice
[167, 177]. MSL is unique among many antioxidants and therefore investigating its
mechanism of action in retrovirus-mediated NDs is highly significant. MSL is a phthalazine
dione redox-buffering compound (Figure 5) with a proven non-toxic quality that modulates
intracellular redox status by being able to accept and donate electrons and by scavenging free
radicals, especially superoxide and peroxinitrite. The position of the amine group on the MSL
phenolic ring enables MSL to enter cells. In cells and in animals MSL has both antioxidant
and anti-inflammatory effects. MSL scavenges free radicals (ROS and RNS) and converts
their energy into light (luminol and H
2
O). This process is reversible, and therefore the MSL
molecule can be recycled (reusable). Luminol is also a well-recognized iron chelator and
thereby preventing iron-catalyzed oxidative stress.
Experiments in mice with oxidative stress related diseases reveal that MSL: 1) is
relatively nontoxic, well absorbed and rapidly excreted upon systemic administration [243,
244]; 2) can balance disordered redox states in stressed cells by resetting proper redox
potentials. This is accomplished by redox buffering actions, by its ability to scavenge free
radicals and upregulate Nrf2 for antioxidant defense [245]; 3) decreases intracellular ROS
levels in primary astrocyte cultures infected with ts1 [167]; 4) reacts with ONOO
-
to prevent
protein nitration and oxidation in microglia cells (Qiang and Wong, unpublished data), and
reduces markers of lipid peroxidation in CNS and thymus of ts1-infected mice; 5) prevents
microglia-induced neuronal damage [246]; 6) restores mitochondrial membrane potential and
mitochondrial-induced cell death in ts1-infected astrocytes and brain slice culture
(unpublished data); 7) upregulates neuroprotective proteins such as vascular endothelial
growth factor (VEGF) and Bcl2, an anti-apoptotic factor [247]; and finally, prevents oxidative
stress not only in the CNS [167], but also in the thymus and intestine of ts1-infected mice
[168, 169].
Since cystine/cysteine availability is rate limiting for GSH production in cells, it is
possible that drugs that replenish cysteine are crucial for full protection of ts1-infected
astrocytes. As a thiol amide, NAC-amide (AD4) is less toxic than NAC and can penetrate the
BBB, cross cell membrane and supply cysteine to cells to overcome GSH depletion under
oxidative stress [241]. Therefore, this effective, penetrative and nontoxic thiol provider could
be suitable for long-term use in treating chronic NDs. AD4 has been shown to be
neuroprotective in three different mouse models of NDs [242, 248, 249]. AD4 has also been
shown to delay ts1-induced ND (Kim and Wong, unpublished data).
As mentioned above, oxidative stress is also associated with neuropathogenesis of A-T.
Several studies by others have shown that anitoxidants, such as NAC [250], EUK-189 [251],
Tempol [252] and 5-carboxy-1,1,3,3-tetramethylisoindolin-2-yloxyl (CTMIO) [253] improve
motor deficits in Atm
-/-
mice. We have also administered MSL to Atm
-/-
mice via drinking
water and found that MSL also improves motor performance in these mice (unpublished
data). These results suggest that oxidative stress is closely linked to ND in A-T, and that
antioxidant treatment could be an effective therapeutic approach for ND in A-T.
2. Using Chemical Chaperones and Proteostasis Regulators for Disease
Intervention
Cellular proteins face constant challenges to their homeostasis or proteostasis (refers to
the stability, conformation, integrity, location and function of individual proteins making up
the proteome of the cell). Defects in proteostasis occurring as a result of ER and oxidative
stress may contribute to many diseases, including NDs, immunodeficiency, atherosclerosis
[254] and cancer. Chemical or molecular resolution of ER stress can prevent misfolded
protein accumulation and facilitate protein secretion. For example, certain small molecules
that act as proteostasis regulators have been shown to ameliorate proteostasis dysregulation in
some of the most challenging diseases [255]. Administration of chemical chaperone
phenylbutyrate (PBA), which improves ER folding capacity and trafficking, reduces ER
stress and restores glucose homeostasis in a mouse model of type 2 diabetes [256]. PBA also
restores proteostasis of the misfolding-prone cystic fibrosis transductance regulator, so it is
currently being tested in clinical trials to treat cystic fibrosis [255]. Proteostasis regulators that
selectively target the folding of viral proteins have been suggested as antiviral drugs that may
prevent evolution of drug resistance [257]. Our published data indicate that PBA reduces the
accumulation of gPr80
env
in ts1-infected astrocytes and reduces the levels of the ER stress-
related chaperone BiP [Kuang et al, Neurochem Int 2010]. As a result, levels of the survival
factor Bcl2 increases and the apoptotic markers Bax and caspase 3 decrease allowing
extended survival of ts1-infected cells. PBA also delays the onset of ND in ts1-infected mice
[Kuang et al 2010 Neurochem Int [258]. The advantage of chemical chaperones is that they
are not disease specific, but instead that they target a common pathway to many diseases (i.e.
proteostasis imbalance). Thus, restoration of proteostasis, mediated by proteostasis regulators,
may ameliorate some of the most important diseases of our era, including HAD and AIDS.
Interestingly, small molecules have been identified that target HIV envelope proteins for ER-
associated protein degradation, and they have been shown to be effective for HIV therapy
[259]. Taken into consideration of the different effects of MSL, AD4 and PBA (Figure 6) a
combined treatment with these three drugs with different targets may provide better
protection relative to treatment with any one of the drugs alone, not only for survival but also
in the major marker analysis.

Figure 6.
3. Potential Therapeutic Intervention Using NSC Transplantation and
Antioxidants
Since self-renewal and proliferation of neural stem cells are defective in ATM deficiency,
a NSC-based treatment may be beneficial to this genetic mutation-related ND. NSCs possess
a range of actions that can be potentially used for therapy [260]. They are capable of self-
renewal, and can differentiate into cells of astroglial and neuronal lineages in the CNS [261].
In addition, they can readily proliferate ex vivo, and when transplanted into diseased brains,
where they migrate and differentiate according to cues from host tissues, appear to capable of
affecting host cells in the recipient brain.
Recent studies imply that NSCs may hold promise for therapeutic treatment of human
genetic diseases resulting in NDs, such as in A-T. The first study involves the nervous (nr)
mutant mice, in which, like in ATM deficiency, the Purkinje neurons (PN) become abnormal
and dysfunctional and a majority of these cells die by the fifth week [262]. By transplanting
normal NSCs into the cerebellum of nr mutant mice, PN function is repaired by the
transplanted NSCs, not just by cell replacement, but also by rectifying their gene expression
and restoring defective molecular homeostasis due to the gene defect. In another study,
intracranial transplantation of normal NSCs was used to treat mice in a model of the human
neurodegenerative disease called Sandhoff disease [260]. This study shows that the
transplantation of normal NSCs into disease brains delays disease onset, preserves motor
function, and prolongs survival of the diseased mice. These two studies show that NSCs may
have a broad repertoire of therapeutic actions, of which neuronal replacement is but one.
Thus, NSCs may also help in formulating a rational multi-factorial strategy, including
combination with antioxidants, for treatment of NDs.
Conclusion
This chapter focuses on two animal models of NDs that show some commonality
involving oxidative stress, ER stress, and mitochondrial dysfunction. These findings also
suggest that ROS signals can engage specific pathways to generate their effects in different
cell types in the CNS. This is not only true for these two particular models, but increasing
bodies of evidence suggest that multiple human NDs and other diseases are affected or
regulated by this culmination of cellular stress. Thus, the inevitable problem in almost all
NDs is accumulation of both ROS with impaired redox homeostasis and accumulation of
abnormal misfolded proteins with stress on ER and mitochondrial functions. Activation of
inflammatory response, apoptotic pathways, and cell death is the ultimate result.
Understanding theses multifaceted pathways and the intricate parts involved with this
mechanism will be beneficial for us to develop basic therapeutic strategies against ND in
general. We can target the oxidative stress, or ER stress, or mitochondrial stress directly and
many therapies may need a combination of these. Small molecule nontoxic drugs that can
effectively pass the blood brain barrier and alleviate these stresses may be extremely
beneficial in the maintenance and treatment of several NDs and promote neuronal cell
survival. We have shown that treatment with the redox-active small molecules, the
phthalazine dione (MSL) fully prevent the neurodegenerative syndrome induced by infection
with the murine retrovirus mutant ts1. Both the oxidative stress and the ER stress with
excessive accumulation of viral mutated proteins in ER as well as the neuronal loss and
gliosis were fully prevented by these treatments. In the murine model of A-T, we have also
observed that the global oxidative stress with neuronal damage and the accompanying
gliosisis can also be fully prevented by treatment with MSL. Potential therapy treatment using
drugs that supply cysteine to cells to overcome GSH depletion under oxidative stress, as well
as chemical chaperone treatment against NDs are discussed. Further understanding of the
mechanism of action of these drugs singly or in combination can more specifically target
these drugs for more effective treatment. Finally although many questions remain to be fully
addressed, stem cell-based therapy represents a potential highly rewarding treatment of
genetic-related NDs. Therefore further research into this area may provide rationale for new
therapies in the future.
Acknowledgments
The authors thank Drs.Virginia Scofield and Mingshan Yan as well as Dr. Joanne Ajmo
and Mr. Mark Henry from Bach Pharma for their helpful support and discussion. The authors
also thank Ms. Shawna Johnson for her assistance of the preparation of this chapter. This
work was supported by NIH Grants MH071583 and NS043984 (awarded to Dr. Paul Wong)
the University of Texas MD Anderson Cancer Center Support Grant CA16672. Support was
also provided by the Longevity Foundation of Austin, Texas and The A-T Childrens Project
of Deerborne Florida.
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Chapter 2
Mechanisms of the Motoneuron
Stress Response and Its Relevance
in Neurodegeneration
Mac B. Robinson, David J. Gifondorwa and Carol Milligan
*
Dept. of Neurobiology and Anatomy, Wake Forest University
School of Medicine, Winston-Salem, North Carolina, USA
Abstract
Preserving motoneuron viability and function during disease or after traumatic injury
is an intense area of research focusing on both the molecular mechanisms of degeneration
and therapeutic interventions to prevent it. Understanding how motoneurons sense and
respond to injury or pathology may help us identify potential targets for therapeutic
intervention. The motoneuron stress response or heat stress response (HSR) has been an
area of investigation spanning now well over a decade and has explored the role of heat
shock protein (HSP) expression during physiological stress and in animal models of
neurodegenerative disease. What we have found from these studies is that, in the midst of
a physiological stress, motoneurons rarely activate a classical stress response as
characterized by increased expression of Hsp70. It has been proposed that this lack of
stress response activation could contribute to pathological motoneuron dysfunction and
degeneration. Understanding the molecular mechanisms responsible for this phenomenon
may provide insights as to why motoneurons are the pathological hallmark in
amyotrophic lateral sclerosis (ALS) and other neurodegenerative conditions.

Keywords: motor neuron, stress response, heat shock proteins, amyotrophic lateral sclerosis,
neurodegenerative disease, heat shock protein 70.
Mac B. Robinson, David J. Gifondorwa and Carol Milligan 46
Introduction
The ability of a cell to properly sense and respond to stress is critical for the cells and
indeed the organisms ultimate survival. One way cells respond to stress is through the
activation of intracellular signaling pathways that, through a series of subsequent
phosphorylation events, influences macromolecular interactions and de novo protein synthesis
that are meant to abrogate the deleterious effects of the stress, or in some cases exacerbate
them; therefore, facilitating the death of the cell. One beneficial response that is influenced by
these intracellular signaling pathways is the heat stress response (HSR). Heat shock proteins
(HSPs), the main product of the HSR, protect cellular proteins from damage by acting as
molecular chaperones; binding to and refolding the damaged proteins, hence preserving their
function. Alternatively, Hsps may facilitate the removal of irreparably damaged proteins
preventing their toxic accumulation (Luders et al., 2000; Kaushik and Cuervo, 2006). While
initially discovered in response to elevations in the thermal environment (Ritossa, 1962;
Ritossa, 1996), the HSR is activated in response to other stresses including injury, oxidative
stress, exposure to heavy metals, antibiotic treatment, infection, and UV radiation exposure
(Schlesinger, 1990; Welch, 1992; Morimoto et al., 1997; Kregel, 2002). Since its discovery, a
field of research has developed focusing on the products and mechanisms of the HSR in
virtually all realms of the biological sciences.
Work in various cell types following numerous insults has provided results leading to an
understanding of some of the mechanisms involved in the HSR (see figure 1). With continued
investigations, however, a number of examples have been found that suggest that the HSR is
not as homogenous as once thought. It is now known that not all cells respond to a particular
stress in the same manner, where the response can vary from eliciting a partial response to not
exhibiting a stress response at all (Morimoto and Fodor, 1984; Manzerra and Brown, 1992;
Mathur et al., 1994; Tacchini et al., 1995; Marcuccilli et al., 1996; Goldbaum and Richter-
Landsberg, 2001; Kaarniranta et al., 2002; Kalmar et al., 2002; Batulan et al., 2003; Robinson
et al., 2005). The extent to which neurons, and specifically motoneurons mount an HSR is
variable and atypical (Manzerra and Brown, 1992; Kalmar et al., 2002; Batulan et al., 2003;
Robinson et al., 2005). The molecular events modulating this lack of response or altered
response have yet to be elucidated, but current data suggest a number of possibilities
including but not limited to aberrant regulation of the intracellular signaling cascades
associated with activation of the HSR, insufficient HSF activation and stress buffering.
The clinical relevance of this line of study is certainly one of the more compelling
examples given the pathology of motoneuron diseases including amyotrophic lateral sclerosis
(ALS) that can include protein aggregation and increased ROS, all of which can lead to
motoneuron cell death, but can be ameliorated to some extent by HSPs. It has been proposed
that, if motoneurons were able to readily initiate a full HSR, it may positively influence the
outcome of motoneurons during disease (Okado-Matsumoto and Fridovich, 2002). Indeed, as
will be discussed, attempts to modulate the HSR have shown promise in mitigating the
pathology in animal models of ALS and may be a promising therapeutic avenue. However,
the effect of HSR modulation at this point is global and not targeted directly at the
motoneuron; therefore, discussion will also be made of the importance of the knowledge we
gain from these studies and where the protective effect may be derived with treatments that
induce the stress response.
Mechanisms of the Motoneuron Stress Response and Its Relevance 47

Figure 1. During the cells basal state or unstressed condition, the heat shock transcription factor, HSF,
and heat shock proteins, mainly Hsp70 and Hsp90 reside in the cytoplasm in a heterocomplex. This
relationship renders the HSF in an inactive monomeric state. However, at the outset of a stress, this
complex dissociates, most likely due intracellular signal and protein misfolding. With the HSF/HSP
complex broken the HSF is free to trimerize and translocate to the nucleus. The prototypical heat stress
response is characterized by the activation of a number of signal transduction pathways. These
molecules can act to phosphorylate the HSF trimer, most likely while bound to the heat shock element
in the nucleus. This inductive phosphorylation creates a transcriptionally competent HSF trimer.
Transcription of HSPs ensues, increasing the free pool of HSPs and potentially mitigating the stress on
a number of levels from refolding, protecting or degrading irreparably damaged proteins to intercepting
both apoptotic and non-apoptotic cell death pathways. Mechanisms of termination are still a matter of
debate; however, there is recurring evidence of regulation by the inducible HSPs, especially Hsp70
and/or Hsp90 in conjunction with p23. Motoneuron HSR dysfunction may occur on a number of levels.
Signaling activation in motoneurons appears to be dampened compared to other cell types.
Additionally, when it is activated, it appears unique. Activation of p38 appears to be a repeatable event
during neurodegeneration and may be an attempt to activate Hsp25 to modulate antioxidant defenses.
Dampened signaling could result in a lack of inducible phosphorylation. In support of this,
overexpression of wtHSF does not facilitate activation of the response. Furthermore, increased basal
levels of Hsc70 may act as an endogenous stress buffer to instantly protect motoneurons from most
stress, allowing the large cell to conserve much needed resources in the midst of a stressor. See
Manzerra and Brown, 1992; Batulan, et al. 2003; Morimoto, 1993; Pirkkala, et al. 2001; Saleh, et
al.2000; Beere, et al. 2000, and Ruchalski, et al. 2006.
Signaling Influences on the Stress Response
There are 4 main steps involved in the activation of the HSR: 1) release of the heat shock
transcription factor (HSF) from multi-chaperone complexes, 2) homotrimerization and
nuclear translocation of HSF, 3) binding of HSF to the heat shock element (HSE), and 4)
transcriptional activation of HSF by phosphorylation and transcription of heat stress genes.
Some of these steps appear to be independent events in the activation of heat stress genes and
can be uncoupled through the use of certain chemicals (Jurivich et al., 1992). Additionally,
intracellular signaling molecules play a critical role in the activation and repression of HSF.
HSR activation and subsequent production of Hsps is a transcriptionally regulated event
though the kinetics of the activation can vary between cell types. Additionally, at least one
cell line has shown a translational mechanism of activation (Kaarniranta et al., 2002), but this
seems an exceedingly rare event. The transcription factor responsible for HSP activation is
the HSF. Mammals have 3 HSFs, HSF1, HSF-2 and HSF-4 with HSF-1 being the stress
inducible HSF and HSF-2 being involved in development (Pirkkala et al., 2001). HSF-4
appears to be expressed in a cell type specific manner. These proteins can be regulated by
alternative splicing and as recent reports indicate, by heterocomplexing within the HSF
family (Ostling, et al. 2007). Most organisms harbor the stress activated HSF, HSF-1, some
other stress activated HSF, or an HSF (Drosophila only has one) that can function in all
aspects of cell development and stress (Birch-Machin et al., 2005). Other proposed stress
activated HSFs, like HSF-3 in the embryonic chick, are most likely regulated similarly. HSF-
1 and HSPs are in heterocomplex when the cell is in a basal, unstressed state. This complex
prevents the homotrimerization step of HSF-1 activation (Santoro, 2000; Pirkkala et al.,
2001). The molecular mechanisms that dissociate this complex are not completely
understood, but presumably, once damage occurs to cellular proteins, the free pool of Hsps
becomes depleted with chaperones being bound to misfolded or damaged proteins (Morimoto,
1993). This depletion triggers a release of chaperones from the HSF/HSP complexes
liberating HSF-1. Subsequently, HSF-1, through what is proposed to be a conserved redox
mechanism, associates into trimers. (Zhong et al., 1998; Ahn and Thiele, 2003). These trimers
translocate to the nucleus and associate with the heat shock elements (HSEs) of stress
inducible genes containing the consensus sequence nGAAnnTTCnnGAAn (Morimoto, 1993).
This process seems not only highly conserved, but also reproducible under a number of
circumstances. Purified HSF-1 can trimerize and bind to the HSE in vitro in response to
increased calcium, heat, or reactive oxygen species (ROS), (Mosser et al., 1990) suggesting
this initial step is fairly liberal in its execution to provide ample response to cover a diversity
of insults. Additionally, a recent report has described a role for HSF-2 in stress induced HSR
activation. Traditionally, thought of as an HSF that was critical during development rather
than mitigating stress, this HSF may associate with HSF1 to modulate expression of heat
stress inducible genes (Ostling et al., 2007). HSF1 associating with other HSFs is not unheard
of. In avian cells, HSF3 and HSF1 have been shown to be in association, though the role of
this association is unclear, but most likely a regulatory event (Nakai et al., 1995; Tanabe et
al., 1998). However, this may be relegated to embryonic and undifferentiated cells. Another
report indicates that in the mature avian system HSF3 may be utlized by mature blood cells
while HSF1 is the main player in the brain (Shabtay and Arad, 2006). This would suggest
that, in addition to cell type and transcriptional factors controlling HSF activation, regulation
may also occur on a developmental level. One of the most interesting examples of a complex
family of HSFs lies in Arabidopsis thaliana, an organism with nearly 21 HSFs organized in to
3 groups and 14 classes (Nover et al., 2001). A recent study increased that number to 22 and
also found 25 HSFs in rice (Guo, et al. 2008).
While physical translocation to the nucleus and binding of HSF to the HSE are obvious
critical steps, this is a transcriptionally impotent arrangement and further modification
through phosphorylative modification of serine residues in the HSF-1 regulatory domain is
needed to fully activate the system. For such regulation, one looks toward the many
intracellular kinases within the cell responsible for phosphorylative modifications and
generation of intracellular signals. Regulation of HSF-1 activity may be attributed to a
number of intracellular kinases including, but not limited to extracellular signal regulated
kinase (ERK), phosphatidylinositol 3 kinase (PI-3K)/protein kinase B (Akt), glycogen
synthase kinase -3beta (GSK-3B), calmodulin dependent kinases II and IV (CAMKII and
CAMKIV), p38 mitogen activated protein kinase (MAPK) and c-jun N-terminal kinase (JNK)
(Bijur and Jope, 2000; Park and Liu, 2001; Pirkkala et al., 2001; Taylor et al., 2007). Serines
303, 307, and 363 are thought to be constitutively phosphorylated and exert a repressive
effect on HSF-1. However, phosphorylation of serine 230 appears to be associated with
transcriptional competence (Pirkkala et al., 2001). The activity of PI-3K and p38 may result
in positive regulation of the HSR. Negative regulation, however, comes from a number of
sources including ERK, GSK-3B, and CAMKII suggesting a fairly tight regulation of the
activation of the response and an even tighter redundant negative regulation (Soncin et al.,
2000). In addition to phosphorylation, sumoylation of lysine 298 on HSF also appears to be
critical in activating the response (Hong et al. 2001). Interestingly, the repressive
phosphorylation site S303 must be phosphorylated for this activating sumoylation to occur
(Hietakangas, et al. 2003).
The idea of stress kinase activation (pJNK and p38) and subsequent, linear activation of
the HSR provides interesting discussion simply because, at least in the case of JNK, the
relationship is not linear. Indeed, JNK is thought to be involved as a negative regulator of
HSF activity. Overexpression of JNK1 and JNK2 negatively regulates HSF-1 activity.
Paradoxically, overexpression of JNK1 and JNK2 also induces the expression of reporter
constructs carrying an Hsp70 promoter. (Park and Liu, 2001). Yet, SP600125, a JNK
phosphorylation inhibitor, can decrease the amounts of hyperphosphorylated HSF-1 (Park and
Liu, 2001; Kim et al., 2005). These contrasting results with JNK manipulation make this
kinase an interesting subject in the study of the HSR. Future experiments utilizing JNK
knock-out mice may be very informative in assigning JNKs ultimate role in activation of the
response. The MAPK, p38 is also an important component in the response. For one, it may be
a first line of defense given a triggering event of the response is oxidative stress and Hsp27
expression has the ability to modulate other antioxidant molecules (Arrigo, 2001).
Additionally, p38 may be required for activation of pAkt that may fully activate the response
(Mustafi, et al. 2009). This would suggest that the stress response, at least in some cell types,
may be a biphasic event. This data, and other data to be discussed, suggest a level of
complexity previously not thought of in terms of the levels of activation.
Once the HSF-1 trimer is activated, Hsp production proceeds. The termination of the
response in regulated on some level by the induced chaperones themselves (Mosser et al.,
1993; Lee and Schoffl, 1996; Satyal et al., 1998). It appears levels of the inducible Hsp70
may be an important factor in limiting the intensity of the response. Indeed, overexpression of
Hsp70 prior to heat stress results in a dampened response. Once free pools of HSPs
accumulate to high levels, they being to associate with free HSF-1. In addition to other
proteins, namely HSBP1, the HSF1 trimer is dephophorylated, released from the HSE and
complexed with chaperones returning the cell to a basal state.
Heat Shock Transcription Factors and Regulation of the
HSR in the Nervous System
The heat shock transcription factors (HSFs) appear to be responsible for regulating Hsp
expression and the HSR. While there is conserved identity in protein sequence, the HSFs are
not functionally interchangeable, and regulation of HSF activity is not consistent for each. For
example, There are four HSFs, and all four have demonstrated roles in the nervous system.
HSF1 becomes functional as a trimer, whereas HSF2 resides as a nonfunctional dimer then
trimerizes under the appropriate stimulus (There are four characterized HSFs). HSF1 appears
to regulate stress induced expression of HSPs. HSF1 null mice develop and are born alive,
and endogenous expression of HSPs is consistent with wild-type animals. The animals or
cells isolated from the animals; however, cannot mount an HSR in response to heat stress.
HSF3 is unique to avians and appears to function cooperatively with avian HSF1. HSF2 does
not appear to be involved in increased expression of Hsps in response to stress, but rather may
dictate developmental expression of the proteins. In the CNS, HSF1 null adult mice exhibit
progressive loss of myelin and astrogliosis. These events are enhanced when HSF2 or 4 are
also knocked-out (Homma et al, 2007). In rodents, HSF1 and HSF2 are expressed in neurons,
astrocytes and oligodendrocytes with HSF2 appearing critical for CNS development (Walsh
et al., 1997; Stacchiotti et al., 1999; Wang et al., 2003; Chang et al., 2006). HSF4 is expressed
in the developing CNS and certain neuronal populations (Hu and Mivechi, 2003), and
mutations in the DNA binding domain of HSF4 are associated with inherited cataract
formation (Bu et al., 2002). The specific roles of each HSF are unclear at this point; however,
it appears as if each regulates distinct gene expression during development or in response to
stressful stimuli. The genetic regulation is further enhanced when HSFs work in concert or in
opposition (reviewed in Akerfelt et al., 2007). Furthermore, it is not known if the CNS
abnormalities observed in the mutant mice are because of insufficient HSP or other gene
expression.
The Motoneuron Stress Response
Examination of motoneuron signaling and the HSR are associated with some unique
challenges. The main hurdle is the difficulty in culturing motoneurons in sufficient amounts
to perform the biochemical and molecular analysis to fully characterize motoneuron signaling
and the motoneuron HSR. However, using immunological approaches and in situ
hybridization in vivo and in vitro systems have provided a relatively informative picture of
how motoneurons respond to loss of trophic support, axotomy, hyperthermia, and in
neurodegenerative disease (Manzerra and Brown, 1992; Kalmar et al., 2002; Batulan et al.,
2003; Batulan et al., 2005; Batulan et al., 2006). As for in vitro approaches, some
investigators have used dissociated spinal cord or spinal cord slice cultures (Batulan et al.,
2003; Batulan et al., 2005; Batulan et al., 2006; Taylor et al., 2007). This has allowed for
some extrapolation as to how certain processes may be executed; however, experiments to
fully examine the mechanistic features of process in specific cell types, namely motoneurons
are difficult in these systems.
Another approach is to use a culture of purified motoneurons. While the cultures allow
for more biochemical analysis including western blots, RNA analysis, and subcellular
fractionations, they are very time-consuming in order to collect sufficient material for
analysis. Many investigators utilize chick motoneuron cultures because they are amenable in
yielding sufficient material for analysis and are more economically feasible than similar
experiments using mammalian systems. With regard to the HSR, however, interpretations of
results must consider that heat shock factor-3 (HSF-3) appears to be the main stress inducible
transcription factor used in the embryonic avian system, rather than heat shock factor-1 (HSF-
1) that is used in mammals. The significance of the use of HSF1 vs. HSF3 is not known
because there appears to be cooperation between the two factors, atleast in embryonic
development (Nakai et al., 1995; Kawazoe et al., 1999). Intracellular signaling is highly
conserved however, so some of the mechanisms involved in activation the HSR in chick may
be conserved in mammals (Pirkkala et al., 2001). For instance, cultured muscle cells from the
chick and from the mouse appear to respond to heat stress in a similar manner albeit at a
different threshold (unpublished observations).

Figure 2. HSFs regulate gene expression independent of HSR genes during development and for
physiological homeostasis. Heat Shock Transcription Factors, especially HSF1 are thought to be key
regulators of the HSR and expression of Hsps. These factors are also key regulators of other gene
expression specific to developmental and physiological events as illustrated in the figure (reviewed in
Akerfelt et al., 2007)

Figure 3. Diagram to illustrate the complexity of the cellular stress response. Motoneurons, like many
other cell types encounter potential toxic changes in the extracellular environment. These changes result
in alterations in plasma membrane structure (1), signal transduction pathways (2), generation of
intracellular ROS and Ca
+2
imbalances (3), and protein denaturation or unfolding (4). The response to
these challenges include increased expression of HSR genes, including Hsps regulated in part by
interactions between the HSFs and other binding proteins (5). The Hsps are critical to refolding of
denatured proteins (6). Denatured proteins not refolded by Hsps are degraded by the ubiquitin-
proteasome pathway (7). When this pathway become damaged, perhaps by being overloaded with
denatured proteins, protein aggregation can occur (8). Protein aggregation may contribute to damage to
intracellular organelles, further compromising the cell. Additionally, intracellular ROS and Ca+2
imbalances can contribute to mitochondrial and ER stress (red arrows). The ER can respond with the
unfolded protein response (9) that may contribute to increased expression of HSR proteins; however, it
also results in an overall decrease in protein translation. See Pirkkala et al., 2001, Boyce and Yuan,
2006 and Lindholm et al., 2006 and Prahlad and Morimoto, 2009 for reviews
Some observations of induction or non-induction of a motoneuron HSR suggest that
HSF1 may be inappropriately or not phosphorylated sufficiently to confer transcriptional
competence (Batulan et al., 2003). Another theory is that alternate signaling mechanisms exist
for specific Hsp regulation in motoneurons. Constitutively active CAMKIV in motoneurons
appears to regulate Hsp70 expression, whereas this kinase has no effect on Hsp70 expression
in fibroblasts (Taylor et al., 2007). Surprisingly, co-injection of a construct encoding wtHSF1,
in addition to CA-CAMKIV, abolished this effect. This would suggest that CAMKIV may be
activating Hsp70 expression independent of HSF1.
Motoneurons are one of the largest cells in an organism and therefore thought to have a
higher metabolic demand than most cells. During embryonic and neonatal development,
motoneuron survival is dependent on target derived trophic support and in the absence of
trophic support they die (Oppenheim et al., 1988). Motoneurons, especially those that appear
to be susceptible in ALS do not express Ca
+2
binding proteins calbindin-D28k and
parvalbumin, suggesting that they have a diminished ability to buffer cytosolic Ca
+2
(Shaw
and Eggett, 2000). Motoneurons also express the Ca
+2
permeable AMPA receptors. They also
appear to be very vulnerable to mitochondrial dysfunction (reviewed in von Lowinski and
Keller, 2005). Given these facts, one would expect this particular cell to have the HSR
molecular machinery poised for activation.
Motoneurons in culture without trophic support die; however, it takes approximately 16
hours for cell death associated events to occur (Li et al., 2001). If availability of trophic
support is so critical for survival, one might reason that during the initial 16 hours, the cells
may mount a HSR in an attempt to promote survival. This however, does not appear to be the
case (Robinson et al., 2005). Additionally, motoneurons barely initiate an HSR in response to
normal heat stress and no response to H
2
O
2
treatment (Robinson et al., 2005; 2007).
Furthermore, stressed induced induction of the HSR in motoneurons does not appear to be
protective. For example, after exposure at 45
o
C motoneurons will increase expression of
Hsp27, 40 and 90, but not 70 and therefore are not considered to mount a HSR (Robinson et
al., 2005). If however, the cells are exposed to 50
o
C, all Hsps, including 70 have increased
expression. Despite the increased expression of Hsp70, there is no protection conferred when
the cells are exposed to a subsequent stress such as H
2
O
2
(Robinson et al., 2007).
Motoneurons express high amounts of endogenous Hsc70 and this has been proposed as a
reason why motoneurons seem resistant to HSR activation (Manzerra and Brown, 1996).
Many functions of both the inducible and constitutively expressed proteins are redundant and
the ability of free Hsc70 to dampen the stress response may be one of those redundant
functions. Additionally, increased Hsc70 could act as a stress buffer. It could make sense for a
cell the size of a motoneuron to have a large amount of free chaperone available for use so not
to deplete resources for the physiological needs of the cell. Interestingly, one molecular event
observed during the HSR, translocation of Hsc70 to the nucleus, is retained in motoneurons
(Manzerra and Brown, 1996). Therefore, motoneurons still recognize and, in part, respond to
stress in what would be a typical manner early in the execution of the HSR, yet a molecular
trigger is not occurring to sufficiently activate the HSR system.
The Heat Stress Response in Motoneuron Injury and Pathology
Probably the most common in vivo motoneuron injury model is sciatic nerve axotomy.
Motoneurons, like DRG, the other neuronal component affected by axotomy, respond through
the activation of JNK, p38, MAPK and PI-3K after axotomy (Murashov et al., 2001; Yang et
al., 2006). Additionally, downstream effectors like c-jun exhibit increased phosphorylation
(Brecht et al., 1997). What does this mean for Hsp expression? It appears that in the case of
axotomy, Hsp27 or Hsp25 and Hsp90, but not Hsp70 are upregulated in the motoneuron
(Murashov et al., 2001; Kalmar et al., 2002; Tidwell et al., 2004). Interestingly, p38 appears
to be a critical link to this stress response in that Hsp25 expression can be inhibited by the
p38 inhibitor, SB203580 (Murashov et al., 2001). These data clearly show that motoneurons
do respond to injury and Hsp expression can be increased. However, Hsp70 appears to not be
a component of this response.
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder affecting both upper
and lower motoneurons, resulting in progressive muscle weakening and loss of motoneuron
function, ultimately leading to paralysis and death within 3-5 years of diagnosis. The
pathology of the disease can be mimicked in transgenic mice harboring a mutation in the
Cu/Zn superoxide dismutase-1 (SOD-1) gene. Altered Hsp levels during the disease process
have been observed; however these changes do not, in most cases, appear to coincide with
any overt signaling changes.
Aggregated proteins may act as a chaperone sink and may be one mechanism by which
neurodegerative diseases may alter HSP levels (Okado-Matsumoto and Fridovich, 2002). The
free pools of HSPs are depleted by misfolded or damaged proteins and sequestered in the
pathological aggregates. Hsp70 is a component of these aggregates as are other chaperones
including glucose regulated protein 78/BiP (GRP78), Hsp25 and Hsp 105. (Yamashita et al.,
2007) (Maatkamp et al., 2004; Strey et al., 2004). Individual chaperone levels can decrease as
much as 50 %. Despite this there appears to be no compensatory activation of the response to
restore the free pools of chaperone resulting in an arguably pathological situation. These data
punctuate the fact that despite cellular stresses that would likely cause the demise of the
neuron, motoneurons seem to be refractory to activating a stress response.
In the mutant SOD1 mouse model there are numerous events that suggest activation of an
HSR. Stresses including mitochondrial swelling and vacuolization and mutant SOD1
inclusions/aggregates and toxicity providing ample opportunity for cellular dysfunction and
cell stress (Manfredi and Xu, 2005). While an overt HSR including increased expression of
Hsp70 does not appear to be activated, an unfolded protein response has been suggested to
play a role. The endoplasmic reticulum (ER) and Golgi are thought to be potential targets of
mutant SOD1 toxicity by its accumulation of misfolded mutant SOD1 that then initiates an
ER stress response that may contribute to pathogenesis in ALS (Tobisawa et al., 2003; Atkin
et al., 2006; Kikuchi et al., 2006; Nagata et al., 2007; Urushitana et al., 2008). Although these
previous studies have focused on the putative role of ER stress in the progression vs. the onset
of disease in ALS mice, a recent study has provided evidence consistent with ER stress being
a contributor to early disease onset in mouse models of ALS as well as playing a role in the
selective early vulnerability of motoneurons (Saxena et al., 2009).
The HSR as a Therapeutic
Overexpression of individual chaperones, particularly Hsp70, was one of the first
experimental measures used to assess the therapeutic role for Hsps. The emphasis on the
Hsp70 family of proteins stems from its abundance during the HSR and the multitude of
effects these proteins have on not only cell death pathways, but also many physiological
processes such as pathological aggregation (Sharp et al., 1999; Muchowski et al., 2000;
Muchowski, 2002). Additionally, therapeutic preconditioning, a means by which one stresses
cells to increase the levels of HSPs in the cell, but maintain cell viability, has also been used
to demonstrate the beneficial effects of the stress response under conditions which would
normally be cytotoxic. Overexpression of mutant superoxide dismutase-1 (mSOD1) and
Hsp70 in the same cell show increased survival and reduced cellular aggregation formation
(Bruening et al., 1999). These and other studies have contributed to a wealth of literature
demonstrating the protective affects of Hsp70 and providing some hope that modulating the
levels of Hsp70 may be beneficial in particular neurodegenerative diseases. However, when
mSOD1 mice are crossed with mice that ubiquitously overexpress Hsp70 no benefit was
observed (Liu et al., 2005). Overexpression of another potent anti-apoptotic chaperone,
Hsp27, has also been investigated and this chaperone also did not increase their lifespan
(Krishnan et al., 2008). However, a recent study from our laboratory using purified Hsp70
showed a benefit to mSOD1 G93A mice when Hsp70 was given by intraperitoneal injection
(Gifondorwa et al., 2007). This raises the common question of the method of administration
and presents the possibility of an unknown effect exerted by extracellular Hsp70. Another
question is whether overexpression of a singular chaperone is sufficient to rescue the mSOD1
phenotype. In support of multiple chaperone overexpression, when Hsp70 and Hsp27 are
overexpressed together a delay in pathology in the G93A mouse is observed (Patel et al.,
2005). Additionally, a recent study using arimoclomol, a hydroxyl-amine derivative and
coinducer of the HSR, to increase multiple Hsps at the time of disease onset in the mSOD1
G93A did result in some rescue of motor function and pathology (Kalmar et al., 2008).
Additionally, this study used a sufficient number of animals to properly control for
background noise influencing the results, a recent criticism of past mSOD1 mouse studies
(Benatar, 2007). The arimoclomal study suggests that not only may regulation of multiple
Hsps be critical, but intervention with the proper pharmacological treatment at disease onset
may be plausible.
This leads us to an area of study that has gained momentum recently and that is the use of
molecules that can alter cell physiology in a way to make a cell more responsive to stress or
allow a stress response to be activated pharmacologically. The molecules that perform this
function are known as coinducers. A number of molecules have been shown to be coinducers
of the HSR including antibiotics, hydroxylamines, alcohols, and non-steriodal anti-
inflammatory drugs (NSAIDS) (Jurivich et al., 1992; Kalmar et al., 2002; Kieran et al., 2004;
Batulan et al., 2006; Salehi et al., 2006). The overexpression of multiple Hsps by the use of
coinducers in a primary cell culture model of ALS has been shown to be highly
neuroprotective in some instances (Batulan et al., 2006). However, some of these molecules
can be toxic to motoneurons so care must be taken in the risk/benefit analysis in drug choice.
Given this, co-inducers are promising pharmacological intervention in the treatment of
neurodegenerative disease. Interestingly, a recent report indicates that HSF1, the inducible
transcription factor for Hsps, is a target of Riluzole, the only FDA approved drug to treat ALS
(Yang et al., 2008). Whether this targeting will have any effect on an affected motoneuron is
unclear. While the study shows Riluzole can activate a reporter construct driven by an Hsp70
promoter and may increase levels of Hsp70 under heat stress conditions in HELA cells, the
motoneuron is clearly a unique cell and it relation to a cell line mechanism may be fairly
disparate.
Discussion
Our current understanding of the motoneuron HSR suggests that these cells do not mount
a response that is sufficient or beneficial for protecting the cell. This logic may be faulty,
however; because our understanding of the mechanisms regulating HSRs in motoneurons and
other cells is not complete. Our definition of HSR in motoneurons is therefore limited and
perhaps incorrect. Signal transduction regulation of HSF activation/inactivation varies
between cells, and even within the same cell under different conditions. Furthermore, the
HSFs regulate expression not only of genes involved in the response to potentially toxic
stimuli, but also genes required for cell maintenance and developmental, differentiation
events (Westerheide and Morimoto, 2005; Akerfelt et al., 2007; Morimoto, 2008). In fact, a
recent study in Drosophila, an organism with only one HSF, shows that during heat stress the
HSF targets nearly 200 genes (Birch-Machin et al., 2005). Additionally, Hsp expression and
function appears to be developmentally regulated and cell type specific. For example, in
rodents, Hsp70 expression is not induced until the third postnatal week. In chick
lymphocytes show increased expression of all Hsps while reticulocytes only show increased
Hsp70. Furthermore, in quail Hsp25 is not produced in myotubes, but is expressed in
undifferentiated myoblasts (reviewed in Linquist 1986). It is also possible and perhaps
probable, that a motoneuron HSR may differ depending on if the potentially toxic stimuli is
delivered extracellularly (e.g., H
2
0
2
administration glutamate toxicity) or intracellularly (e.g.,
overexpressing of a mutant protein). Therefore, while examining the expression of HSPs in
the stress response is a good first step, clearly we have a long way to go until we fully
understand the scope and necessity of the protein constituents activated by a cell with
multiple HSFs. Nonetheless, careful characterization of these responses in individual cell
populations and the role these responses play in neurodegenerative disorders is the critical
first step if therapeutic approahes are going to be developed.
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In: Neurodegeneration: Theory, Disorders ISBN: 978-1-61761-119-3
Chapter 3
Methylene Blue Induces Mitochondrial
Complex IV and Improves Cognitive
Function and Grip Strength in old Mice
Afshin Gharib
2,3
and Hani Atamna
1
*
1
Department of Basic Sciences, Neuroscience, The Commonwealth
Medical College, Scranton, PA 18510
2
Children's Hospital Oakland Research Institute (CHORI), CA 94609.
3
Dominican University of California, San Rafael, CA 94901
I. Abstract
Methylene blue (MB) is very effective in delaying cellular senescence and enhancing
mitochondrial activity of primary human embryonic fibroblasts. At nanomolar
concentrations, MB increased the activity of mitochondrial cytochrome c oxidase
(complex IV), heme synthesis, cell resistance to oxidants, and oxygen consumption. MB
is the most effective among the many agents that has been are reported to delay cellular
senescence. We extended these in vitro findings to the investigation of the effect of long-
term intake of MB in old mice. We administered MB, in the drinking water (250 M), to
old mice for 90 days. In vivo, MB prevented the age-related decline in cognitive function
and spatial memory. MB also prevented the age-related decline in grip strength.
Interestingly, MB resulted in 100 % and 50 % increases in complex IV activities in the
brains and hearts of old mice, respectively. The age-related decline in protein content of
the brain was prevented by MB. We also found a 39 % decrease in brain monoamine
oxidase (MAO) activity in old mice treated with MB while aging or MB did not affect the
activity of brain NQO1. Our findings suggest that the in vitro model for cell senescence
may be used for fast and reliable screening for mitochondria-protecting candidate agents
before testing in animal models. The study also demonstrates simultaneous enhancement

*
Corresponding author: Assistant Professor of Biochemistry&Neuroscience, Department of Basic Sciences , The
Commonwealth Medical College, Tobin Hall, 501 Madison Avenue, Scranton, PA 18510, Office: (570) 504-
9643, hatamna@tcmedc.org
Afshin Gharib and Hani Atamna 64
of mitochondrial function, improvement of the cognitive function, and improvement of
grip strength in old mice by a drug. Since these are three major concerns in human aging,
MB may be a useful agent for delaying neurodegeneration and physical impairments
associated with aging.

KeyWords: Mitochondria, aging, brain, methylene blue, senescence, complex IV, Alz-
heimers diseases.
II. Introduction
II.a. Mitochondria and Aging
Physical and cognitive impairments in age-related disorders are often ascribed to
impaired mitochondrial function [1-3]. Impaired mitochondrial function interferes with
energy and intermediary metabolism, increases the production of oxidants, and the risk for
tissue dysfunction. The aging brain has a limited capacity for self repair, increased
mitochondrial dysfunction, impairment in energy metabolism, and oxidative stress [4]. For
example, the decline in the activity of mitochondrial complex IV, energy hypometabolism,
and increased oxidative stress are associated with the early signs of Alzheimers disease
(AD). Therefore, mitochondria-protecting agents may be potential drugs to prevent or delay
age-related neurodegernation (e.g., Alzheimers disease).
Physical and cognitive declines in age-related disorders are widespread medical problems
with mounting social and economic implications [5]. Like humans [6], aging rodents show an
age-associated decline in spatial memory in addition to declines in muscle strength and
physical independence [7]. These declines are linked, in part, to age-associated changes in
mitochondria in neuronal and muscle cells leading to impaired hippocampal or muscular
functions, respectively [4, 8].
II.b. Aging and Cognition
In humans [9, 10] and rodents [11, 12] aging is associated with changes in both muscle
strength and performance on a variety of cognitive tasks. A long established test for muscle
strength in rodents is to measure the grip strength of the animal as it holds on to a bar attached
to a strain gauge [13]. With age, grip strength declines [11].
In rodents, the age-associated change in cognitive ability has been particularly well
studied in terms of spatial memory. The standard test for spatial memory in rats and mice is
the Morris water maze [14], in which the animal is placed in a pool of water and has to locate
a hidden platform using the spatial cues located around the pool and around the testing room.
Over a number of trials, cognitively unimpaired animals become faster at finding the
platform. There is a well established decline in water maze performance with age [15].
Methylene Blue Induces Mitochondrial Complex IV and Improves 65
II.c. Methylene Blue; new use for an Old Drug
Research interest on aging is directed at finding pharmacological or nutriceutical agents
that could be used to improve the quality of life in the elderly by delaying the onset of age-
related neurodegenration and physical disability due to dementia, sarcopenia, or other factors
[3, 16]. We recently showed that Methylene blue (MB) improves mitochondrial function and
delays cell senescence by interacting with the electron transport complexes of mitochondria
[17]. MB extended the life span of primary cells at nanomolar concentrations by more than
50% [17], which makes MB the most effective among the many agents reported to delay cell
senescence [18].
Methylene blue has been in clinical use for about a hundred years to treat a variety of
pathological conditions and diseases [19]. One of the most common uses is the chronic
treatment of congenital methemoglobinemia, which is due to methemoglobin reductase
deficiency. MB is also used to treat methemoglobinemia caused by cyanide, CO, or nitrate
poisoning [19]. Recent clinical uses for MB include preventing the side effects of
chemotherapy (e.g., ifosfamide-induced encaphelopathy [20, 21]), and preventing
hypotension in septic shock [22, 23]. MB is also used in the treatment of some psychiatric
disorders because of its anxiolytic and antidepressant properties [24-26].
MB has been shown to protect against endotoxin-induced lung injury, bacterial
lipopolysaccharide-induced fever [27, 28], cyclosporin injury to the kidney [29], and
streptozotocin injury to the pancreas [30]. MB also protects from ischemic-reperfusion injury
[31], radiation [32, 33], and enhances |-oxidation of long chain fatty acids [34]. MB inhibits
the aggregation of amyloid-| peptide [35] and choline esterase [36]; both are implicated in
Alzheimers disease. A single high dose of MB improves the escape response in rats and
increases the activity of cytochrome c oxidase (complex IV) by 25 % in the brain [37, 38].
MB also protects from methylmalonate-induced seizures [39] and lowers retinal injury
induced by rotenone [40]. MB has no side effects when used at the clinically recommended
dose. Although when exposed to high intensity of UV light MB causes oxidative damage to
isolated DNA, no such toxicity has been shown in humans [41], presumably because it
requires high exposure to UV and most MB in vivo is in the reduced form of MBH
2
, which
does not have photodynamic activity [42].
MB has been proposed to act by inhibiting the NO-activating soluble guanylate cyclase
(sGC) [43] (though the basal activity of sGC is not affected), inhibiting nitric oxide synthase
(NOS) [44], inhibiting monoamine oxidase (MAO) [45], or acting as an antioxidant precursor
[46]. These effects of MB were measured at concentrations greater than 10 M. However,
recent studies showed effects of MB that are not consistent with the above proposed
mechanisms [17, 47]. These discrepancies may be caused by MB exhibiting different effects
at different concentrations (>10 M vs., nM concentrations). We have proposed a new
mechanism that may explain, in part, some of the biological actions of MB that we observed
when using MB at nM concentrations [17]. This mechanism requires MB cycling between
oxidized (MB) and reduced (MBH
2
) forms (Scheme 1).


Scheme 1: The oxidized (MB) and reduced (MBH
2
) forms of Methylene blue.
II.d. Objectives of the Current Study
The objectives of the study we describe here are to test if MB exhibits positive effects on
specific organs and activities in old mice. Furthermore, we wanted to also examine if an in
vitro finding from cell culture [17] can be extended to aging in vivo. Using old mice, we
demonstrate that MB exhibits mitochondria-protecting activity in vivo, which might be
relevant for preventing specific age-related diseases. Furthermore, our findings suggest that in
vitro models of cell senescence may be useful to evaluate specific potential mitochondria
protecting agents.
III. Experimental Procedures
III.a. Experimental Design
We used male C57BL/6 mice (Harlan, NIA). The Childrens Hospital Oakland Research
Institute (CHORI) approved the protocol for the experimentation using MB and mice.
Methylene blue was purchased from Fluka (through Sigma, St. Louis, MO) and purified by
crystallization as described in [48]. In a pilot experiment directed at examining if MB can be
administered in drinking water for extended period of time and determining the highest
tolerated concentration of MB (an 100, 250, 500, or 1000 M) by old mice, we found that 250
M added to drinking water was the best tolerated dose. In addition, we found that the MB at
this dose improved the grip strength in old mice (data not shown). We then performed a

Reduction
(H
3
C)
2
N
Methylene Blue (MB)
Leucomethylene Blue (colorless)
N
S
N
(H
3
C)
2
N
(CH
3
)
2
+
N
H
(CH
3
)
2
+
S
N
H
second larger study using 25 old (starting at age 21 months) and ten young (starting at age 3
months) mice. Animals were housed in groups of 45 and maintained on a 12/12 h light/dark
cycle. Because the mice had been on a chow diet, all mice were switched to an AIN93 diet for
a month before starting the treatment with MB. At this time, 12 old mice (now 22 months old)
were maintained on the AIN93 diet and water (control group, old), while the remaining 13
were maintained for three months on the AIN93 diet and water supplemented with 250 M
MB (MB group). Ten mice from the old control group and 9 mice from the MB group
survived until the end of the study. All the young mice (now 4 months) were given the AIN93
diet without MB and used as controls (control group, young). All mice had free access to food
and water. All mice were tested for grip strength for 4 days (2 trials per day) after 2 month of
treatment. Mice were also given spatial memory tests in the water-maze after 2 months of
treatment. All behavioral testing occurred during the light phase.
III.b. Measuring Cognitive Function
Spatial memory is frequently tested in rodents using the Morris Water Maze. The pool
consists of a circular plastic tank (1.5 m diameter by 0.5 m height) with a removable circular
white platform (13.5 cm diameter). The water (25 C) is made opaque by the addition of a
non-toxic, water-soluble white dye. The platform is 30 cm above the floor of the pool and the
water level is 1 cm above the platform. The pool is divided into four equally sized imaginary
quadrants and the platform is placed approximately 30 cm from the pool wall in one of the
quadrants. A digital camera suspended over the pool and computer software (Columbus
Instruments, VideoMex-V) recorded in real time the distance, speed, and location of the
animal during the swim trial. Animals were trained for 6 days. During each day of training,
animals received four trials. For each trial, the animal was released with its head facing the
opposite pool wall from one of four possible quadrant boundary lines. Each mouse was
allowed to swim until 1) locating the platform or 2) 60 seconds passed without finding the
platform. The time to locate the platform was recorded. Mice that failed to locate the platform
were carried and placed on the platform. All mice were allowed to stay on the platform at the
end of each trial for 30 seconds. After the 4
th
trial, mice were housed in a cage which rested
on heating pads 10 20 minutes (with access to drinking water) until the animal was dry.
After 6 days of training, a single 60 second probe trial was given. During this trial, the
platform was removed and the total distance swam in the platform quadrant as well as the
number of times the animal swam into the specific area that used to contain the platform was
recorded. These two measures reflect the animals memory for where the platform was
located. The better the memory, the more often the animal should enter the exact location of
the platform, and the more the animal should swim around in the quadrant in which the
platform had been located.
III.C. Measuring Grip-Strength
The peak force a mouse exerted by the forelimbs was measured using a grip strength
meter (Columbus Instruments). The grip strength meter consists of a steel frame which
supports a steel shaft. A push-pull strain gauge in the horizontal attitude is attached to the top
of the steel shaft. A waffle style grip plate is attached to the end of the push-pull strain gauge.
Each mouse was tested twice a day for 4 days before the treatment, 1 month after, and again
after 2 months of treatment. Mice were grasped firmly at a point near the base of the tail and
held above the grip plate. Rodents reflexively sprawl, extending the limbs and flexing the
head and body upward. The mouse was lowered toward the grip plate until it firmly grasped
the plate. The mouse was gently pulled away from the grip plate until it released. The tension
force of the forelimbs and compression force of the hind limbs was measured in Kilogram
units.
III.D. Assay for Cytochrome C Oxidase (Complex IV)
After the completion of all testing on live mice, all mice were sacrificed, their brains and
hearts were collected and were snap frozen in liquid nitrogen, and immediately stored at -80
C. Brains and hearts were homogenized in ice-cold PBS/1mM EDTA/0.1 % triton-x100/anti-
proteases, aliquoted and stored at -80 C. The homogenates were later used for further
investigation. The activity of cytochrome c oxidase (complex IV) was measured by following
the oxidation of reduced cytochrome c at 550 nm. Briefly, commercial cytochrome c from
horse heart (Sigma, St. Louis, MO) is mostly oxidized. The substrate for complex IV is the
reduced form of cytochrome c. Thus, reduced cytochrome c was prepared by incubating
cytochrome c with excess ascorbic acid. Reduced cytochrome c was then separated from
ascorbic acid on P-2 gel filtration resin (Bio-Rad, Hercules, CA) and the concentration was
determined using the mM excitation coefficient of 19.6 mM
-1
cm
-1
for the difference between
reduced and oxidized cytochrome c. Reduced cytochrome c was used to assay complex IV
activity in the brain and heart homogenates as described previously [49]. Briefly, the assay
homogenate was potassium phosphate buffer (20 mM, pH 7) and n-dodecyl-|-D-maltoside
(0.45 mM). Cytochrome c was added to the assay buffer and nonenzymatic oxidation was
measured and subtracted from the oxidation rate following the addition of the homogenate
(50-100 g protein). The rate of complex IV activity was calculated and normalized to mg
protein.
III.E. Assay for NQO1
NQO1 activity was measured as previously described [50] using DCPIP as an electron
acceptor. The reaction buffer consisted of 25 mM Tris-HCl with 2 mM EDTA, pH 7.5. A
known amount of the protein (100-150 g) from the homogenate was added to the reaction
buffer containing 200 M NADPH. DCPIP was added at final concentration of 40 M, and
the reduction was monitored at room temperature using the decline in 600 nm absorbance and
700 nm as background. The inhibition by dicoumarol prepared in DMSO (final concentration
20 M) as indication for the specificity of the reaction catalyzed by NQO1. The mM
extinction coefficient 21 mM
-1
cm
-1
was used to calculate the activity of NQO1.
III.F. Assay for Monoamine Oxidase (MAO)
MAO was assayed using Amplex Red/monoamine oxidase/HRP assay kit from
Molecular Probes (Invitrogen, Carlsbad, CA). The substrate for MAO was benzylamine,
which upon oxidation produces H
2
O
2
. H
2
O
2
is then used by HRP to oxidize Amplex red to
resorufin. Fluorescence microplate reader was used to measure the production of resorufin
using excitation at 560 10 nm and fluorescence detection at 590 10 nm. Total MAO was
measured in this assay. We did not distinguish between the activity of MAO-A and MAO-B.
III.G. Quantification of MB in the Brain
The steady state level of MB in the brain was quantified in the homogenates using
LC/MS with electrospray ionization (ESI) [51], with modification. We used 1,9-
dimethylmethylene blue as an internal standard (instead of methylene violet [51], which was
added to each extracted sample and to MB standard. Brain homogenate from MB-treated
mice was extracted by acetonitrile, 0.1 M 1,9-dimethylmethylene blue was added, and liquid
chromatography was performed using a Shimadzu HPLC system (Shimadzu, Columbia, MA)
and an Aquasil C18 50 x 2.1 mm column (Thermo, Torrance, CA). The column was operated
at ambient temperature. The mobile phase was acetonitrile-methanol-ammonium formate
(35:53:12 v/v) containing 20 mM ammonium formate at a flow rate of 0.2 ml/min. Each
sample solution was injected via an autosampler at an injection volume of 10 L and eluted
using an isocratic mobile phase from 0 to 20 min. Sample solutions were kept at 4 C in the
autosampler before injection. The eluate was monitored using a Waters Micromass Quattro
LC triple quadrupole mass spectrometer (Waters, Milford, MA) equipped with an
electrospray ionization (ESI) source. Selected reaction monitoring (SRM) measurements were
performed at 1.2 kV multiplier voltage. SRM transitions monitored in the positive ion mode
were m/z 284.1 m/z 268.1 for methylene blue and m/z 312.25 m/z 296.3 for 1,9-
dimethylmethylene blue (internal standard). Masslynx and Quanlynx software (Waters,
Milford, MA) were used for system control and data processing. The source temperature and
capillary temperature were kept at 130 C and 350 C. The optimum cone voltage, extractor
voltage, and spray voltage were set to 45 V, 3 V, and 3.5 kV, respectively.
III.H. Protein Assay
The protein concentration of each brain homogenate was determined using the Bradford
quantification reagent (Bio-Rad, Hercules, CA) and fat free BSA as standard. Each brain was
homogenized in 5 ml homogenization buffer (ice-cold PBS/1mM EDTA/anti-proteases).
III.I. Statistical Analyses
Graphing, regression, and statistical analysis were performed using Prism 5.0 (GraphPad,
San Diego, CA). For comparison between the groups one-way ANOVA or other tests were
used. Significance was considered at an alpha level of p = 0.05.
IV. Results
IV.A. the Effect of MB on Body Weight and Intake of Food and Water
At the end of the three months of treatment with MB, no significant difference was found
among the groups in body weight or water intake (Figure 1). Food intake, on the other hand,
significantly decreased in MB-treated old mice. This effect of MB on food intake suggests
that MB may work as a calorie restriction (CR) mimetic; an agent that could mimic the
benefits of CR [52]. However, loss of body weight, which is a constant byproduct of CR, did
not happen in MB-treated mice. Therefore, the effect of MB on food intake is possibly a
result of an MB effect on appetite. MB enhances |-oxidation and fat metabolism [34], which
may contribute to this effect of MB. Although, we do not have at this point a definite
explanation, it might be due to combination of the above listed factors in conjunction to MBs
effect on mitochondria.

Figure 1 (Continued)

Figure 1. The effect of Methylene blue on body weight and food and water intake in old mice.
Old mice (22 months) were treated with MB in drinking water (250M) for three months. Control
groups of old and young mice were maintained on drinking water only until the end of the
experiments (see Experimental Procedures). Body weight of each mouse was measured on weekly
bases, while food and water intake were measured every week for each cage (3-5 mice per cage)
and the average of daily intake for each mouse was calculated. Data presented are meansem
(n=10 young, 10 old, and 9 old+MB). One-way ANOVA (Fisher's LSD Multiple Comparison
Test) was used to compare the groups.
IV.B. MB Restores the age-Related Decline in Cognitive
Function and in Grip Strength
The final age-related decline in the spatial memory of old mice was about 30%
(P<0.001), as demonstrated by the increase in the time (in seconds) that the old mice needed
to find the location of the hidden platform (Figure 2 top). This increase in time to find the
platform did not occur in the old mice treated with MB. When comparing the cognitive
performance of old and young groups using the water maze test, the age-related decline in
vision may contribute to the decline seen in the old group. In our experiment both groups of
interest were old (MB-treated and control), suggesting that the vision limitation seems
unlikely to explain our findings.
After 6 days of training, the platform was removed on the 7
th
day and a single probe trial
was given. The number of times the animals entered the area where the platform used to be
(Figure 2 middle) and the distance the animal swam in the quadrant the platform used to be in
(Figure 2 bottom) were recorded. The old mice entered the platform area less (P<0.05) and
swam less in the platform quadrant (P<0.05) than either young animals or old animals treated
with MB. There was no difference on these measures between MB treated animals and young
animals.


Figure 2. The effect of Methylene blue on the age-related decline in spatial memory in mice.
Top- Old mice (22 months) were treated with MB in drinking water (250M) for three months.
Control groups of old and young mice were maintained on drinking water only until the end of the
experiments. Spatial memory was measured using water-maze test as described in the
Experimental Procedures. The time to platform increases in old mice not treated with MB. The
time to platform is similar between the young mice old mice treated with MB. Data presented are
meansem (n=10 young, 10 old, and 9 old+MB). One-way ANOVA (Fisher's LSD Multiple
Comparison Test) was used to compare the groups.
Middle- The effect of methylene blue on the age-related deficit in searching for the platform in the
probe trial. The number of entries into to the area occupied by the platform on probe trials
decreases in old mice not treated with MB. The number of entries into the platform area is similar
between the young mice old mice treated with MB. Data presented are meansem (n=10 young, 10
old, and 9 old+MB). One-way ANOVA (Fisher's LSD Multiple Comparison Test) was used to
compare the groups.
Bottom- The effect of methylene blue on the age-related deficit in searching in the platform
quadrant during the probe trial. The distance (in cm) swam in the quadrant the platform had been
in on probe trials (B) decreases in old mice not treated with MB. The distance swam in the
platform quadrant is similar between the young mice old mice treated with MB. Data presented are
meansem (n=10 young, 10 old, and 9 old+MB). One-way ANOVA (Fisher's LSD Multiple
Comparison Test) was used to compare the groups.
We also tested the effect of MB on the grip strength as a measure for the possible effect
of MB on muscle strength in general. The age-related decline in the grip strength applied by
the front limb was about 20% (P<0.001). This decline did not occur in old mice treated with
MB (Figure 3).

Figure 3. The effect of Methylene blue on the age-related decline in grip strength in mice.
experiments. Grip strength was measured using water-maze test as described in the Experimental
Procedures. The tension force of the forelimbs (in Kilogram units) is lower in old mice not treated
with MB. The tension force is similar between the young mice old mice treated with MB. Data
presented are meansem (n=10 young, 10 old, and 9 old+MB). One-way ANOVA (Fisher's LSD
Multiple Comparison Test) was used to compare the groups.

The data presented in figures 2B through 3 do not distinguish between the possibilities
that MB slows the decline in cognitive function with age or reverses it. The reason for this is
that the cognitive function of the old mice, as compared to the young, was not established at
the beginning of the treatment with MB in this experiment. The positive effect that MB has
on old mice is obvious, however the exact mechanism of the effect of MB will be clarified in
future studies.
IV.C. MB Crosses the Blood Brain Barrier
The steady-state level of MB in the brain following treatment with MB is 128 nM (0.51
0.25 pmols/mg protein) as measured by LC-MS-ESI [51]. As expected, no MB was detected
in the brains of the control mice. Thus, although MB reaches the brain, the concentration is
quite low. However, since it is within the 100 nM concentration range we found to be optimal
for delaying cell senescence and protecting the mitochondria in cultured cells [17], it is
reasonable to assume that MB is active in the brain at a nanomolar range concentration.
IV.D. MB Reverses the age-Related Decline in
Protein Content of the Brain
Following brain homogenization, we were surprised to find that the protein content
significantly declines with age. Interestingly, the age-related decline in protein content was
prevented by MB. MB restores protein content in the brain to levels similar to young mouse
brain (Figure 4, one way ANOVA). A 5-15 % decline in protein content in human brain has
been reported previously [53, 54].

Figure 4. The effect of Methylene blue on the age-related decline in brain protein content in mice.
experiments. Brain homogenates were prepared as described in the Experimental Procedures.
Protein was measured using Bradford based assay. Fatty acid free BSA was used as standard. One-
way ANOVA (Tukey's Multiple Comparison Test) was used to compare the groups. Groups with
different letters are significantly different. Data presented are meanSD (n=10 young, 10 old, and
9 old+MB).
IV.F. MB Increases the Activities of Brain and Heart Mitochondrial Complex
IV by 100 And 50 %, Respectively
A previous in vitro study showed that MB enhances mitochondrial function and increases
complex IV [17]. Thus, we examined the effect of MB cytochrome c oxidase (complex IV) in
vivo. We found that the activity of complex IV in the brain of old mice treated with MB was
doubled (Figure 5). We also measured the effect of MB on complex IV from the heart.
Interestingly, MB significantly increased the activity of complex IV in the heart of old mice
by 50% (Figure 6). These effects indicate that MB is active in tissues other than the brain.

Figure 5. The effect of Methylene blue on the activity of complex IV in the brain of old mice.
experiments. Brain homogenates were prepared and brains complex IV activity was measured as
described in the Experimental Procedures. One-way ANOVA (Newman-Keuls Multiple
Comparison Test) was used to compare the groups. Groups with different letters are significantly
different. Data presented are meansd (n=10 young, 10 old, and 9 old+MB).

Figure 6. The effect of Methylene blue on the activity of complex IV in the heart of old mice.
experiments. Heart homogenates were prepared and hearts complex IV activity was measured as
described in the Experimental Procedures. One-way ANOVA (Tukey's Multiple Comparison Test)
was used to compare the groups. Groups with different letters are significantly different. Data
presented are meansd (n=10 young, 10 old, and 9 old+MB).

The activity of complex IV in the brain homogenates did not change significantly in old
vs. young mice. The effect of normal aging on complex IV decline is not conclusive [55-58].
However, the decline in complex IV in age-related disorders, such as Alzheimers disease,
has been established [59]. Thus, the induction of complex IV by MB may be useful for
delaying the decline in complex IV in Alzheimers disease which may offer clinical benefits
for Alzheimers patients.
One possible explanation for the lack of effect of age on complex IV in our experiment
could be that the old group was not old enough. Alternatively, a possible disconnects between
physiologic age and chronic age may explain the lack of difference with age in complex IV
activity. Such disconnect is not uncommon and could sometimes mask significant changes
with age or interesting patterns of aging.
IV.G. MB Lowers the Activity of Monoamine
Oxidase in the Brain of old Mice
MB caused a significant decrease in the activity of MAO in the mouse brain (Figure 7).
This decrease is likely due to direct inhibition by MB [60]. Interestingly, in our experiment,
mice exhibit a 30 % age-related decline in the MAO in the brain, which was further enhanced
by MB (Figure 7). In general, MAO activity in mice seems to decline at middle age and
slightly increase after 25 months of age [61]. MAO activity in human brain increases with age
[61-63]. The increase in MAO in the aging human brain may lower the bioactive amines (e.g.,
dopamine) and may increase the risk of age-related dementia [63, 64]. The inhibition of MAO
by MB is consistent with previous findings [45].

Figure 7. The effect of Methylene blue on the activity of monoamine oxidases in the brain of old mice.

experiments. Brain homogenates were prepared and brains monoamine oxidase activity was
measured using Amplex Red /HRP assay kit as described in the Experimental Procedures. One-
way ANOVA (Tukey's Multiple Comparison Test) was used to compare the groups. Groups with
different letters are significantly different. Data presented are meansd (n=10 young, 10 old, and 9
old+MB).
IV.H. MB Did not Affect the Activity of NQO1
The activity of NQO1 (NAD (P)H quinone oxidoreductase) on the other hand was not
affected by chronic treatment with MB, suggesting that it has no effect on NQO1 activity
(Figure 8). NQO1 is a detoxification enzyme that functions to prevent one electron reduction
of quinones resulting in the production of radical species. Thus, the preservation of NQO1
indicates that MB likely does not interfere with the detoxification system in the cell. This
finding is contrary to what we previously proposed [17], where we used a reporter gene
system to study the effect of MB on NQO1. In the previous experiments we used HepG2 cells
(transformed cells), which may explain, in part, the different responses to MB in the two
experiments.

Figure 8. The effect of Methylene blue on the activity of NQO1 in the brain of old mice.

experiments. Brain homogenates were prepared and brains NQO1 activity was measured using
Amplex Red /HRP assay kit as described in the Experimental Procedures. One-way ANOVA
(Tukey's Multiple Comparison Test) was used to compare the groups. Groups with different letters
are significantly different. Data presented are meansem (n=10 young, 10 old, and 9 old+MB).
V. Discussion
We demonstrated that chronic treatment with MB improves spatial memory and muscle
strength in old mice. In the water maze, not only did the young animals and MB-treated
animals find the platform more quickly than old mice, they also swam more in the quadrant of
the platform and entered the area the platform had been located in more often when the
platform was removed in the probe trial. These differences in performance support our
conclusion that the young and MB-treated animals had a better memory of where the platform
had been located than the old animals. Previous research on the effects of MB on cognitive
function in rodents have been limited to young animals, using short term (single day)
treatment with MB [37, 38, 65]. Our findings suggest that low chronic dose of MB can have a
positive impact on performance in a spatial memory task in old animals. In future studies, a
memory task that is independent of motor ability should be used to better tease apart the
decline in muscle strength and memory.
MBs concentration in the brain at the end of the three month treatment was 0.51 0.25
pmols/mg protein indicating that MB crosses the Blood Brain Barrier. The ability of MB to
cross the Blood Brain Barrier following i.v. injection also has been demonstrated in previous
studies [66]. These observations are encouraging since they indicate that a low chronic dose
of MB administered in drinking water reaches the brain, enhances mitochondrial function,
and protects against the age-related memory impairment in old mice. MB also prevents the
decline in brain-protein content in old mice. A decrease of 5-15 % in protein content in the
aging human brain (ages from 30 to 90 years old) has been previously described [53]. The
activity of MAO, which increases with age in the human, was decreased by MB. The increase
MAO with age in humans may interfere with brain concentrations of bioactive amines
including serotonin and the catecholamines. Interestingly, MAO is also involved in producing
hydrogen peroxide, which may further contribute to mitochondrial impairment with age.
Thus, modulating the activity of MAO by MB may also contribute to the effect that treatment
with MB has on the aging brain.
The most intriguing finding of this study is that low chronic levels of MB increase the
levels of complex IV in brain and heart. This increase in the activity of complex IV is
consistent with the effect of MB on IMR90 cells in vitro [17], where the level of complex IV
also increased. Complex IV is found in 5 fold excess over the other electron transport
complexes of the mitochondria [67], which may indicate the physiological significance of
excess complex IV. However, there is no definitive answer yet as to the physiologic
significance of the excess in complex IV. The following discussion may provide some
insights. Energy production by mitochondria relies on four electron transport complexes
(ETC), which are complex I, II, III, and IV. Electron transfer through each one of the
complexes starts at complex I, which catalyzes a two-electron oxidation of NADH, and
continues until water is formed by complex IV (Scheme 2). Complex IV consumes more than
95% of the O
2
that reaches the cell. The production of O
2
-free radicals is enhanced by the
stalling of electrons upstream of complex IV; at complexes I and III [68, 69]. Complex I and
complex III are two sites responsible for the production of free radicals by non-specific
transfer of electron to O
2
[70]. Thus, induction of complex IV by MB may play a key role in
lowering the steady-state concentration of intracellular O
2
and, as a result, lowering the
production of oxidants by the mitochondria.

Scheme 2: The electron transport chain (ETC) of the mitochondria.
The four complexes are: complex I (ETC I), complex II (ETC II), complex III (ETC III), and
complex IV (ETC IV) in addition to ATP synthase (i.e., complex V). The electron transfer through
each of the ETCs starts at ETC I, which catalyzes two electrons by the oxidation of NADH and
continues until water is formed on ETC IV. Coenzyme Q serves as low molecular weight electron
carrier from ETCs I and II to III. Cytochrome c (cyt c) serves as electron carrier from ETC III to
ETC IV.

In attempt to understand how MB induces complex IV, we used our experimental
findings in conjunction with previous studies on MB and proposed a molecular mechanism to
explain, in part, MBs effect on complex IV [17]. Briefly, we propose that MBH
2
and MB
serve as electron carriers between several dehydrogenases and heme-proteins (e.g.,
cytochrome c) (Scheme 1). Complex IV in turn recycles the reduced cytochrome c (Scheme
NADH ETC I Coenzyme Q ETC III
cyt c ETC IV (complex IV) O
2
H
2
O
FADH
2
ETC II
2), which trigger the induction of additional complex IV. This mechanism is explained in
more detail in [17].
We do not yet know the molecular mechanism that leads to improving cognitive function
or muscle strength in old mice. We are proposing a possible mechanism based on emerging
research from various studies. The ETC complexes are biochemically and physiologically
connected and directed at energy production. High levels of complex IV correlate with
neuronal metabolic activity [71] and with cognitive performance [72, 73]. Thus, we predict
that the induction of complex IV by MB will increase the cellular energy charge. An increase
in energy charge and the metabolic activity of neurons improves learning and memory
retention. Furthermore, learning and memory retention require adequate neuroplasticity and
neurogenesis. Recent research showed that neurogenesis occurs within the adult brain (e.g.,
dentate gyrus of the hippocampus) [74-77]. Interestingly, an age-related decline in
neurogenesis of the adult brain [78-82] and muscle endogenous progenitor cells [83] has been
demonstrated. This decline may be attributed, in part, to senescence of the progenitor cells or
senescence of the somatic cells that are surrounding the progenitor cells (thus indirectly
affecting the normal function of the endogenous progenitor cells) [83]. Since, MB is very
effective in delaying cell senescence in vitro [17], it may retain its anti-senescence action also
in vivo (as it retained its effect on mitochondria) and restore the function of neuronal
progenitor cells. MB may improve the mitochondrial activity and energy metabolism in
neuronal progenitor cells. Pharmacological approaches that are directed at inducing
neurogenesis hold promise for preventing or delaying age-related disorders [77, 84-86]. Thus,
MB could in part, improve memory and muscle function as part of such an approach.
MB prevents the decline in complex IV, which is a key cytopathology of Alzheimers
disease (AD). Thus, by increasing the brain reserve of complex IV, we propose that MB could
delay or slow the age-related decline in complex IV, thus preserving mitochondrial function,
energy metabolism, and memory retention in AD. Due to its effect on heart complex IV, MB
may improve physical activity and delay the onset of sarcopenia. Thus, MB could be used to
improve the quality of life of the elderly. MB is a drug with an extended medical and safety
record in humans; thus, FDA approval for its use in clinical trials in connection to aging and
age-related disorders should not be denied on safety grounds (discussed in [17]).
Conclusions
We proposed mitochondria-based approach to prevent or delay the onset of the age-
related degenerative disorders (e.g., Alzheimer, sarcopenia). This strategy is directed at
enhancing mitochondrial activity using low chronic dose of MB to increase the level of
complex IV. Energy deficiency and increase free radical production in Alzheimer and aging
may be contributed by mitochondrial function. In vitro and in vivo MB exerts its effect at very
low (nM) concentration, which in conjunction with its safety record in human further
minimizes any risk of side effects of chronic exposure to MB. MB can also induce heme
synthesis, thus, it may improve iron metabolism and preventing heme deficiency, which are
key cytopathologies in Alzheimer.
Acknowledgment
The authors would like to thank the Bruce and Giovanna Ames Foundation and the
American Federation for Aging Research (AFAR) for supporting this research. We are
grateful to Prof. Gerald Litwack and Drs. William Phillips and William Zhering for
commenting on the manuscript. We also would like to thank Gordon Wu, Tina Ghirarduzzi,
Tegan Hall, and Lara Corkrey for technical assistance.
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Chapter 4
Receptor Specific Features
of Excitotoxicity Induced
Neurodegeneration
Sergei M. Antonov
*
and Dmitrii A. Sibarov
Sechenov Institute of Evolutionary Physiology and Biochemistry
RAS, Saint-Petersburg, Russia
Abstract
Excitotoxicity is a term that describes the neuronal death caused by neurotoxic
effects of glutamate, which is the most abundant excitatory neurotransmitter in the
vertebrate central nervous system. Glutamate is well known to be involved in cognitive
functions like learning and memory, but its excessive accumulation in extracellular space
can lead to neuronal damages and eventual cell death via necrosis and apoptosis. As a
result excitotoxicity contributes to pathogenesis of numerous neurodegenerative diseases.
Both normal function and pathological action imply an activation of the same glutamate
receptors particularly of NMDA- (N-methyl-D-aspartate), AMPA- (-anino-3-hydroxyl-
5-methyl-4-isoxazole-propionate) and KA- (kainate) subtypes.
Many achievements in the mechanisms of neurodegeneration were obtained using
different experimental approaches on primary neuronal cultures. Double successive
acridine orange and ethidium bromide staining combined with confocal microscopy
offers fast, easy, sensitive and reproducible method by which necrosis and apoptosis can
be recognized and quantified in a population of living neurons. Together with
immunostaining they provide many research advantages and allow analysis of protein
expression patterns.
The growing quantity of evidence reveals the diversity of apoptosis cascades.
Whereas our data show the same profiles of excitotoxicity for NMDA and KA, we found
receptor subtype specific differences in neuronal death mechanisms. For example,
apoptosis caused by prolonged NMDA receptors activation develops through the caspase-
independent cascades via release of apoptosis inducing factor (AIF) from mitochondria

*
Corresponding author: antonov@iephb.ru, dsibarov@gmail.com
Sergei M. Antonov and Dmitrii A. Sibarov 88
and its direct action on nuclear chromatin. In contrast AMPA and KA receptors mediated
apoptosis includes caspase-dependent pathway.
On the basis of our data and literature the chapter will review the contemporary state
of research concerning the aspects of excitotoxicity mechanisms discussed above.
Introduction
Glutamate receptor (GluR) is the main subject of investigations, concerning
excitotoxicity and neuroprotection. Functional disregulation of neuronal metabolism, resulted
from overactivation of GluRs is thought to cause neuronal death and to underline a wide
range of central nervous system (CNS) disorders like spinal cord and brain injuries, stroke,
neurodegenerative diseases, etc. Loss of cerebral blood flow causes massive neuronal
depolarization and Glu accumulation in extracellular space [Lipton, 1999]. The prolonged
presence of Glu released from neurons and glial cells by non-quantum secretion [Antonov,
Magazanik, 1988] and activation of ionotropic GluRs have extensive consequences for
neuron functioning, starting with an increase in intracellular Ca
2+
concentration, imbalance of
transmembrane gradients of the main electrogenic ions (Na
+
and K
+
), and an activation of
various intracellular cascades, and ending with destruction of the plasma membrane or
nuclear apparatus of neurons [Choi 1987, 1988; Jonston, 1994, Oiney, 1994; Schoepp,
Sacaan, 1994; Hatanaka et al, 1996]. Massive cytoplasmic calcium ions accumulation is
thought to be one of the most important activators of various cell death mechanisms.
Its important to note, that a great body of facts concerning Glu stimulated neuronal
disfunction were obtained in neuronal culture models. Cell cultures provide uniform cell
composition and highly controllable extracellular environment which is favorable to study
fine intracellular mechanisms. However, we must take into account that this model focuses on
neurons alone and ignores tissue control. Other brain tissue cell types like astrocytes and
oligodendrocytes can promote or prevent excitotoxic neuronal death. Actrocytes not only
regulate neuronal ion balance, but can also maintain low extracellular Glu levels due to
uptake with glial GLAST and GLT-1 transporters [Rothstein et al, 1994]. Glial cells express
all kinds of ionotropic GluRs and respond to Glu stimulation with neurotrophic factors
secretion like BDNF (brain-derived neurotrophic factor).
Glu induced excitotoxic neuronal death involves activation of at least three types of
receptors: NMDARs (selective agonist N-methyl-D-aspartate, NMDA), AMPARs (selective
agonist -anino-3-hydroxyl-5-methyl-4-isoxazole-propionate, AMPA) or KARs (selective
agonist kainite, KA). Because KA is not strictly selective to KARs and can activate both
KARs and AMPARs, below we mention these receptors together.
NMDARs are highly permeable to Ca
2+
, as well as to Na
+
and K
+
. An accumulation of
free Ca
2+
inside the cell triggers multiple predominantly Ca
2+
-dependent mechanisms of cell
death usually ending as apoptosis [Khodorov, 2004]. Apoptosis, or programmed cell death,
plays an enormous role in the development and formation of organs, as well as in the
functioning of rapidly renewing tissues [Abrams et al, 1993; Jonston, 1994]. However, in
nervous tissue, where there is virtually no regeneration, although there are now some data on
de novo neuron formation from glial cell precursors [Skachkov et al, 2006], apoptosis is the
key factor in the pathogenesis of many nervous system disorders, along with necrosis
[Jonston, 1994].
Receptor Specific Features of Excitotoxicity Induced Neurodegeneration 89
In general, AMPARs and KARs are permeable to Na
+
and K
+
, and much less permeable
to Ca
2+
than NMDARs. The impermeability of AMPARs and KARs to Ca
2+
is determined by
the presence of GluR2 subunit [Burnashev et al, 1996; Dingledine et al, 1999]. GluR2 is
expressed in all brain areas and neuronal subsets [Sun et al., 2005] except motor neurons and
interneurons having comparatively low GluR2 expression [Magazanik et al, 1997], which
makes them selectively vulnerable to AMPAR-mediated excitotoxicity. Prolonged activation
of AMPARs and KARs elevates intracellular Na
+
an contributes to cell swelling [Choi, 1987,
1988], which increases the probability of necrotic cell death. On this basis, we expect the
distinctive features of NMDARs and AMPARs/KARs to underlie the different ratio of
necrotic and apoptotic neuronal death while neurodegeneration caused by selective activation
of particular receptor types using NMDA or KA.

Figure 1. Simplified diagram of caspase-dependent and caspase-independent pathways of apoptosis. In
response to different stress types protein P53 becomes activated and induces Bax expression and
translocation to mitochondrial membrane. Bax promotes opening of mitochondrial permeability
transition pores (PTP). It allows AIF and Cytochrome-C to be released to cytoplasm. AIF represents
caspase-independent pathway and can directly cause DNA fragmentation. Cyt-C (caspase-dependent
pathway) activates caspases phosphorilation cascade ending at Caspase-3, causing DNA fragmentation.
Proapoptotic Bax protein promotes Cyt-C and AIF exit to cytoplasm. Antiapoptotic protein Bcl-2 has
an opposite function
Although the cell death during apoptosis and necrosis have clear morphological and
biochemical differences [Cafforio et al, 1996; Philpott et al., 1996], most methods do not
allow the simultaneous identification of apoptosis and necrosis in living tissue, are very
laborious and require preliminary biochemical processing, fixation, or resuspension of the
study material [Gavrieli et al, 1992]. The development of a vital kit for rapid analysis of the
cellular composition of neurons in tissue cultures [Mironova et al, 2007] allows the automatic
quantification of cell populations to be performed for the presence of apoptosis and necrosis.
It greatly facilitates the study of neurodegenerative process dynamics.
Its well known, that the development of apoptosis in various tissues can occur via two
basic mechanisms (Figure 1): the caspase-dependent cascade and a cascade not involving
caspases, i.e., via the direct action of apoptosis-inducing factor (AIF) on cell nuclei [Hong et
al, 2004]. Inactive procaspases always exist in cytoplasm. Procaspases activation requires
proteolytic splitting of proenzyme into two subunits and further cleavage of N-terminal ends
[Ermshaw et al., 1999]. Subunits are then assemble into active oligomers. Initial procaspase
proteolysis can be done by various proteases, including other caspases. Caspase independent
mechanism can be triggered via hyperproduction of proapoptotic proteins like Bax and Bak,
which can induce apoptotic cell death even in the presence of caspase inhibitors [Xiang et al,
1996, McCarthy et al, 1997]. An appearance of caspase 8, which activates procaspases 3, 6
and 7 is enough to start apoptosis. The key enzyme in the caspase-dependent pathway is
caspase 3 (Cas-3), activation of which leads to irreversible destruction of nuclear DNA.
Apoptosis can be reversed before an activation of Cas-3 with Cas-8. There are several
proteins promoting and preventing caspases activity at this moment [Kidd, 1998; Adams,
Cory, 1998; Reed et al, 1998; Huppertz et al 1999; Gross et al, 1999]. These proteins include
inhibitors of apoptosis like A1, Bcl-2, Bcl-W, Bcl-XL, Brag-1, Mcl-1 and NR13, and
proapoptotic proteins Bad, Bak, Bax, Bcl-XS, Bid, Bik, Bim, Hrk, Mtd. Most of these
proteins belong to Bcl-2 family of proteins which is evolutionary conservative. For example
in sponges Geodia cydomium and Suberites domuncula, homologous proteins are involved in
morphogenesis process [Adams, Cory, 1998].
This raises the question about the contribution of different cell death mechanisms to
apoptosis evoked by selective activation of different neuronal GluR subtypes. Identification
of the receptor specificities of apoptosis pathways simplifies the search of intracellular targets
for new neuroprotective agents.
The goals of this work, performed on primary neuronal cultures from rat cortex were: to
evaluate necrosis/apoptosis ratio during exposure to selective GluR agonists - NMDA and
KA, as well as the Glu itself; to study potential neuroprotective properties of selective
NMDAR, AMPAR and KAR antagonists, and to elucidate possible diversity of apoptotic
cascades determining excitotoxicity triggered by excessive activation of GluR subtypes.
Methods
Experiments were performed at room temperature (20-22
o
C) on primary cultures of
neurons from embryonic rat brain cortex. All procedures using animals were in accordance to
FELASA recommendations and were approved by the local Institutional Animal Care and
Use Committee. The method used for preparing cultures has been described in detail
previously [Antonov, Johnson, 1996; Mironova, Lukina, 2004]. Briefly, pregnant Wistar rats
were sacrificed by CO
2
inhalation 16 days after conception. Cortices were obtained by aseptic
dissection, incubated for 30-40 min at 37
o
C in growth medium containing 83% minimum
essential medium (MEM), 25 mM Hepes, 2 mM L-glutamine, 10% D-glucose plus 0.03%
trypsin, and then dispersed mechanically by trituration with a fire-polished glass Pasteur
pipettes. Cells were pelleted by light centrifugation (410 x g, 5 min at 25
o
C), the supernatant
discarded and the cell pellet resuspended in pre-warmed Hanks solution. For better washout
of reagents this procedure was repeated. A plating suspension at a density of 130,000 cells per
ml was prepared in growth medium. This density was optimal for the neuronal survival, the
neuronal network formation and further experimental manipulations with the cultures. The
suspension of cells was plated onto 15 mm diameter glass coverslips that had been coated
with poly-D-lysine in 35 mm plastic Petri dishes. Cultures were kept at 37
o
C in a humidified
5% CO
2
-containing atmosphere. The growth medium was refreshed twice a week.
Cells were used for experiments after culturing 7 days in vitro (DIV). Directly before the
experiments, coverslips with neuronal cultures were placed in the bathing salt solution. Since
extracellular Mg
2+
, which blocks NMDAR channels [Antonov, Johnson, 1999], has
neuroprotective effects on young neurons [Antonov et al, 2006], Mg
2+
-free bathing salt
solution was used of the following composition: 140 mM NaCl, 2.8 mM KCl, 1.0 mM CaCl
2
,
10 mM Hepes; pH was adjusted to 7.2 7.4 with NaOH. To initiate excitotoxic insults 3 mM
Glu, 30 M NMDA and 30 M KA were added to the bathing salt solution. In the case of
NMDA in all experiments 30 M glycine was coapplied, as a coagonist of NMDAR
[Johnson, Ascher, 1987]. When effects of antagonists and modulators were studied the
compounds were coapplied with the GluR agonists. Measurements of the proportion of dead
cells among whole cell population were performed after 120 and 240 min treatment with the
compounds.
Cell viability was determined utilizing the vital fluorescence assay. Confocal images
captured after 120 and 240 min of incubation with compounds were subjected for automatic
cell counting after staining of all nuclei with acridine orange and dead cell nuclei with
ethidium bromide. First, cells were treated with 0.001% acridine orange for 30 s. After
complete washout of contaminating acridine orange cells were exposed to 0.002% ethidium
bromide for 30 s. This procedure was applied directly before every measurement. As a result
in confocal microscopy experiments the nuclei of live neurons, labeled with acridine orange,
looked green and the nuclei of injured neurons, labeled with ethidium bromide, looked red
(Figure 2, A) [Pulliam et al, 1998; Mironova et al, 2007]. In the absence of correlated pixels
(Figure 2) the cell viability was estimated by the ratio of green pixels (the number) to the total
number of lightened pixels (red plus green, Figure 2, B). If some population of neurons
exhibited apoptotic transformations their nuclei looked yellow-orange, revealing the
colocalization of fluorescence in green and red spectral regions (Figure 3). In this case the
fractions of live, apoptotic and necrotic cells were calculated on the basis of correlation plot
(Figure 3) as the ratio of green, yellow-orange and red pixels to the total number of lightened
pixels (the sum of green, yellow-orange and red), correspondently. To improve the validity of
cell viability measurements, 3 non-overlapping images from a single coverslip were pooled to
calculate the mean value for this particular coverslip, which represented a single experimental
measurement.

Fluorescence images were captured using a Leica (Leica Microsystems, Heidelberg,
Germany) TCS SL scanning laser confocal microscope (upright) equipped with argon laser of
50 mW (excitation wavelengths 458, 476, 488 and 514 nm, approximately 10 mW each).
Cultures were viewed with a 40x water objective (HCX APO L 40x/0.80, Leica
Microsystems, Heidelberg, Germany). To resolve fine details an additional electronic zoon
with a factor of 1.5 3.5 was used. Since ethidium bromide has a second peak of excitation,
which is consistent with the excitation of acridine orange, both acridine orange and ethidium
bromide could be visualized using the same laser line. For two-channel imaging of acridine
orange and ethidium bromide, neuronal cultures were excited with 488 nm laser line, which
could be varied between 0.1 10 mW by means of a neutral density filter. The emitted
fluorescence was acquired at 500 560 nm (green region of spectrum, for acridine orange)
and > 600 nm (red region of spectrum, for ethidium bromide) and collected simultaneously
using separate photo multiplier tubes. Microscope settings were adjusted so that imaging
conditions for both channels were kept equal and constant. To improve signal-to-noise ratio 6
scans (512 x 512 pixel array) were averaged at each optical section. The confocal images
from both channels were merged using standard Leica software and program ImageJ
(http://rsb.info.nih.gov). In order to quantify colocalized and non-colocalized fluorescence the
correlation plot, which sorts values of given pixels in the first image as the x-coordinate and
values of corresponding pixels in the second image as the y-coordinate, was generated for
each of measurements. On the resulting image non-correlated pixels looked green and red and
were attributed to live and necrotic neurons, correspondently. Correlated pixels looked
yellow-orange and were attributed to nuclei of apoptotic neurons.
Apoptotic proteins were identified immunochemically using mouse monoclonal
antibodies to proapoptotic proteins P53 (dilution 1:400), AIF (dilution 1:1000), Cas-3
(dilution 1:100) and Bax (dilution 1:200). Monoclonal antibodies were visualized using
secondary antibodies conjugated with the fluorochromes FITC, Phr, and Cy3, diluted 1:150.
Images were recorded using a Leica TCS SL confocal scanning microscope. Fluorochromes
were excited by light at a wavelength of 488 nm (for FITC), 488 nm (for Phr), and 514 nm
(for Cy3). Emission was recorded in the green spectral region (emission peak at 525 nm) for
FITC, in the red spectral region (emission peak at 620 nm) for Phr, and in the yellow spectral
region (emission peak at 570 nm) for Cy3. All antibodies were obtained from Sigma.
Before fixation, neurons on coverslips were treated with neurotoxic factors (Glu, NMDA
or KA) separately or in combination with antagonists for 240 min. Cells were then fixed with
4% paraformaldehyde solution in PBS (phosphate-buffered saline) for 30 min. After fixation,
cells were washed out twice with PBS (15 min x 2). Before treatment with BSA (bovine
serum albumin, 2%), cells were incubated with Triton X-100 (0.2%) for 15 min, washed out
with PBS, and exposed to monoclonal antibodies for 12 h at 4C. After washing out to
remove primary antibodies, fluorochrome-conjugated secondary antibodies were added.
Reactions with secondary antibodies lasted 40 min at a room temperature of 23C. Before
recording of data, antibody-bound preparations were fixed on slides with Moviol glue to
prevent fading of fluorochromes.
All drugs were kept frozen in stock solutions at concentrations of 1 100 mM and were
diluted to the required concentrations before use. All chemical reagents were obtained from
Sigma Chemical Co. (St. Louis, MO, USA).
Data were analyzed statistically using Excel, Origin 6.1, and SigmaPlot 8. All histograms
and Table 1 show means standard errors of mean (s.e.m.). Students two-tailed t-test and
ANOVA were used for statistical comparisons.
Results
Double sequential staining with acridine orange and ethidium bromide allowed cells
dying by apoptosis or necrosis and living cells to be discriminated [Mironova et al, 2007]. In
course of exposing of neuronal cultures to 3 mM Glu for 120 min green nuclei of live neurons
and red nuclei of injured neurons were observed (Figure 2, A). The intensity correlation plot
(Figure 2, B) reveals non-overlapping areas of red and green pixels. An absence of
colocalization between green and red fluorescence points on necrotic type as a mechanism of
neuronal death under these particular conditions. This data suggests that during 120 min
excitotoxic insult cells die by necrosis. To induce apoptosis a longer treatment by Glu or its
agonists is required.

Figure 2. Algorithm of recognition and automatic quantication of live and dead neurons using the
sequential acridine orange and ethidium bromide confocal microscopy assay. (A) Neurons after
exposure of neuronal culture to 3 mM Glu during 120 min. Dead neurons are labeled with ethidium
bromide and have red nuclei. Live neurons are labeled with acridine orange and have green nuclei. (B)
The correlation plot for the image A. The absence of correlation between images obtained in green and
red spectral regions demonstrates the lack of apoptotic nuclei. Dashed lines indicate thresholds, chosen
empirically, that separates visible uorescence from dark pixels

Figure 3. Distribution and automatic counting of living and dead cortical neurons in cultures treated
with 3 mM glutamate for 240 min. Vital sequential staining with acridine orange and ethidium bromide
using confocal microscopy is shown

Three groups of nuclei can be distinguished: live neurons (example marked with green circle are visible
only in green spectral region, left panel); neurons dying by necrosis (marked with red circle are visible
only in red spectral region, intermediate panel), and apoptotic neurons (orange circle mark can be seen
in both parts of the spectrum. The right image represents the result of merging red and green spectral
regions). Frame size is 139 139 m. The correlation plot shows correlated pixels or colocalization of
fluorescence between the images recorded in the green and red spectral region. Correlating pixels are
orange. The dotted lines show the empirically selected limits discriminating lighting-up pixels from
non-lighting-up pixels. The proportion of live cells (37% for this experiment) was evaluated by the ratio
of the number of green pixels to the total number of pixels lighting up.
Figure 3 shows images captured in the green and red spectral regions after exposure to 3
mM Glu for 240 min. The combined image shows the most typical situation, in which
neurons can be discriminated into three classes on the basis of their nuclei. The nuclei of live
neurons are visible only in the green part of the spectrum. They are detected by staining with
and fluorescence of acridine orange. Yellow-orange nuclei on combined image (Figure 3) are
seen in both the green and red areas and are detected because of the shift of acridine orange
fluorescence towards the red spectral region as a result of nucleus acidification while
apoptosis. [Mpoke, Wolfe, 1997]. Red nuclei are seen only in the red part of the spectrum,
and are the nuclei of neurons with plasma membrane destruction, i.e., cells which have died
by necrosis and which are detected by the fluorescence of ethidium bromide.
Table 1. Comparison of the proportions of necrotic, apoptotic, and living neurons by
activation of different types of glutamate receptors for 240 min. Values marked with *
are significantly different from the control (p < 0.05, Students two-tailed t test)
Treatment Necrosis, % Apoptosis,
%
Living cells,
%
Number of
experiments
Control 3 1 15 5 82 6 4
30 M NMDA 15 1* 45 9* 40 10* 5
30 M NMDA + 50 M AP5 3 1 16 4 81 5 4
30 M kainite 16 2* 52 5* 32 6* 5
30 M kainate + 30 M CNQX 1 0.5 16 4 83 4 4
3 mM Glu 35 8* 31 3 34 5* 4
3 mM Glu + 30 M CNQX 19 6* 16 13 65 8 4
3 mM Glu + 50 M AP5 49 13* 17 3 34 6* 4
The proportion of neurons in each of three physiological states can be determined as
ratios of the numbers of correlating pixels (lighting up in both parts of the spectrum) and non-
correlating pixels (lighting up in only one part of the spectrum) to the total number of pixels
lighting up. In the experiment shown in Figure 3, 3 mM Glu induced the apoptotic processes
in 39% of neurons; 24% died via necrosis and 37% of neurons remained live. Average data
are shown in Table 1. Under the control conditions (incubation in the bathing salt solution for
240 min) only 3% of necrotic and 15% of apoptotic neurons were found. Significantly
increased number of necrotic and apoptotic cells at the cost of decreased number of live
neurons illustrates the strong neurotoxic action of Glu. Obviously, neurodegeneration
triggered by excitotoxic effects of Glu has two components. Neuronal death by necrosis
occurred rather quickly and was accompanied by delayed activation of apoptotic process.
The first step in necrotic cell death is usually cell swelling. Multiple cell swellings or
varicosities along dendrites were observed after treatment of neurons with 30 NMDA.
The dynamics of varicosities swelling can be seen on Figure 4. The process of cell swelling in
response to NMDA application is possibly related to activity of volume-sensitive chloride
channels (VSOR Cl
-
) [Inoue and Okada, 2007]. VSOR Cl
-
channels play a great role in cell
volume regulation, but cause neuronal body and processes swelling after prolonged NMDA
excitotoxic insult, which leads to necrotic cell death. Despite the data about the mechanisms
of NMDA induced necrosis [Inoue and Okada, 2007], information about necrotic processes
caused by KA is scanty.

Figure 4. Confocal microscopy images, demonstrating the dynamics of varicosities formation in
neuronal cell culture continuously exposed to 30 NMDA. Vital staining with ANEPPS. Arrows
indicate the place of single varicosity growth
Selective agonists of different Glu receptor subtypes can be utilizes to assess the
particular receptor type that determines the major contribution to neurodegeneration induced
by natural neurotransmitters. In order to evaluate the functional role of NMDARs and
AMPAs/KARs in Glu induced excitotoxic stress, we used the approach described above. We
have analyzed the effects of 3 mM Glu in combination with 50 M AP5, a selective
competitive antagonist of NMDARs, and 30 M CNQX, a selective antagonist of
AMPARs/KARs [Dingledine et al, 1999]. CNQX was found to provide highly significant
protection of neurons against the toxic action of 3 mM Glu: the viability of neurons in the
presence of CNQX increased, on average, from 34% to 65% (Figure 5). On the contrary, AP5
did not have any neuroprotective action suggesting that NMDARs are not involved in the
transmission of 3 mM Glu excitotoxicity. This observation clearly demonstrates that an
activation of AMPARs/KARs provide a dominant contribution to the neurotoxic effect of 3
mM Glu, which is consistent with data obtained using trypan blue as an indicator of necrosis
[Mironova et al, 2006].

Figure 5. Effects of 240-min exposure of glutamate receptor agonists and antagonists on ratio of live,
necrotic and apoptotic neurons in whole cellular population. (Glu) 3mM glutamate; (NMDA) 30 M
NMDA; (KA) 30 M KA; (CNQX) 30 M CNQX, (AP-5) 50 M AP-5. * The data significantly differ
from corresponding values, obtained in the absence of antagonists (p < 0.05, Students two-tailed t test).

The ability to induce neurodegeneration by activation of AMPARs/KARs was supported
by experiments using KA a selective agonist of these receptors. After 240 min exposure to
30 M KA 16% necrotic neurons and 52% of neurons dying by apoptosis ware observed (see
Table 1, Figure 5). KA produced a statistically significant reduction in the proportion of live
cells and increased the proportions of necrotic and apoptotic cells in comparison to the control
(see Table 1). When KA was coapplied with CNQX, the neurotoxic effect was significantly
weakened; the proportion of apoptotic and necrotic cells decreased, on average, to 16% and
1% respectively (Figure 5), and did not significantly differ from the control values (see Table
1).
The role of NMDARs in induction of neurodegenerative processes in neuron cultures was
evaluated by studying the neurotoxic action of 30 M NMDA. All experiments with NMDA
were performed in the presence of a saturating concentration of glycine (30 M) which is a
coagonist of NMDARs [Johnson, Ascher, 1987; Dingledine et al, 1990]. The data are
presented on figure 5. Activation of NMDARs for 240 min induced neurodegeneration mostly
via apoptosis (45%) rather than necrosis (15%). The data obtained in the presence of NMDA
with respect of all necrosis, apoptosis and live neurons were significantly different from those
of the control (see Table 1). NMDA induced neurodegeneration was completely prevented by
an addition of AP5 (Figure 5). The ratio of apoptotic, necrotic, and live cells obtained after
application of 30 M NMDA combined with 50 M AP5 did not significantly differ from the
control (see Table 1). This demonstrates the neuroprotective properties of NMDAR
antagonists.
At this stage of our study we employed a simple procedure using vital fluorescence stains
acridin orange and ethidium bromide. Recently it has been demonstrated the double
sequential acridine orange and ethidium bromide staining combined with the confocal
microscopy offers an express, fast, easy, sensitive and reproducible method by which
necrosis, apoptosis and live neurons can be recognized and automatically quantified in a
population of living cells [Mironova et al., 2007]. The utility of this assay has a broad
experimental potential ranging from the analysis of dynamics and mechanisms of cell death to
the pharmacological studies on a variety of live preparations.
The data suggests that either NMDARs or AMPARs/KARs can mediate excitotoxicity.
Excessive and selective activation of these Glu receptor types results in the development of
neurodegeneration via both mechanisms, necrosis and apoptosis, although apoptosis is
predominant. Overall, the neurotoxic action of KA was more pronounced than of NMDA, as
the total number of necrotic and apoptotic neurons was greater when excitotoxic insults were
induced by KA. Selective antagonists produced neuroprotective effects, preventing the
neuronal death by necrosis as well as the induction of apoptotic processes. Long-lasting
presence in the media of the natural neurotransmitter, Glu, which at concentrations used in
our experiments, presumably, open all NMDARs, AMPARs and KARs, led to substantially
more remarkable neurodegeneration in comparison with the selective agonists of particular
receptor types (Figure 5). In case of Glu the necrotic component was noticeably larger, as the
quantity of necrotic neurons was significantly greater when Glu was applied as a neurotoxic
agent, than those obtained after NMDA or KA exposure (p<0.05 for both comparisons,
Students two-tailed t test). In consistence with previous studies [Mironova et al, 2006;
Mironova et al, 2007] our experiments favor the conclusion that onset of necrosis requires
much less prolonged Glu receptor agonists exposure then of apoptosis. As could be seen from
Figure 2, 120 min agonist exposures caused rundown of live neuron because of necrosis. At
this period of time no visible features of apoptosis were found. The development of apoptosis
required a longer agonist treatment so, as a large portion of neurons exhibited apoptotic
features after 240 min agonist exposures. In conclusion, Glu and selective agonists of Glu
receptors are triggering neurodegeneration, which mechanistic pattern changes in time: at the
beginning of excitotoxic insults neurons predominantly die via necrosis, to start apoptosis a
longer period of time is required.
The vital fluorescent assay applied to neuron cultures provides an estimate of integral
pattern of apoptosis and can not provide any information about the particular intracellular
cascades involved in this pathology process. The particular apoptotic pathways that
participate the neurodegeneration evoked by different Glu receptor agonists (3 mM Glu, 30
M NMDA and 30 M KA) were analyzed in immunocytochemical experiments. We studied
an expression patterns of two apoptotic proteins P53 and Bax and the key proteins of two
basic apoptotic cascades AIF (apoptosis inducing factor) and Cas-3 (caspase 3). Neuron
cultures were incubated in the bathing salt solution supplemented with one of the agonists for
240 min. Cells then were fixed and the proteins of interest were visualized using immune
reactions with monoclonal antibodies.
Each of the agonists induced an increase of the number of cells that were immunopositive
to proapoptotic protein P53 (Figure 6, A). After exposure of neuronal cultures to any agonist
the number of neurons expressing P53 was significantly greater than those under the control
conditions (Figure 6, A), though the proportion of P53-positive cells exhibited no significant
difference between agonists (p>0.05, ANOVA). P53 is known to be involved in apoptosis
regardless of its particular cascades. This is a protein which is involved in the reparation when
DNA damages are present, and P53 expression increases as the cell progresses along the
apoptosis [Bates, Vousden, 1999; Miller et al, 2000]. In the case of our experiments P53
expression level in neuron cultures increased during excitotoxic stress caused by any agonist
and could be taken as an integral measure of the apoptosis intensity. This is supported by an
agreement in assessments of apoptosis either by P53 immune-positive cell count in fixed
tissues and the vital fluorescence assay. For example, the proportion of apoptotic neurons
among whole cellular population in course of treatment with 30 M NMDA (240 min) were
45 9% (n = 5) and 40 10% (n = 4) obtained using the vital fluorescence assay and the
reaction for P53, respectively, and did not differ significantly (p > 0.76, Students two-tailed t
test).
The same experimental protocol as for P53 has been used to study another proapoptotic
protein Bax, which demonstarated similar to P53 expression profile. Numerous Bax positive
cells were found after treatment of neurons with 30 M NMDA, 30 M KA and 3 mM Glu
(Figure 6, B). Glu was the most effective inductor of Bax expression, since in its presence
nearly 90% of neurons were Bax-positive. Selective activation of NMDARs or KARs was
less effective, causing substantial increase of Bax-positive neurons up to 50% (Figure 6, B). It
is well evaluated, that an elevation of Bax expression observed during apoptosis is
independent of involved apoptotic cascade (caspase-dependent or caspase-independent)
[Saikumar et al, 1998]. Bax translocation to mitochondrial membrane is critical for these
processes. With this respect an estimation of P53 and Bax expression can serve as two
reliable marks of apoptosis onset. These data suggest that any type of Glu receptors can
mediate neurotoxic insult, that promotes Bax expression.
Unlike P53 and Bax, AIF and Cas-3 are key molecules mediating caspase-independent
(AIF) or caspase-dependent (Cas-3) apoptotic cascades. AIF or Cas-3 inmunostaining allows
recognition of particular cascades involved in excitotoxicity. Under the control conditions
AIF protein was present in the cytoplasm of most neurons and only a few (on average 6%)
contained AIF in the nucleus (Figure 6, C). Treatment with 30 M NMDA for 240 min
produced a statistically significant increase in the proportion of cells containing AIF in the
nucleus (p < 0.001, Figure 6, C). The effects of 30 M KA and 3 mM Glu on AIF expression
did not differ, but were significantly weaker than 30 M NMDA induced increase of AIF
expression.
An elevation of Cas-3 expression depended on Glu mimetic used (Figure 6, D). The
histogram in Figure 6, D summarizes the results obtained. The quantity of neurons exhibiting
high nuclear content of Cas-3 during 30 M NMDA exposure did not differ from the control
level. The number of cells containing nuclear Cas-3 in the presence of 3 mM Glu and 30 M
KA were significantly greater than in the control (Figure 6, D).

Figure 6. Apoptotic proteins P53, Bax, AIF and Cas-3 expression in response to glutamate, NMDA and
KA for 240 min exposure. (A) immunostaining for P53 after exposure to 30 M KA; (B)
immunostaining for Bax after exposure to 30 M NMDA; (C) immunostaining for AIF after exposure
to 30 M; (D) immunostaining for Cas-3 after exposure to 30 M kainate. Immunoreactive neurons are
indicated by arrows. Histograms presented below the images show percentage of neurons
immunopositive for P53, Bax, AIF and Cas-3. (Intact) incubation in the bathing salt solution; (NMDA)
treatment with 30 M NMDA; (KA) treatment with 30 M kainate; (Glu) treatment with 3 mM
glutamate. * The data differ significantly from the data obtained under the control condition (p < 0.05,
Students two-tailed t-test)
In fact, 30 M NMDA was successful to increase the quantity of AIF-positive cells, but
did not affect the expression of Cas-3. In opposite, 3 mM Glu and 30 M KA, whose effects
are realized by AMPARs and KARs, increased the number of Cas-3-positive cells, but did not
affect AIF nuclear expression (Figure 6). In two sets of our experiments performed to
evaluate the apoptosis (immunostaining for P53 and Bax in fixed culture and the vital
fluorescence assay) regardless the methods NMDA and KA induced similar apoptosis
intensity. Observed difference of expression pattern for Cas-3 and AIF in the case of NMDA
and KA allows us to suspect that NMDARs-mediated apoptosis predominantly involves AIF-
dependent cascades, while AMPARs and KARs operate via the Cas-3-dependent apoptosis
pathway.

Conclusion
It is generally accepted that research to investigate the nature of processes and the extent
of changes in cells and cellular structures under different physiological states, including
pathogenesis, provides complex understanding of morphological and functional processes
occurring in development, normal functioning, regeneration and neurodegeneration of CNS.
A variety of stains [Noraberg, 1999; Patterson, 1979] and biochemical methods [Bergmeyer,
Bernt, 1974; Koh, Choi, 1978; Uliasz, Hewett, 2000] have recently become available whose
combination allows an assessment of the whole cellular cycle from the cell appearance till the
death. Each of these approaches has its own advantages and disadvantages.
Analysis of neurodegeneration processes in our studies was addressed by the recognition
of neuron death using sequential staining with acridine orange and ethidium bromide. The
characteristics, suitability for experiments on living tissues, effectiveness, and reliability of
this method of identifying necrosis and apoptosis have recently been described in detail
[Mironova et al, 2007]. The method is based on the ability of acridine orange to penetrate
membranes and stain the nuclei of living cells; ethidium bromide can only detect cells with a
disintegrated plasma membrane, staining their nuclei [Pulliam, 1998]. In addition, acridine
orange allows apoptosis to be detected because of the difference in staining of apoptotic and
living nuclei which has previously been used to identify apoptosis in cell populations of
Drosophila embryos [Abrams et al, 1993; White et al, 1994], in Tetrahymena, and chick
chondrocytes [Mpoke, Wolfe, 1997]. Many studies demonstrated that apoptosis is
accompanied by acidification of cells [Gottlieb et al, 1995; Li, Eastman, 1995], that appears
as a result of nuclear disintegration and is associated with decreases in pH due to fusion of
nuclei with lysosomes [Mpoke, Wolfe, 1997]. The difference in staining of living and
apoptotic nuclei results from a displacement of acridine orange emission to the red spectral
region while acidification is occurring during apoptosis [Zelenin, 1966].
Experiments have demonstrated that prolonged exposure of neurons to the GluR agonists
induces neurodegeneration, with substantial contributions being made by both apoptosis and
necrosis. Unlike Glu, the neurotoxic actions of NMDA and KA mainly induce apoptosis
rather than necrosis. This is not surprising because NMDA and KA are specific agonists of a
particular receptor type, whereas Glu can simultaneously activate any Glu receptors [Gibb,
Colquhoun, 1992]. Furthermore, the large contribution of necrosis to neurodegeneration
evoked by 3 mM Glu can also be explained by the fact that it is a natural neurotransmitter
and, therefore, has broad physiological effects which are not restricted by the activation of
ionotropic GluRs only. In the continuous presence in extracellular media, Glu may interfere
and interrupt the functioning of neuronal and glial Glu transporters, presynaptic structures by
the interaction with metabotropic Glu receptors as well as induce an impairment of the
transmembrane ionic gradients at the least for Na
+
, K
+
and Ca
2+
[Antonov, Magazanik, 1988;
Antonov, 2001]. The Glu concentration producing the neurotoxic effects in our experiments
on primary cortical neuron cultures of 7 DIV was rather high. This concentration was chosen
since a lower one (1 mM) had no particular effect throughout 240 min experiment [Mironova
et al, 2006]. This would appear to be associated with the receptor expression level, as the
effective (in terms of excitotoxicity) Glu concentrations for neurons of 14 DIV were almost
100-orders of magnitude lower than for 7 DIV [Mironova et al, 2007]. These lead us to the
conclusion that the endogenous agonist at high concentrations is more effective in the
induction of necrosis than its synthetic analogs. The opposite situation was observed in
relation to the promotion of apoptosis.
Overall our study on rat cerebral cortical neurons of 7 DIV revealed two components of
neurotoxic Glu action. One component was associated with the induction of necrotic cell
death, while the other was associated with triggering of apoptotic mechanisms. Both
components were clearly recognized for selective and natural agonists of Glu receptors rising
up the possibility of separate characterization of their pharmacological and neurochemical
properties.
Neurodegeneration induced by NMDA and KA was completely abolished (to the control
level) by the competitive NMDAR antagonist - AP5 and the competitive AMPAR and KAR
antagonist CNQX, respectively. This observation suggests that selective activation of any
receptor type is sufficient to trigger apoptosis that is consistent with previously published data
[Xiao et al, 2001; Wise-Faberowski et al, 2006]. This also demonstrates the potential of
selective antagonists of Glu receptors revealing different mechanisms of action, including
non-competitive inhibitors [Antonov, Johnson, 1996; Antonov et al, 1998], as neuroprotective
agents that are able to prevent neuronal injuries and the development of apoptotic processes.
The neurotoxic action of Glu was slightly reduced by CNQX, as a significant increase in
neuronal viability was found in the presence of CNQX, although no significant decrease in
necrosis and apoptosis was observed. At the same time, the effects of 3 mM Glu were
completely resistant to AP5, a specific antagonist of NMDARs. These suggest that the
neurotoxic effects on neurons of 7 DIV induced by 3 mM Glu are presumably mediated by an
activation of non-NMDA receptors [Mironova et al, 2006].
We identified cells expressing proapoptotic peptides P53, Bax, AIF and Cas-3 using
immunocytochemical methods. P53 is known to be a universal protein which is indirectly
involved in all apoptotic mechanisms. In our experiments, P53 expression is the same when
selective agonists or Glu were used, which is consistent with other data [Miller et al, 2000].
Under similar conditions, the number of neurons expressing P53 coincided well with the
apoptosis estimates obtained with the vital fluorescence assay. Immunostaining for P53 is
often utilized as universal marker for apoptosis, since an expression of this protein is
proportional to the degree of cell genome damages [Bates, Vousden, 1999]. In the case of
moderate genome damages, the cell division stops, DNA reparation occurs, and the cell
survives. In conditions of extreme genome fragmentation, when DNA can no longer be
repaired, receptor- and Cyt-C - dependent apoptotic cascades activate caspases. The first data
concerning the involvement of caspases in the neuronal death were obtained from studies of
their inhibitor P53 in cultures of substantia nigra neurons [Rabizadeh et al, 1993]. In these
cells, P53 blocked caspases, which resulted in inhibition of apoptosis induced by
hypoglycemia, Ca
2+
excess and deprivation of neurotrophic factors. An activation of caspases
is evidently one of the possible mechanisms of neuronal death in neurodegenerative
conditions.
P53 induces expression of proapoptotic Bax protein. In normal conditions small amounts
of Bax exist in the cytoplasm. During apoptosis Bax can be translocated to mitochondrial
membrane where it interacts with proteins of mithochondrial permeability transition pores
(PTP), forcing them to open. Opened PTP are permeable to AIF and Cytochrome-C. An
active involvement of Bax into Glu induced neuronal death has been demonstrated in
experiments with Bax-knockout cortical neurons cultures [Dargusch et al., 2001]. Antibodies
for Bax are now widely used in investigations of neurodegeneration [Zhang, Bhavnani, 2005;
Raghupathi et al., 2003]. We also have some preliminary data that increased expression of
Bax during apoptosis is accompanied with suppressed production of antiapoptotic Bcl-2
protein. The balance between pro- and antiapoptotic Bcl-2 family proteins it thought to be the
key parameter in switching neuronal programmed cell death.
An activation of ionotropic receptors (usually NMDARs) results in increased influx of
Ca
2+
into cells, triggering proteolysis and degradation of cellular structures [Hatanaka et al,
1996; Jonston, 1994]. This process is also accompanied by increased lipid peroxidation and
subsequent development of oxidative stress [Tapia, 1992; Waters, 1995; Boldyrev, 2001].
Although an activation of Glu receptors is evidently accompanied with neuronal oxidative
stress [Waters, 1995], there are nevertheless specific cascades triggering apoptosis whose
involvement depends on activation of a given receptor type. This statement is supported by
our experimental data on AIF and Cas-3 expression. The induction of apoptosis via the
activation of NMDARs was not accompanied with increased Cas-3 expression, though overall
assessment of apoptosis using P53 and the vital fluorescence assay gave high values. On the
contrary the significant increase in the number of neurons expressing AIF was observed.
Induction of apoptosis by KA and Glu led to the significant increase in Cas-3 expression as
compared to the control, though AIF production was substantially lower than in the case of
NMDA application.
It can be suggested that hyperactivation of AMPARs and KARs, which have low Ca
2+

conductance, is associated with impairments to energy metabolism and complex alterations of
mitochondrial and sarcoplasmic reticulum functions [Khodorov, 2004]. Complex
disregulation of mitochondrial membrane permeability is associated with the release of
several apoptogenic factors: Cyt-C and procaspases 2, 3, and 9. Cyt-C catalyzes Cas-3
activation which determined caspase-dependent pathway of apoptosis. The increase in Cas-3
in the experiments with 3 mM Glu and 30 M KA provide evidence that apoptosis was
triggered via the caspase pathway.
Another mechanism regulating apoptotic death occurs when NMDARs are activated.
This mechanism involves the release of AIF, which is translocated from the mitochondrial
membrane to the nucleus, inducing DNA degradation via the caspase-independent pathway
[Yu et al., 2002]. It is supported by observed increase of nuclear AIF presence after 30 M
NMDA excitotoxic insults. The results described in this chapter are indirectly supported by
data that NMDARs mediated apoptosis can not be blocked with caspase inhibitors, but is
prevented in the case of Bcl-2 hyperexpression [Wang et al., 2004].
Thus, we can conclude that apoptosis induced by the NMDARs activation develops
through the caspase-3 - independent pathway that involves direct AIF accumulation in nuclei.
The AMPARs/KARs mediated apoptosis includes a caspase-3 dependent mechanism. An
existence of the receptor specific apoptosis cascades during excitotoxicity reveals new
directions in selective therapy of neurodegenerative states.
The work presented in this chapter is supported by RFBR grants 05-04-49789 and 08-04-
00423.
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Chapter 5
Mitochondrial Uncoupling
Proteins Therapeutic Targets
in Neurodegeneration?
Susana Cardoso, Cristina Carvalho, Snia Correia,
Renato X. Santos, Maria S. Santos

and Paula I. Moreira
*
Center for Neuroscience and Cell Biology,
University of Coimbra, Coimbra, Portugal
Abstract
Uncoupling proteins (UCPs) are mitochondrial inner membrane proteins that uncouple
electron transport from ATP production by dissipating protons across the inner membrane.
UCP1 was the first uncoupling protein described and is present in brown adipose tissue being
involved in the non-shivering thermogenesis. Subsequent studies demonstrated that neurons
express at least three UCPs isoforms including the widely expressed UCP2 and the neuron-
specific UCP4 and UCP5. UCPs control the mitochondrial membrane potential, free radicals
production and calcium homeostasis and thereby influence neuronal function. Given that
mitochondrial energy impairment and free radicals production are thought to be central
players in neurodegeneration, recent data suggest that UCPs may have an important role in
neuroprotection and neuromodulation. The function of neuronal UCPs and their impact on the
central nervous system are attracting an increased interest as potential therapeutic targets in
several disorders including neurodegenerative diseases. Here we will discuss the uncoupling
process as an intrinsic mechanism of mitochondria physiology. The role of UCPs in healthy
and pathological brain conditions will be also considered. Finally, we will discuss UCPs as
potential therapeutic targets in stroke and neurodegenerative diseases.

Keywords: Brain, mitochondria, neurodegenerative diseases, stroke, UCPs

*
Corresponding author: Paula I. Moreira, Center for Neuroscience and Cell Biology, Faculty of Medicine-
Physiology, University of Coimbra, 3000-354 Coimbra, Portugal. venta@ci.uc.pt/ pismoreira @ gmail.com
Susana Cardoso, Cristina Carvalho, Snia Correia et al. 108
I. Introduction
Mitochondria are organelles located in the cytoplasm of all eukaryotic cells and involved
in many processes essential for cells survival and function, including energy production,
calcium homeostasis, redox control, and some metabolic and biosynthetic pathways [1].
Energy is generated as electrons are passed from donors at lower to acceptors at higher redox
potential through various protein complexes. Coupled with electrons transport, protons are
pumped from the matrix outward generating a potential difference across the inner
membrane. The resulting potential energy produced by the proton gradient is used to drive
phosphorylation of ADP to ATP [2].
Uncoupling proteins are a family of mitochondrial anion-carrier proteins located on the
inner mitochondrial membrane, and their primary function is to leak protons from the
intermembrane space into the mitochondrial matrix [3,4]. Through this process, the ATP
synthesis is uncoupled from the electron transport, dissipating energy in the form of heat. The
process of uncoupled respiration was first discovered in brown adipose tissue, where
uncoupling is affected by UCP1, the most well-characterized isoform. In the last few years,
studies brought evidence of the existence of other members of the UCP family that promote
partial uncoupling of oxidation from phosphorylation. These proteins include UCP2, UCP3,
UCP4, and UCP5 (also known as Brain Mitochondrial Carrier Protein 1, BMCP-1) that differ
among each other in tissue distribution and regulation and may have distinctive physiological
roles [3]. The specific role of UCPs has been widely discussed and although there is no
general agreement, there is a strong conviction that these proteins may be involved in the
defense against reactive oxygen species (ROS) therefore protecting or reversing oxidative
damage. The first evidence came from a study made by Ngre-Salvayre and colleagues [5]
revealing that the inhibition of UCP1 activates the formation of ROS in brown fat
mitochondria. Subsequent data suggested that UCP activity may lead to an increase in proton
conductance through the interaction with superoxide [6] or ROS products [7].
The alteration of mitochondrial energy metabolism leads to reduced ATP production,
impaired calcium buffering, and generation of ROS. Indeed, up to 90% of intracellular ROS
are generated in mitochondria during the oxidation of nicotinamide dinucleotide (NADH) and
flavin adenine dinucleotide (FADH
2
) [8]. The generation of ROS is increasingly recognized
as playing an important role in diabetes, obesity, ischemia/reperfusion, neurodegenerative
disorders and aging where mitochondria are both sources and targets of these reactive species
[9-11]. Therefore, it was suggested that an increased ROS production would lead to
uncoupling that in turn would decrease ROS formation, leading to reduced oxidative damage
[12]. Thus, this possible role of neuronal UCPs in neuroprotection and neuromodulation has
been faced as an important way to control and limit the formation of free radicals in several
disease and physiological processes. In this chapter we will begin by introducing the
uncoupling process as an intrinsic mechanism of mitochondria. We will then discuss the role
of neuronal UCPs in normal brain function. Finally, the role of UCPs in neurodegeneration
and stroke will be discussed highlighting their interest as potential targets for therapeutic
intervention in brain diseases.
Mitochondrial Uncoupling Proteins Therapeutic Targets in Neurodegeneration? 109
II. Mitochondrial Physiology and Uncoupling
Mitochondria are intracellular organelles highly efficient in their ability to utilize oxygen
(O
2
) and substrates to produce energy in the form of ATP [13]. The oxidative phosphorylation
system (OXPHOS) system is located in the inner mitochondrial membrane and is composed
by five respiratory chain complexes, NADH ubiquinone oxidoreductase (Complex I),
succinate ubiquinone oxidoreductase (Complex II), ubiquinone-cytochrome c reductase
(Complex III), cytochrome c oxidase (Complex IV) and ATP synthase (Complex V). There
are two electron carriers, ubiquinone (coenzyme Q), located in the inner mitochondrial
membrane and cytochrome c, located in the intermembrane space (Figure 1) [14]. Reducing
equivalents produced in the Krebs cycle and in the -oxidation pass through complexes I to
IV and the energy generated by the electron transfer is used to pump protons from the
mitochondrial matrix into the intermembrane space creating an electrochemical proton
gradient used to drive complex V to generate ATP (Figure 1) [15]. The efficiency in use the
formed H
+
gradient is called coupling. In a perfectly coupled system, protons only enter the
mitochondrial matrix through ATP synthase in the presence of ADP, this form of respiration
is classified as state 3 (O
2
is consumed only in the presence of substrate and ADP). But
mitochondria can also use O
2
even in the absence of ADP, which occurs when protons leak
back into the matrix via a mechanism independent of ATP synthase that will uncouple
respiration from oxidative phosphorylation. O
2
consumption in the absence of ADP or in
totally uncoupled mitochondria is designated to as state 4 of respiration [2]. H
+
gradient can
be consumed by many proteins, like members of the mitochondrial carrier family (ADP/ATP
carrier and glutamate/aspartate carrier) and others like phosphate carrier and other carriers
employing substrate-H
+
symport [16]. Additionally, under normal conditions, a portion of the
created H
+
gradient is consumed by proton backflow to the matrix via non-protein membrane
pores or protein/lipid interfaces [17]. These mechanisms allow H
+
backflow to the matrix
bypassing ATP synthase and thereby provoke protein-mediated respiration uncoupling [18].
Thus, uncoupling is an inherent part of mitochondrial physiology. The process of proton leak
has been suggested to be involved in thermogenesis, regulation of energy metabolism or
carbon fluxes, control of body mass, and attenuation of ROS production [19].
Mitochondrial uncoupling proteins (UCP1, UCP2, UCP3, UCP4, and BMCP-1) that are
located into the inner mitochondrial membrane function as proton carriers and are responsible
for basal proton leak (Figure 2), which in the rat accounts for 10-27% of resting O
2

consumption, depending on the organ studied [19]. UCPs isoforms are activated by free fatty
acids (FFAs), superoxide and inhibited by purine nucleotides [20], possessing some degree of
homology among each other. UCP1 was first described in brown adipose tissue and is
responsible for heat generation in the newborn and may be involved in the normal response to
cold stress in the adult [21]. UCP2 is 59% identical to UCP1 and is widely expressed in
spleen, lung, stomach, white adipose tissue and also in the brain [22]. UCP3 that is mainly
expressed in skeletal muscle [23] possesses approximately 57% and 73% of similarity with
UCP1 and UCP2, respectively. Finally, UCP4 [24] and BMCP-1 [25] are mainly expressed in
the central nervous system being 34% and 30% identical to UCP1, respectively, and BMC1 is
the only UCP found in the parenchymal cells of the liver [8]. All the UCPs share in common
a tripartite structure that consists of three repeats of approximately 100 amino acids, each one
containing two hydrophobic stretches that correspond to transmembrane alpha helices that
span the phospholipids bilayer of the mitochondrial inner membrane. The two attached alpha
helices are linked by a long hydrophilic loop, which is oriented toward the mitochondrial
matrix side. UCPs have a monomer molecular weight of about 30 kDa with both the N- and
the C-terminal ends oriented toward the cytosolic side of the inner mitochondrial membrane.
The functional unit of UCPs is believed to be a homodimer formed by two identical subunits
that contain 12 transmembrane helices [1,3,26].

Figure 1. Schematic representation of the mitochondrial electron transport chain (ETC). ETC is
composed by four protein complexes (I-IV). Electrons from oxidative substrates are transported through
ETC, by the two electron carriers, coenzyme Q that accepts electrons from complexes I and II and
donate them to complex III, and cytochrome c that transfers electrons from complex III to complex IV
leading to oxygen reduction to water. In this process, protons are pumped across the inner
mitochondrial membrane to establish a proton motive force. The energy that is conserved in this proton
gradient drives the synthesis of ATP via complex V (ATP synthase) as protons are transported back
from the intermembrane space into the mitochondrial matrix. UQ: coenzyme Q; Cyt c: cytochrome c;
complex I: NADH ubiquinone oxidoreductase; complex II: succinate ubiquinone oxidoreductase;
complex III: ubiquinone-cytochrome c reductase; complex IV: cytochrome c oxidase; complex V: ATP
synthase.
III. UCPs in the Brain- Effects on
Neuronal Function
Neuronal UCPs specific roles are still under intense debate. Although initially neuronal
UCPs (UCP4 and BMCP-1) were not considered real uncoupling proteins, it is becoming
more clear that these proteins perform important roles in neuronal function. For instance,
using specific antibodies for UCP4 it was found that this protein is present in fetal murine
brain tissues as early as embryonic days 12-14, which coincides with the beginning of
neuronal differentiation [27]. Moreover, UCP4 content decreases as the mice get older being
its highest levels found in the cortex, which suggest a role for UCP4 in neuronal cell
differentiation [27]. Nevertheless, it is important to note that the tissue distribution of
neuronal UCPs mRNA differs between rat and mouse [28]. In this section it will discussed
some possible roles of neuronal UCPs in normal brain function.
Thermogenesis
Energy homeostasis is a highly regulated process and is central to the control of body
weight. UCPs have been identified and implicated as potential regulators of adaptive
thermoregulation, body composition, and metabolism [29]. Furthermore, temperature
differences in various brain regions have been observed in rodents and more recently in
humans, by invasive and non-invasive techniques [30-33]. UCP2 mRNA is expressed
throughout the brain with some variability. A markedly intense expression is found in several
parts of the brain including hypothalamus, thalamus, brainstem, cerebellum and the choroid
plexus [22]. Indeed, evidence reveals a dorsoventral temperature gradient that coincides with
the occurrence of UCP2 and increased proton leak [34]. This may suggest UCP2-induced
mitochondrial uncoupling involvement in thermoregulation of the brain. The mRNA
abundance of UCP4 and UCP5 is modulated by nutritional and temperature manipulations in
a tissue-specific manner [29]. On cold exposure, brain UCP4 rise significantly and remains
elevated from 1 to 24 h, whereas UCP5 mRNA in brain and liver increases at 1 h and remains
elevated through, at least, 6 h [29]. Additionally, body temperature changes were preceded by
a rise in UCP4 (in brain) and UCP5 (in brain and liver) expression [29]. Accordingly, cold
temperature significantly increased UCP4 mRNA levels in rat hippocampal neurons [35].
Thus UCPs may play an important role in thermoregulatory processes induced by cold
exposure. Nevertheless, it was recently demonstrated that in adult mice brain UCP4 highest
levels occur in the cortex, which does not support the thermoregulatory function of UCP4
[27]. Further research is needed in order to clarify UCP4 involvement in thermogenesis
regulation.
Calcium
Mitochondria play important roles in the regulation of cellular calcium (Ca
2+
) dynamics
in physiological situations, including neurotransmitters release, and in pathological states.
Mitochondrial bioenergetics and redox state are also influenced by intracellular Ca
2+
levels
being mitochondria capable of promoting Ca
2+
uptake into the matrix that is driven by
mitochondrial membrane potential (
m
) [36]. Within mitochondrial matrix increased levels
of Ca
2+
can affect mitochondrial ROS release and subsequent oxidative stress. Mitochondrial
dysfunction, resulting from the disruption of calcium homeostasis and the generation of ROS,
is a central player in neuronal dysfunction and death following acute brain insults [37].
Uncoupling agents like 2,4-dinitrophenol (DNP) have been shown to reduce mitochondrial
Ca
2+
increases that occur after quinolinic acid-induced NMDA receptor activation [38]. Also
in an animal model of traumatic injury, DNP treatment reduced by 30% the ability of
mitochondria to sequester Ca
2+
[39]. More recently, Chan and colleagues [40] demonstrated
that human UCP4 expression in PC12 cells regulates mitochondrial Ca
2+
sequestration and
entry but not the release from intracellular stores. Furthermore, UCP4 expression decreased
the magnitude of sustained elevation in intracellular Ca
2+
concentration after cellular Ca
2+

depletion and inhibited mitochondrial Ca
2+
overload and oxidative stress thereby preventing
cell death (Figure 2) [40].
Neuronal ROS Production
A small proportion of the electrons flowing through complexes I and III react with O
2

forming superoxide anion [41] that can be converted into hydrogen peroxide (H
2
O
2
) utilizing
both manganese superoxide dismutase (MnSOD), which is located to the mitochondria and
copper-zinc superoxide dismutase (Cu/ZnSOD) found in the cytosol. H
2
O
2
is promptly
converted to H
2
O via catalase and glutathione peroxidase, but has the potential to be
converted to the highly reactive hydroxyl radical via the Fenton reaction, underlying ROS
neurotoxicity [42].
Within the cells ROS may have a dual role, acting as beneficial or harmful species [15].
In response to certain stimuli cells produce low/moderate levels of ROS that have
physiological functions intervening in several cellular signaling pathways, therefore acting as
second messengers [15,43]. In opposite, excessive ROS formation will attack unsaturated
fatty acids in membranes leading to lipid peroxidation and the production of 4-
hydroxynonenal (HNE) that conjugates to membrane proteins impairing their function [39].
Moreover, situations of increased oxidative stress and mitochondrial Ca
2+
overload promote
the opening of the permeability transition pore (PTP), a situation characterized by the
mitochondrial proton motive force disruption. PTP opening will lead to the release of pro-
apoptotic proteins like cytochrome c, which induce the caspase-mediated apoptosis [15]. In
order to overcome the oxidative insult, cells possess a variety of enzymatic and non-
enzymatic antioxidant defenses. However, if an imbalance between antioxidant defenses and
ROS formation occurs, oxidative damage of cells will happen contributing to the
development of neurodegenerative diseases [44].
Mitochondrial ROS production is closely linked to
m
such that hyperpolarization (high
m
) increases and promotes ROS production [45]. While long-term, complete uncoupling of
mitochondria would be detrimental, it has been postulated that mild uncoupling could be
beneficial since it causes a decrease in ROS production [45]. In rat kidney mitochondria,
superoxide activates UCPs from the matrix side of the mitochondrial inner membrane [46]
while the presence of mitochondrial-targeted antioxidants prevents superoxide-induced
uncoupling [46]. Interestingly, uncoupling is not inhibited by extracellular SOD that cannot
access mitochondrial matrix side, so demonstrating that superoxide generated at the matrix
side did not need to be exported or cycle across the inner membrane to cause uncoupling [46].
Also, UCP3 knockout mice exhibit increased ROS in muscle [47].
Several lines of evidence demonstrate that brain regions that overexpress UCP2 present a
decreased energy coupled efficiency and a reduced
m
[48]. The GDP-stimulated
uncoupling that occurs in the presence of ubiquinone is abolished by SOD suggesting that
UCPs are activated by superoxide [49]. Liu and colleagues [35] demonstrated that UCP4 have
an important role in promoting neuronal survival. Indeed, the authors observed that PC12
cells overexpressing UCP4 present a reduction in mitochondrial oxidative phosphorylation
and ROS production that result from the UCP4-induced metabolic shift associated with
enhanced glucose uptake and glycolysis to compensate for reduced ATP mitochondrial
production [35]. Thus, UCP4 activity makes neural cells less reliant on mitochondrial
respiration for maintenance of energy levels. In order to establish the function of endogenous
UCP4 in neurons, it was performed RNA interference (RNAi) to deplete UCP4 in cultured rat
hippocampal neurons. It was shown that cells transfected with UCP4 RNAi present a
decreased survival rate compared to cells transfected with scrambled RNAi [35]. These
results demonstrate that endogenous UCP4 is required for hippocampal neurons survival.
BMCP-1/UCP5 is present in the cortex, basal ganglia, substantia nigra, cerebellum, and
spinal cord [50]. Data show that neuronal cell lines transfected with BMCP-1/UCP5 had
higher state 4 of respiration and lower
m
, revealing greater mitochondrial uncoupling [50].
In addition, it was also observed a reduction in mitochondrial ROS production [50] (Figure
2). Moreover, the exposure of neurons overexpressing BMCP-1 to linoleic acid further
enhanced state 4 of respiration while the addition of bovine serum albumin prevented this
augmentation [50], supporting the notion of UCPs activation by FFAs. Accordingly, the
reduction of dietary fat in immature animals rapidly reduces neuronal UCPs
expression/activity leading to increased mitochondrial ROS production [51]. These changes
decreased animals resistance to excitotoxic insults resulting in increased neuronal death [51],
thus implicating a neuroprotective role for UCPs in neuronal injury.

Figure 2. Potential neuroprotective mechanism of neuronal UCPs. Mitochondrial uncoupling proteins
(UCPs) function as an uncoupler by acting as a channel for proton entry into the matrix, which then
dissipates the transmembrane potential (
m
) generated by respiratory complexes I through IV. This
reduces the proton motive force leading to the uncoupling of respiration from oxidative
phosphorylation, dissipating energy in the form of heat. UCP-induced
m
reduction in neurons will
reduce ROS produced in mitochondria and prevent mitochondrial calcium overload. UQ: coenzyme Q;
Cyt c: cytochrome c; complex I: NADH ubiquinone oxidoreductase; complex II: succinate ubiquinone
oxidoreductase; complex III: ubiquinone-cytochrome c reductase; complex IV: cytochrome c oxidase;
complex V: ATP synthase

Due to UCPs role in the regulation of energy metabolism, their possible role in diabetes
has been exploited. Diabetes, namely hyperglycemia, leads to an oversupply of electrons in
the ETC that result in mitochondrial membrane hyperpolarization and ROS formation
[42,52]. Although UCP3 expression was thought to occur mainly in the muscle, a recent
finding shows that UCP3 is also normally present in dorsal root ganglion (DRG) neurons
[53]. Moreover, UCP3 levels were decreased in DRG neurons isolated from streptozotocin-
induced diabetic animals while UCP3 overexpression in cultured DRG neurons was able to
prevent glucose-induced mitochondrial hyperpolarization, ROS formation and programmed
cell death induction [53]. Also, the human neuroblastoma cell line SH-SY5Y when exposed
to high concentrations of glucose presented a down-regulation of UCP3 protein expression,
and an increase in
m
and intracellular ROS [54]. In opposite, the addition of insulin-like
growth factor (IGF-1), which positively regulates UCP3 expression [55,56], prevented the
glucose-induced neurite degeneration and UCP3 down-regulation leading to ROS levels and
m
normalization [54].
IV. UCPs in Neurodegeneration and Stroke:
Potential Therapeutic Targets
The brain is extremely sensitive to oxidative damage due to its high O
2
demand, its high
content of oxidisable polyunsaturated fatty acids, the presence of redox-active metals and a
low activity of antioxidant enzymes [10,14,57]. Since oxidative stress increases with age and
mitochondria are both targets and sources of ROS, there is the assumption that mitochondria
have a central role in aging and neurodegenerative disorders [58]. Indeed, despite the fact that
neurodegenerative disorders have disparate clinical features, they are characterized by
mitochondrial dysfunction and oxidative stress [59]. It has been demonstrated that human
UCP2 targeted expression in mitochondria of adult fly neurons promotes an increase in state 4
of respiration, a decrease in ROS production and oxidative damage accompanied with an
extension in life span without the compromise of fertility or physical activity [60]. Due to
UCPs ability to regulate both mitochondrial metabolic efficiency and free radical generation,
they are of special interest in stroke and neurodegenerative pathologies, including
Alzheimers (AD) and Parkinsons (PD) diseases and amyotrophic lateral sclerosis (ALS).
Alzheimers Disease
Alzheimers disease (AD) is a progressive age-dependent neurodegenerative disorder and
the most common form of dementia, accounting for 50-70% of dementia cases. While less of
5% of AD cases are familial and associated with mutations in amyloid precursor protein
(APP) and presenilins 1 and 2 (PS1 and PS2), the majority of AD cases are sporadic in origin
and involve genetic and environmental factors that taken alone are not sufficient to develop
the disease [61]. This neurodegenerative disease is characterized by progressive cognitive
decline and the presence of A plaques and tau neurofibrillary tangles [11,62]. Abnormal
APP processing is believed to play a central role in AD. APP may be metabolized along two
distintic pathways: the amyloidogenic and the non-amyloidogenic pathways. In the latter,
APP is cleaved by an -secretase producing non-amyloidogenic proteins. In the
amyloidogenic pathway APP is cleaved by - and -secretases producing the A peptides
[63]. It has been reported that A oligomers and plaques are potent synaptotoxins, which
block the proteasome function, inhibit mitochondrial activity, increase oxidative stress, and
alter intracellular Ca
2+
levels leading to synaptic dysfunction [64].
Recent data from postmortem brain tissue from AD patients with different degrees of
severity demonstrated that AD is associated with impairments in mitochondrial gene
expression, namely in complex IV of the mitochondrial respiratory chain, increased levels of
p53 gene expression and increased molecular indexes of oxidative stress, such as up-
regulation of nitric oxide synthase (NOS) and NADPH-oxidase (NOX). Additionally, the
authors performed real time quantitative RT-PCR studies and found that in the brain, UCP4
and UCP5 expression is approximately 200 and 800-fold higher, respectively, than UCP 2
expression [65], thus suggesting a protective role of UCP4 and UCP5 against oxidative stress
under normal circumstances. However, in AD brains UCPs gene expression decreased
significantly relative to control brains and the mean levels tended to be lower in AD brains
with higher grades of neurodegeneration [65]. So, the failure to maintain normal levels or
increase the expression of UCPs may potentiate oxidative stress leading to progressive
mitochondrial DNA damage and energy depletion.
Data show increased levels of UCP4 expression in the cortex and hippocampus of mice
undergoing dietary restriction [35,66]. Since dietary restriction has been shown to be
neuroprotective in animal models of AD [67], it was suggested that neuronal UCPs may have
a role against chronic neurodegenerative diseases [37,68].
Parkinsons Disease
PD is the second most common neurodegenerative disorder that begins by causing motor
dysfunction but ultimately affects the mind and personality [62]. This disease is clinically
characterized by progressive rigidity, bradykinesia and tremor and pathologically by the
degeneration of pigmented neurons in the substantia nigra and by the presence of
intraneuronal proteinaceous cytoplasmic inclusions that immunostain for -synuclein and
ubiquitin, designated Lewy Bodies [58,62]. It is well established that oxidative stress and
mitochondrial dysfunction are associated with the degeneration of dopaminergic neurons in
PD.
The involvement of mitochondrial dysfunction in PD arose from the finding that 1-
methyl 4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP), a synthetic opiate, caused
parkinsonism in drug addicted individuals [69]. MPTP is metabolized to MPP
+
in glial cells
and this metabolite inhibits the complex I of the mitochondrial respiratory chain [69]. Horvath
and colleagues [70] have previously shown that CoQ therapy in a PD animal model induces
mitochondrial uncoupling in the substantia nigra preventing MPTP-induced cell death.
Moreover, it seems that this effect is mediated by UCP2 activation that precedes the CoQ
prevention [70]. Later on, Andrews and co-workers [68] demonstrated that dopamine neurons
sensitivity to MPTP is increased in UCP2 knockout mice, whereas UCP2 overexpression
decreased MPTP-induced nigral dopamine cell loss by increasing mitochondrial uncoupling
and decreasing ROS production [68]. Furthermore, electron microscopic analysis revealed
that the substantia nigra from UCP2 knockout mice had significantly lower number of
mitochondria compared with control [68]. Similarly, Conti and colleagues [71] showed that
transgenic mice overexpressing UCP2 in catecholaminergic neurons present increased
uncoupling of their mitochondria, a reduction in oxidative stress and retention of locomotor
functions after MPTP exposition. These results suggest that UCP2 is an essential homeostatic
protein regulating cell survival and vulnerability in the presence of harmful toxins. UCP2 role
in PD was also explored in normal nigrostriatal dopamine function and previous studies show
that mice lacking UCP2 exhibited reduced dopamine turnover in the striatum, reduced
tyrosine hydroxylase immunoreactivity in the substantia nigra, striatum and nucleus
accumbens and reduced dopamine transporter immunoreactivity in the substantia nigra [72].
Moreover, when evaluating those deficits in locomotor function, the authors observed that
UCP2 knockout mice exhibited reduced total movement distance, movement velocity and
increased rest time compared with wild type rats [72], giving the suggestion of UCP2
involvement in the maintenance of normal nigrostriatal dopamine neuronal function.
Recently, the same authors reported that UCP2 mediates ghrelin-induced neuroprotection
against dopaminergic cell loss in the substantia nigra after MPTP treatment through
alterations in mitochondrial respiration, ROS production, and biogenesis [73].
Neuronal UCP5 role was investigated in a neuroblastoma cell line exposed to MPTP [74].
The authors observed that UCP5 knockdown increased caspase 3 levels and cell death. UCP5
knockdown also increased cytotoxicity induced by low doses of MPTP but had no effects in
oxidative stress and membrane depolarization [74]. However, in the presence of high doses of
MPTP, UCP5 knockdown exacerbated cytotoxicity, and increased oxidative stress and
mitochondrial membrane polarization [74], demonstrating the importance of UCP5in
oxidative stress-induced neurodegeneration.
Amyotrophic Lateral Sclerosis
ALS is the most common adult-onset motor neuron disease [75] resulting in weakness,
paralysis and subsequent death. Approximately 90% of the cases are sporadic and the
remaining 10% are familial [58]. Mitochondrial and bioenergetic defects are widely
implicated in ALS being reported alterations in mitochondrial structure, number and
localization in motor neurons and skeletal muscle [76]. Additionally, the presence of mutant
SOD1 within motor neuron causes alterations of the mitochondrial respiratory chain [77],
namely in mitochondrial complexes II and IV [78]. Other studies show an involvement of
ROS in the pathology of ALS [79]. Therefore, there is a strong notion that mitochondrial
dysfunction may play a critical role in ALS pathology.
Motor neurons have high energy demands, which make them particularly vulnerable to
the adverse effects of mitochondrial impairment. When metabolically compromised, motor
neurons become unable to maintain membrane potential, resulting in the opening of voltage
dependent NMDA glutamate receptors and excessive calcium influx [80]. Indeed, it has been
shown that glutamate levels are increased in the cerebrospinal fluid of half of ALS patients
[81], this effect being associated with the loss of spinal motor neurons [82,83].
Neuronal UCPs are expressed in the mice spinal cord and their levels do not seem to be
affected in SOD1 transgenic mice at different ages [84]. Nevertheless, UCP3 mRNA and
protein levels are selectively increased in ALS skeletal muscle, both in animal models and in
human biopsies [84]. On the other hand, other studies suggest that transient mitochondrial
uncoupling can confer neuroprotection following spinal cord injuries [39]. For instance, rats
administrated with DNP, a mitochondrial uncoupler, present less tissue loss, improved
behavioral outcomes and a concomitant reduction in mitochondrial oxidative damage, Ca
2+

loading and dysfunction after spinal cord injury [39,85]. Further research is needed in order to
clarify the role of UCPs in ALS.
Stroke
Acute brain injury, caused by stroke and trauma, is a major cause of morbidity and
mortality in the industrialized countries. During a stroke a drastic reduction in blood supply to
neurons occurs resulting in cellular hypoxia and glucose deprivation, which impair oxidative
phosphorylation and ATP formation [13]. In experimental models of stroke, ischemic
neuronal damage includes excitotoxicity, Ca
2+
influx and accumulation within mitochondria,
superoxide production, and subsequent oxidative stress, p53 and Bax expression, PTP
opening, cytochrome c release and caspases 9 and 3 activation [86]. Therefore, mitochondrial
dysfunction appears as a common pathway in neuronal cell death in acute brain injury.
Mitochondrial UCPs reduce mitochondrial ROS production by dissipating the hydrogen
ion gradient across the inner mitochondrial membrane. Mattiason and co-workers [87] found
that cortical neurons overexpressing UCP2 are protected against oxygen-glucose deprivation
(OGD)-induced cell death. Furthermore, transgenic mouse overexpressing human UCP2
protein subjected to ischemic preconditioning are protected after a severe ischemic insult
presenting enhanced neurological recovery [87]. The authors found that UCP2 promote a shift
in hydrogen peroxide release from the mitochondrial matrix to the extramitochondrial space,
where it can be degraded by some antioxidant enzymes [87]. Recently, Liu and colleagues
[88] also demonstrated that ischemic preconditioning caused increased expression of UCP2 in
rat hippocampus that conferred protection against ischemia/reperfusion injury. Treatment
with SOD at the time of ischemia preconditioning attenuated the increase of UCP2 staining,
therefore implying a role for superoxide-induced UCP2 expression [88]. Interestingly, another
previous work in UCP2 knockout mice demonstrate that deficiency in UCP2 results in
increased resistance to cerebral ischemia that is associated with reduced oxidative injury and
an increase of the cerebral neuronal anti-oxidant state [89]. Moreover, authors show that
UCP2 mRNA induction was temporally associated with changes of mitochondrial glutathione
levels following ischemia in mice. The authors suggested that a chronic adaptation to the lack
of UCP2 occurs, which may contribute to the reduction of ischemic injury and levels of lipid
peroxidation in UCP2 knockout mice [89]. Therefore, interventions aimed to maintain
mitochondrial homeostasis may provide new directions to prevent or attenuate acute central
system injury [37]. It has been also reported an increased UCP2 and UCP5 expression in
ischemic lesions in brain slice sections prepared from embolic stroke and multiple infarction
brains [90]. Moreover, UCP5 expression in the lesions was higher in multiple infarction cases
than in embolic stroke cases suggesting that UCP5 may respond to repetitive ischemic
stresses or have a long-term effect [90]. So, under ischemic conditions brains may develop
adaptive mechanisms involving an increased expression of UCP2 and UCP5.
Although more studies are needed to elucidate the mechanisms underlying the protective
roles of UCPs, the above-discussed studies show that UCPs are attracting an increased
interest as potential therapeutic targets in several brain disorders.
Conclusion
Similar to UCP1, also neuronal UCPs (UCP2, UCP4 and BMCP-1) appear to modulate
proton leak through the inner mitochondrial membrane decreasing the mitochondrial
electrochemical potential. However, while the main UCP1 function is to produce heat to
maintain body temperature, neuronal UCPs biological function seems to be different and
include the control of ROS production.
Neurodegenerative disorders are characterized by a progressive decline in neurological
function and neuronal cell death and despite the fact that these disorders have disparate
clinical features, they are characterized by mitochondrial dysfunction and oxidative stress.
Research in neuronal UCPs is still an emerging field where future analyses of such proteins
are needed. Nevertheless, the results obtained in experimental models indicate that their
possible neuroprotector and neuromodulator role could be a promising avenue to develop
better therapies to prevent or ameliorate stroke and neurodegenerative disorders.
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Chapter 6
Targeting Caspases in Neonatal
Hypoxic-Ischemic Brain Injury and
Traumatic Brain Injury
Xin Wang
*1
, Rachna Pandya
1
, Jiemin Yao
2,4
, He Ma
3
and Jianmin Li
2

1
Laboratory of Neuroapoptosis Drug Discovery, Department of Neurosurgery, Brigham
and Womens Hospital, Harvard Medical School, Boston, Massachusetts, USA
2
Department of Neurosurgery, Thi rd Affiliated Hospital,
3
Department of Anesthesiology, Tumor Hospital, Guangxi Medical University,
Nanning, Guangxi China
4
Department of Medicine-Infectious Disease, The University of Texas Health Science
Center at San Antonio, San Antonio, Texas , USA
Abstract
Mounting evidence implicates apoptosis in the pathogenesis of both acute and
chronic neurological disorders. The caspase family of cysteine proteases plays a central
role in the initiation and execution of neuronal apoptosis. So far the caspase family has
been expanded to 18 cysteine protease members. About two decades of investigation
involving the caspase family has produced a wealth of information. Studies indicate that
targeting the caspase family can prevent neuronal cell death in neurological disorders.
This chapter will discuss the role of the caspase family in experimental models of
neonatal hypoxia-ischemia brain injury and traumatic brain injury in vivo and in vitro, as
well as in human neonatal hypoxic-ischemic encephalopathy and traumatic brain injury.
Given that elucidation of the roles of individual caspases could yield multiple points of
possible therapeutic intervention, from the drug discovery and treatment perspective, the
review will summarize what is currently known about the beneficial effects of targeting
caspases using a variety of treatments against neonatal hypoxia-ischemia brain injury and

* Address correspondence to correspondence author:Xin Wang, Ph.D., Brigham and Womens Hospital, Harvard
Medical School, Department of Neurosurgery, Boston, Massachusetts 02115, USA, Phone: (617) 732-4186,
Fax: (617) 732-6767, E-mail: xwang@rics.bwh.harvard.edu
Xin Wang, Rachna Pandya, Jiemin Yao et al. 126
traumatic brain injury. It will focus on commonalities in the inhibition of caspase in the
cell death receptor pathway, the mitochondrial death pathway and the endoplasmic
reticulum death pathway.
1. Introduction
Caspases, a family of 18 enzymes, play a pivotal role in various neurological diseases of
neonates and adults including neonatal hypoxic-ischemic encephalopathy (H-IE) and
traumatic brain injury (TBI), respectively. In acute neurological diseases due to ischemia,
necrosis is responsible for cell death in the core of the lesion; however, at the penumbra of the
lesion apoptosis mediates cell death. Caspases are chronically activated in this area of
ischemic/hypoxic lesion, eventually leading to cell death. Thus caspase inhibition is a
possible approach to treatment of acute neurological diseases[1].
Caspases, first identified in the nematode Caenorhabditis elegans[2], are evolutionarily
conserved in many multicellular organisms. Genes for Ced-3, ced-4, egl-1, and ced-9 are
identified as loci controlling these enzymes[3]. In 1996 the nomenclature Caspase was
coined based on their structure and function (as cysteine-dependent aspartate specific
proteases), and numbers were given according to hierarchical discovery of each caspase. They
are the central mediators in apoptosis or programmed cell death, through which the body
regulates the number of cells in an organ[4]. Functionally caspases are zymogens containing
an N-terminal prodomain. Their mature enzymes are heterotetramers of two subunits of ~20
kDa and two subunits of ~10 kDa each[5, 6]. The active enzyme is obtained via
endoproteolytic cleavage and exists either as a dimer or monomer. These active enzymes,
obtained through cleavage at the Asp 297 site, recognize tetrapeptide motifs and cleave their
substrates on the carboxyl end of aspartate residues. All caspase recognition sequences
contain Asp at -1 position. However, the specificity of the individual caspase is determined by
positions -2, -3, and -4[7].
Caspases can be broadly classified as either effector caspases (3, 6, 7), which have short
N-terminal domains and are activated by proteolysis of other caspases, or initiator caspases
(1, 2, 4, 5, and 8-13), which have long N-terminal domains and are activated by non-
proteolytic signaling molecules[4, 5]. Caspase-14, which has been found in cornifying
epithelia such as skin[8], is the only purely non-apoptotic caspase with a role in cytokine
maturation[1]. Eckhart et al. identifies and characterizes caspase-15 as a mammalian caspase
with proapoptotic activity[9]. Eckhart et al. also demonstrates that caspase-16 is most similar
in sequence to caspase-14, caspase-17 to caspase-3, and caspase-18 to caspase-8[10].
The cell death receptor pathway is activated by the ligation of Fas (CD 95/Apo1), the
TNFR1 homologue, with the cognate ligand. The death inducing signal complex (DISC) is
formed[11] by the DD (Death Domain) in the receptor as well as the adaptor molecule
(FADD) and the DED (Death Effector Domain) in the adaptor molecule and procaspase-8,
eventually leading to activation of caspase-8. Caspase-8 further activates effector caspases (3,
6, 7), which carry out limited proteolyis. The mitochondrial death pathway is activated by
mitochondrial permeabilization leading to release of proapoptotic factors cytochrome c (cyto.
c), Smac and apoptosis inducing factor (AIF)[12]. An apoptosome of procaspase-9, cyto. c,
and Apaf-1 is formed eventually activating caspase-9. These in turn activate the effector
caspases (3, 6, 7). Caspase-12 is located in the endoplasmic reticulum (ER) and is activated
Targeting Caspases in Neo-Natal 127
by ER stress, calcium influx, and accumulation of excessive proteins in the ER[13], leading to
activation of the ER death pathway, which in turn acts on the effector caspases. Caspase-3 (32
kDa) is one of the primary protease executioners of apoptosis and is activated by intra-chain
proteolytic cleavage, which generates a large subunit (17 kDa) and a small subunit (12 kDa).
Typically, after apoptotic stimuli, the level of cleaved caspase-3 increases while that of pro-
caspase-3 decreases. The effector caspases act on the protein kinases, other signal
transduction proteins, DNA repair proteins, chromatin modifying proteins, and nuclear matrix
proteins which eventually hails the death of the cell. Activation of CAD (caspase activated
DNAse) via caspase-3 leads to DNA fragmentation. In addition, the effector caspases cleave a
variety of molecules including poly ADP-ribose polymerase (PARP), protein kinase, spectrin,
actin, and DNA-dependent protein kinase. Rip2 is the upstream modulator of pro-caspase-1 in
the apoptotic cascade that acts on Bid downstream to execute cell death[14]. The final
mechanism of caspase regulation is proteasome degradation and inhibitors of apoptosis
proteins (IAPs) seem to parcipate in clearing the active caspases[15].
2. Targeting Caspases in Neonatal Hypoxia-
Ischemia Brain Injury and Hypoxic-Ischemic
Encephalopathy
2.1. Neonatal Hypoxia-Ischemia Brain Injury and Hypoxic-Ischemic
Encephalopathy
The incidence of hypoxic-ischemic encephalopathy (H-IE) is 1-8 cases per 1000 births in
the United States. Perinatal asphyxia, stroke, and intraventricular hemorrhage are general
causes of neonatal brain injury, with hypoxia-ischemia as the final common pathway. In
severe neonatal hypoxia-ischemia brain injury (HIBI), the mortality rate is 25-50%. Those
who survive have a substantial risk of permanent disability such as mental retardation, motor
impairment, behavioral and cognitive disabilities, and seizures. Currently no treatment for
neonatal HIBI is available, and only general supportive measures are used to prevent further
morbidity. Hence there is an unmet need to discover novel therapeutics for H-IE.
Various techniques reliably identify neurons rendered apoptotic by multiple mechanisms
of brain injuries such as caspase assays[16], TUNEL (terminal deoxynucleotidyl transferase
dUTP nick end labeling) staining[17], Flourojade B staining[16], DNA fragmentation
assay[18], and Annexin V staining[19]. In vivo the Rice-Vanucci model demonstrates the
effect of hypoxia-ischemia (H-I) injury on experimental animals. P7 day pups of different rat
or mouse species are used, as their neural development simulates that of human neonates of
36-37 weeks gestation. Levines procedure is used, consisting of unilateral carotid artery
ligation, and the pups are made hypoxic with 8-10% of O
2
and equilibrated N
2
in a warm bath
of 37C for a specified period[20, 21]. The pups are sacrificed later to study the effects.
Primary cerebellar granular neurons[22] and mouse primary cortical neuron (PCN)[23] are
used as in vitro models.
HIBI and H-IE can be diagnosed by various imaging modalities. In less experienced
hands cranial ultrasonography, magnetic resonance imaging (MRI) techniques and pulse
sequences such as T1 and T2 weighted images remains the modality of choice for neonatal
imaging. However, in tertiary care centers, diffusion weighted imaging (DWI) and magnegtic
resonance spectroscopy (MRS) are conventionally used[24]. Early use of positron emission
tomography imaging in detecting brain injury due to perinatal and intrauterine insults can
provide new insights in prognosis and instituting early therapy[25]. These imaging modalities
in association with EEG help prognosticate the outcome of H-I injury in the neonate.
2.2. The Activation of Caspases in Neonatal HIBI and H-IE
Studies have shown that neonatal HIBI is mediated by apoptosis[26-32] and that caspases
are vital to mediate this injury. We and other researchers found that initiator caspase-8 may
directly process and activate effector caspase-3, or indirectly activate caspase-3 through
cleaving and activating the cytosolic BID, which promotes the release of cyto. c and feedback
activation of caspase-9 and -3[14, 33]. The FAS-mediated cell death receptor pathway,
leading to activation of caspase-8, plays an important role in H-I neurodegeneration[34].
Neuronal loss due to H-I injury is associated with the cleavage of initiator caspase-8 and -
9[33]. Caspase-10 activation occurs primarily in the ischemia/reperfusion models[35].
Caspase-2[36] and caspase-3[37, 38] are activated extensively in the immature brain after H-I
injury. Cleaved effectors caspase-7 and -3 are activated in neurons in the

ipsilateral
hippocampus after H-I injury[39]. Caspase-1 is an apical mediator of neuronal cell death, as
shown by in vitro hypoxia and in vivo ischemia models[14]. Caspase-1 mRNA was increased
in neonatal rats after HIBI with a time frame consistent with brain injury[40]. Caspase-1
converts pro-interleukin-1| (IL-1|) to IL-1|, which mediates hypoxic ischemic brain damage
in experimental animals and human neonates[41], and ICE deficient mice are resistant to mild
to moderate HIBI. Post-mortem studies of neonatal brain in cases of pontosubicular necrosis
have shown caspase-3 activation in human perinatal HIBI[42]. Caspase-12 is involved in
apoptosis induced by ER stress[43] or Bax shuttling[44] in neonatal HIBI rats.
2.3. Targeting Caspases in Treatment of Neonatal HIBI and H-IE
Neonatal HIBI is an evolving process that develops (after a certain delay) mainly due to
neuronal apoptosis mediated by caspases. After the primary insult there is a complete
recovery, and the secondary energy failure, which causes the lesion, occurs after about a
delay of 6 to 48 hours. This presents a therapeutic window during which the
neurodegeneration leading to H-IE could be prevented via a host of potential targets for
intervention[45]. In vivo and in vitro studies have shown that inhibition of the caspase family
prevents cell death in neurodegenerative diseases, and treatments targeting caspases may
potentially play a neuroprotective role in neonatal HIBI[26] and in human perinatal H-IE.
Herein, we review the current literature on a variety of treatments for neonatal HIBI and H-IE
via caspase inhibition (Fig. 1 and Table I).


Figure 1 Treatments targeting individual caspases in neonatal HIBI and TBI. Various treatments act on
caspases in the cell death receptor pathway, mitochondrial death pathway and ER death pathway in
neonatal HIBI and TBI. Treatments are labeled red for both HIBI and TBI, and Blue for HIBI and H-IE
only, and green for TBI only.
Table I. Treatments for HIBI and H-IE by inhibition of the caspases. Targeting caspases
with different treatments in neonatal HIBI reduces brain tissue loss and reduces infarct
volume in various brain areas. Different species of experimental animals are tabulated.
CO: cortex, HC: hippocampus, TH: thalamus, ST: striatum, CrC: corpus callosum, CC:
cerebral cortex, SN: subthalamic nuclei, IC: internal capsule, EC: external capsule, AN:
amygdaloid nucleaus.

Treatments Targeting te Mitochondrial Cell Death Pathway in HIBI and H-IE
Since caspases play a major role in neuronal apoptosis, caspase inhibitors can effectively
protect neurons from inappropriate apoptosis and help in prolonging cell survival. Indeed,
caspase inhibition has proven efficacious in the treatment of neonatal HIBI and H-IE
involving apoptosis. M826 is a selective caspase-3 inhibitor that can block almost all the
activities of caspase-3 by intracerebroventricular injection[23]. A third generation dipeptidyl,
broad spectrum pan-caspase inhibitor quinoline-Val-Asp(Ome)-CH2-O-phenoxy (Q-VD-
OPh) has a carboxy terminal phenoxy group conjugated to the amino acids valine and
aspartate. It has proved to be neuroprotective on a gender specific basis (females are more
protected than males), given either before or after ischemia[46]. It inhibits all the three
pathways of caspase mediation (-3/9, 8/10 and 12[47, 48]) and reduces brain injury due to H-
I. BAF (boc-aspartyl(OMe)-fluoromethylketone), a pan-caspase inhibitor either alone or with
hypothermia reduces apoptosis and protects against neuronal damage in HI models[49].
However, of a variety of tested strategies, hypothermia, is the only intervention that has
translated to some clinical benefit in newborn babies[50]. Hypothermic medicine with either
selective head cooling or systemic body cooling to 30-34C rectal temperature has proven
beneficial in neonatal HIBI in some way inhibiting the expression of caspases. The effect of
hypothermia depends on timing of initiation, depth and duration of cooling after resuscitation.
More pronounced protection is attained when cooling is started during ischemia.
Intraischemic hypothermia[51] or post-ischemic hypothermia has proven to be of therapeutic
value. The infarct size, extent of neuronal loss, and severity of brain injury are significantly
reduced in the hypothermic group compared to the control group. Mild hypothermia (34C)
decreases expression of caspase-3 mRNA and lowers caspase-3 enzyme activity[52].
Prolonged hypothermia for about 72 hours post-H-I decreases caspase-3 in the parietal cortex
and hippocampus[53]. Systemic hypothermia at 30C beginning immediately post-H-I and
lasting for 10 hours decreases caspase-2 and -3 in the cortex and dentate gyrus[36]. These
results indicate that hypothermia may act at least partially through inhibition of the intrinsic
pathway of caspase activation in the neonatal brain, thereby preventing apoptotic cell death.
The library of the Neurodegeneration Drug Screening Consortium of 1,040 compounds
assembled by the National Institute of Neurological Disorders and Stroke includes
minocycline, doxycycline, nicotinamide, and melatonin.
Minocycline, a semisynthetic derivative of tetracycline, is a FDA approved antibiotic
used for decades in the treatment of various infectious diseases. We and other groups have
reported that minocycline has proven effective in neurodegenerative diseases[1, 54-56]. If
administered either immediately before or after H-I insult, it provides near complete
neuroprotection in the neonatal brain by decreasing caspase-3 levels and thus preventing from
neuronal injury due to apoptosis[57]. Doxycycline (another FDA approved tetracycline),
given either before or immediately after H-I insult to experimental neonates, not only reduces
caspase-3, but also reduces microgliosis, and hence prevents cell death. It reduces caspase-3
activation in a time-dependent manner. This prosurvival effect is seen in most vulnerable
regions of the brain such as CA1, dentate gyrus, cortex and striatum[58]. Nicotinamide
(vitamin B3), a precursor of NAD and a form of niacin, plays a vital role in cell survival and
cell growth. Nicotinamide confers effective protection against HIBI in adult rats and has also
proven effective in the neonatal rats[59]. Its proven pharmacological actions include reducing
lipid peroxidation, reducing PARP[60], inhibiting apoptosis[60] and preventing ATP
depletion[61]. It improves motor coordination and reduces the brain injury. Nicotinamide can
also control the mitochondrial permeabilization and prevent cyto. c release, which reduces
caspase-3 and -9 activity. Melatonin is a free radical scavenger and antioxidant. We have
demonstrated that melatonin targets caspases in a number of neurological disorders including
ischemic brain injury[62, 63]. It has been shown to play a role in the sleep-wake cycle and
circadian rhythm[64]. It inhibits cyto. c release and loss of mitochondrial membrane
potential[63]. When administered to pregnant spiny mice, melatonin reduces CNS
inflammation and apoptosis in pups in a H-IE model[65]. Melatonin administered before and
after H-I provides protection against brain injury as well as long-term improvement in
behavioral asymmetry and learning deficits induced by H-I[66].
Because free radicals play an important role in mediation of HIBI, drugs that act as free
radical scavengers and antioxidants can be neuroprotective. Edaravone, a free radical
scavenger and antioxidant that also inhibits lipid peroxidation, is shown to improve motor
functions in clinical trials of acute cerebral infarction. It is reported that edaravone also
confers neuroprotection in H-I brain damage in newborn rats[67] through impeding apoptotic,
necrotic, and mitochondrial mechanisms. This effect was dose- and time-dependent[68]. N-
acetylcysteine (NAC) is a precursor of glutathione, a potent antioxidant, and a free radical
scavenger. NAC is widely used as a mucolytic agent and as an antidote for paracetamol
poisoning[69]. It is a precursor of glutathione, a potent antioxidant, and a free radical
scavenger. Antenatal infection and H-I have a synergistic effect in neurodegeneration. NAC
protects against lipopolysacharide-sensitised H-I and inhibits caspase-3 and -1 activation[70].
Caffeic acid phenethyl ester is another antioxidant, an inflammatory and antiviral agent with
immunomodulatory functions. It inhibits neurotoxicity due to induced nitric oxide (NO)
synthase and caspase-1 expression in vivo and in vitro, and it also prevents Ca2+-induced
cytochrome c release and blocks caspase-3 activation in isolated brain mitochondria[71, 72].
NO plays a role in neuronal apoptosis by generating reactive nitrogen species, which causes
nitration of lipids, DNA, and proteins, leading to neuronal cell damage[73]. Polyphenols have
antioxidant properties[74]. Pomegranate polyphenols and resveratrol, when given to the
dams, reduce activation of caspase-3 in the hippocampus of neonatal rats after H-I[75].
Another polyphenol amentoflavone reduces brain tissue loss due to H-I injury. It blocks
caspase-3 activation in rats[76]. Pyrrolidine dithiocarbamate (PDTC) is an antioxidant, that
confers neuroprotection and reduces cleaved caspase-3 expression in the PDTC-treated
rats[77]. Though hydrogen gas neutralizes free radicals and reduces oxidative stress, its
clinical use as a gas poses issues regarding safety and convenience. When saturated hydrogen
saline is administrated intraperitoneally immediately and 8 hours post HI, it reduces the
infarct ratio and improves long-term neurological and neurobehavioral function. It reduces the
levels of malondialdehyde (a marker of oxidative damage), Iba-1, and caspase-3[78].
Hyperbaric oxygen preconditioning provides protection to various organs including the brain
in neonatal H-IE[79]. It reduces infarct ratio and increases survival. Suppression of anti-
apoptotic pathways of caspase-3 and -9 activities play a role in this neuroprotection[79].
PAF (platelet-activating factor) is a lipid-mediator, released from various cells such as
platelets, monocytes, macrophages, endothelial cells, and neutrophils[80]. PAF
concentrations increase during ischemia and oxidative stress and have a proinflammatory
effect[81], mediating neurotoxicity and neuronal degeneration. Thus PAF antagonist can be
neuroprotective in HIBI. Indeed, the PAF antagonist ABT-491 reduces caspase-3 activity and
TUNEL staining in experimental animals[82]. The use of NMDA receptor antagonist MK-
801 reduces caspase-3 activation as shown by western blot. Brain injury, caspase-3 activation,
and DNA fragmentation are all reduced in the cerebral cortex showing that NMDA plays a
role in apoptotic mechanisms in HIBI[83].
The role of neurotrophic factors in apoptosis during development is important. BDNF is a
neurotrophic factor that blocks caspase-3 activation in neonatal HIBI. Intracerebroventricular
injection of BDNF prevented activation of caspase-3 in vivo[84] and protected the brain from
tissue loss. Insulin-like growth factor-1 (IGF-1) is another neurotrophic factor that plays an
important role in neuronal cell survival. It reduces phosphorylation of Akt and GSK3| after
H-I injury, while caspase-3 and -9 activity is significantly reduced but caspase-8 or -1 activity
is not affected[85]. Additionally, FGF 1 is neurotrophic factor. In transgenic rat expressing
FGF-1, apoptosis is markedly reduced via decreased caspase-9, caspase-3 and its substrate
PARP in rat pups and in PCNs[86]. It also blocks the H-I induced reduction of anti-apoptotic
and survival promoting protein XIAP expression, thus providing neuroprotection[86].
Trapidil is an antiplatelet agent that acts as a phosphodiesterase inhibitor and a
competitive inhibitor of platelet derieved growth factor (PDGF)[87]. Besides its biological
effects in vasodilation and stimulating prostacyclin, trapidil plays a role in inhibiting neuronal
apoptosis in a neonatal rat model of HIBI by noticeably reducing caspase-3 levels[87]. 2-,
iminobiotin is an inhibitor of neuronal and inducible nitric oxide synthase. It is
neuroprotecgtive by improving the cerebral energy state, decreasing vasogenic edema, and
inhibiting neuronal apoptosis in experimental animals. It can be intravenously administered
after H-I and is neuroprotective 24 hours after the insult[88]. Mixed lineage kinases are
expressed in the neuronal cells and CEP-1347 is their semisynthetic inhibitor. Mitogen
activated protein kinase activates mixed lineage kinases, leading to phosphorylation of c-jun,
which in turn causes cyto. c release and activation of the caspases. CEP-1347 reduces
neonatal brain damage and apoptosis, as shown by reduction in caspase-3 activity[17].
Hormone has been shown to be neuroprotective. Hexarelin is a peptide with a potent
ability to stimulate growth hormone secretion. Hexarelin reduces caspase-3 activity in
cerebral cortex of ipsilateral hemisphere by intracerebroventricular injection, and increases
Akt and pGSK3| phosphorylation[89]. Erythropoietin (EPO) belongs to the cytokine
superfamily and has traditionally been viewed as a hematopoiesis-regulating hormone. The
receptors of EPO are present in central nervous system. It offers protection and reduces
caspase-3 activation in neonatal mice after H-I injury[90]. Deferoxamine displays anti-
oxidative actions, and EPO confers anti-apoptotic and anti-inflammatory effects, each has
been shown to provide neuroprotection in neonatal rodent models of brain injury.
Furthermore, intraperitoneal administration of deferoxamine and/or EPO, in rats reduces the
number of cleaved caspase-3 positive cells[91]. Though 17 | estradiol, a circulating
hormone, attenuates neonatal HIBI, however caspase-dependent pathways play a little
role[92].
Both extrinsic and intrinsic apoptotic pathways mediate the neuroprotective effects of
Hsp70 overexpression in neonatal H-I[33]. The direct binding of Hsp70 to Apaf-1 may be one
of the mechanism that reduces caspase-9 cleavage, thus reducing apoptosis. Apaf-1
interacting protein (AIP), containing N-terminal caspase recruiting domain, binds to Apaf-1
thereby inhibiting the formation of apoptosome and caspase-3 and -9 activation following H-
I. Transgenic mice overexpressing AIP (Tg-AIP mice) prevent H-I brain injury while
intraperitoneal administration of TAT-AIP fusion protein confers neuroprotection and
attenuates activation of caspase-3 and -9[93]. Bcl-xl, located mainly in the mitochondrial
membrane, is an important anti-apoptotic molecule. A fusion protein with TAT (forming
TAT-Bcl-xl) is protective against cell damage in the neonatal rat brain. This has been shown
with the reduction of caspase-3 and -9 assays after intraperitoneal injection of the fusion
protein in experimental animals[94]. On the other hand, mice deficient in interleukin-1
converting enzyme (ICE) are resistant to neonatal HIBI, and caspase-1 activity significantly
contributes to the progression of neonatal HIBI[95]. Neonatal mice

deficient in Atg7 gene
show nearly complete protection from both

H-I-induced caspase-3 activation and neuron
death[39]. Endogenous BAX plays a role in regulating cell death in the CNS following
neonatal H-I, Bax -/- mice had significantly decreased caspase-3 activation as compared to
bax expressing mice following H-I[96]. Additionally, Bax-inhibiting peptide (BIP), a novel
membrane-permeable peptide, which can bind Bax in the cytosol and inhibit its translocation
to the mitochondria, significantly suppresses both the number of TUNEL-positive cells and
the increase in caspases-3 and -9 activities induced by glutamate in cerebellar granule
neurons, a cellular model of HIBI[22].
Studies have shown simvastatin to be neuroprotective in neonatal HIBI[97, 98] via
inhibition of caspase-3, which is independent of calpain activation. Caspase-3 proteolytically
cleaves many substrates including PARP, which is the biochemical hall mark of apoptosis.
PARP cleavage is reduced in simvastatin-treated animals. Early necrotic death due to calpain
activation was not inhibited, however at 48 hours cell death due to caspase-3 activity is
inhibited. In addition, caspase-1 activity, IL-1|, and ICAM-1 mRNA are also reduced due to
simvastatin proving that its neuroprotective activity is due to inhibition of apoptotic cell
death[99].
Treatments Targeting Cell Death Receptor Pathway in HIBI
FLIP (FLICE-like inhibitory protein) acts as an endogenous cytoplasmic decoy for
caspase-8 and provides neuroprotection[100]. Antioxidant status alters the expression of FLIP
and caspase-8 simultaneously in opposite directions[101]. Animals overexpressing
superoxide dismutase had excessive activation of pro-caspase-8, and activated caspase-8
leaded to apoptosis. On the other hand, animals overexpressing glutathione peroxidase have
increased expression of FLIP which provides neuroprotection[101].
Treatments Targeting the ER Pathway in HIBI
Administration of molecular hydrogen after HI insult reduces caspase-12 levels in a time-
dependent manner. This inhibition of caspase-12, leading to inhibition of downstream
apoptotic mechanisms (such as caspase-3 activation), in turn provides neuroprotection by
reducing the infarct volume in cortex and hippocampus[102]. Thus, these experimental
studies of H-I models using various treatments show that targeting caspases in different ways
can protect the neurons from neuronal degeneration and may prevent the mortality and
morbidity associated with H-IE.

3. Targeting Caspases in Traumatic Brain Injury
3.1. Traumatic Brain Injury
The incidence of TBI is approximately one-sixth the total numbers of injuries. About
seven million patients sustain a TBI annually worldwide due to traffic accidents, falls,
assaults, or sports injuries[103]. In the United States, the estimated incidence of TBI is 100
per 100,000 persons, with 52,000 annual deaths. According to the World Health
Organization, TBI will surpass many diseases as the major cause of death and functional
disability by the year 2020[104]. Those sustain and survive a TBI are left with significant
cognitive, behavioral, and communicative disabilities.
Neuronal injury can be detected as soon as 10 minutes after TBI in cerebral cortex,
thalamus, hippocampus, and other regions can be observed[105]. On the other hand,
progressive gray and white matter atrophy and neuronal death can progress for one year
following TBI[106, 107]. The pathophysiological mechanism of CNS injury in the acute
phase and chronic nerve cell damage of TBI have been investigated by us and other
groups[108-111]. It is generally believed, neuronal cell death plays an important
pathophysiologic role in the cascade of CNS cell degeneration and associated neurologic
deficits[106, 112]. Cell death in the CNS following TBI can take the forms of apoptosis and
necrosis[113], and both caspase-dependent and independent apoptotic pathways have been
identified in CNS trauma. Herein, we focus on the current interventional therapies targeting
caspase-mediated apoptosis.
A number of experimental animal models, organotypic cultures and cultured cell models
of TBI have been developed to replicate the characteristics of human head injury, such as
contusion, concussion, and/or diffuse axonal injury. In caspase-mediated TBI studies, animal
models that have been used include controlled cortical injury (CCI)[114], lateral cortical
contusion[115], and fluid-percussion (FP) brain injury[105, 116, 117]. Besides the ex vivo
organotypic hippocampal cultures[118], cellular models include primary mouse[119] or
rat[120] cultures of cerebral cortical neurons, neuronal-glial cultures[121], P2Y2R-1321N1
astrocytic cells[122], nerve growth factor differentiated PC12 cells[123], and septo-
hippocampal cell cultures[124] are used. Several methods similar to those utilized in neonatal
HIBI, have been used to detect apoptosis in TBI including caspase assay[117], TUNEL
staining[125, 126], FJB staining[127], DNA fragmentation analysis[128], ApopTag assay and
Wright staining[120]. Advanced techniques such as computed tomography scan[129] and
MRI[130-132] have been used to identify damage induced by TBI. Furthermore, the more
sensitive magnetic resonance DWI, diffusion tensor imaging (DTI), white matter fiber
tractography (DTT), functional magnetic resonance imaging (fMRI), and MRS have been
also used to investigate TBI[131-136].

3.2. The Activation of Caspases in TBI
The extrinsic pathway of apoptosis has been reported to be involved in TBI. Caspase-8 is
expressed in cortical areas[117, 137] and thalamus[117], while it is shown to be activated in
neurons, astrocytes, and oligodendrocytes[138]. The activation of caspase-8 contributes to
caspase-3-mediated apoptotic cell death in experimental animals after TBI[138]. High levels
of caspase-8 gene induction are observed after cortical impact in rats[139]. In addition,
TNFR1 and TRAF1 are recruited to lipid rafts in rats after TBI. Subsequently, the signaling
complex contains activated caspase-8, thus initiating cell apoptosis[140]. In a nerve growth
factor-differentiated PC12 cell model of TBI, caspase-8 and -3 are activated upon stearic acid
and palmitic acid apoptotic induction[123]. Moreover, using a mouse CCI model in vivo and
primary cultures of cerebral cortical neurons in vitro, caspase-8 and -3 are activated while Fas
is reported to retain its function as a death receptor after TBI. Furthermore, the interactions
between Fas receptor and FADD, pro-caspase-8 and pro-caspase-10, and Fas-RIP-RAIDD-
caspase-2 are apparent after CCI[119].
Our studies suggest that caspase-1 plays a key role in H-I-associated neuronal death[14].
Caspase-1 generates the pro-apoptotic tBid fragment, which plays a role in the release of
mitochondrial apoptogenic factors cyto. c/Smac/AIF. Released cyto. c induces apoptosome
assembly, resulting in caspase-9 and -3 activation[14]. Regarding models of TBI, caspase-1 is
not only activated in the rat model of TBI[114], but also plays an important functional role in
mediating neuronal cell death and dysfunction after TBI in mouse brain[141], while
neuroprotection following TBI is achieved in a transgenic mouse expressing a dominant
negative inhibitor of caspase-1[142]. In addition, caspase-1 mRNA content is increased in
rats after FP-induced TBI[143]. Finally, in a rat FP injury model, TBI induces the activation
of processing of caspase-1 and increases expression of caspase-11[144].
The intrinsic apoptotic pathway has been proposed as one mechanism of cell death after
TBI. Indeed, the release of cyto. c from mitochondria and the activation of caspase-1 and then
-3 in the injured cortex of a CCI rat model of TBI confirms the involvement of the
mitochondrial death pathway[114]. Furthermore, activated caspase-2 has been shown to
trigger mitochondrial apoptotic events by inducing conformational changes in Bax/Bak with
subsequent release of mitochondrial apoptotic factors including cyto. c, AIF, and
endonuclease G[145]. In addition, caspase-2 mRNA expression is increased in the cerebral
cortex of rats after TBI[146]. Initiator caspase-9 is activated in rats after TBI[137, 139, 147].
After acute injury to mature brain, injury-induced cyto. c-specific cleavage of caspase-9 is
reported to be followed by activation of caspase-3 correlated with marked Apaf-1
increases[148]. Moreover, initiator caspase-9 is predominantly expressed in cortical areas and
in the thalamus while caspase-3 is expressed throughout the traumatized cerebral cortex and
hippocampus[117]. Caspase-3 is induced by at least two major initiator pathways: a caspase-
8-mediated extrinsic pathway and a caspase-9-mediated intrinsic pathway. Activation of
caspase-3 after TBI has been widely recognized[117, 139, 143, 147, 149-151]. Caspase-3-
specific spectrin breakdown products (SBDPs) are increased in cerebrospinal fluid after TBI
in rats[152, 153]. Caspase-7, another apoptosis executioner, is generally believed to be
present in only minute amounts in the brain (with highly restricted activity) or is completely
absent. Larner, S. F. et al. demonstrate that caspase-7 is up-regulated and activated after TBI
in rats[154].
Furthermore, Larner, S. F. et al. also report increased expression and processing of
caspase-12 following TBI in rats[155]. Therefore, the caspase-12-mediated ER death pathway
may play a role in rat TBI pathology independent of the cell death receptor or mitochondria
apoptotic pathway. In summary, the caspases are activated in the extrinsic and intrinsic death
pathways and the ER death pathway following TBI in animal models in vivo and cultured cell
models in vitro.
Growing evidence demonstrates that caspases are activated in human TBI. After severe
TBI, caspase-9 and caspase-3 are activated and cyto. c is released in the cerebrospinal fluid of
patients[156-158]. Activated caspase-3 is also reported in specimens of human brain tissue
with TBI[125, 159, 160], while caspase-3 is being investigated as a specific biomarker of
proteolytic damage following TBI[161]. In addition, caspase-7 and its cleavage product, as
well as the cleavage of caspase-1, are increased in human brain tissue[162, 163], caspase-1 is
increased in the ventricular cerebrospinal fluid of TBI patients[164]. Through the analysis of
brain tissue samples from adult patients with severe intracranial hypertension after TBI or
post-mortem TBI brain samples and comparison with post-mortem control brain tissue
samples, caspase-8 mRNA and protein are found to be increased in TBI adult patients while
proteolysis of caspase-8 to 20-kDa fragments is seen only in severe TBI adult patients, and
caspase-8 protein was predominately expressed in neurons[165]. In addition, Fas-pro-
caspase-8 interaction is significantly increased in contused brain samples removed from
severe TBI patients[119]. Taken together, experimental evidence demonstrates that the
caspases in both intrinsic and extrinsic death pathways are activated in human TBI.
3.3. Targeting Caspases in Treatment of TBI
The damage to the traumatized brain includes two phases: the initial irreversible primary
phase being the injury itself, and the secondary phase, which begins at the time of injury and
continues for days, weeks, or longer. Secondary brain injury represents a window of
opportunity in which treatments with neuroprotective properties could be administered[166].
Determination of the specific expression profiles of caspases affected by injury is critical to
develop targeted therapeutics for TBI. We review the current literature on treatments
targeting caspases in TBI, and provide evidence supporting the therapeutic use of caspase
inhibitors and other treatments in the setting of these conditions (Fig. 1 and Table II).
Pan-caspase inhibitor BAF treatment reduces acute cell death in rats after TBI by
inhibiting mitochondrial release of cyto. c, initiator caspase-2, and effector caspase-3[167]. In
addition, post-CCI with intracerebroventricular injection of BAF in mice significantly reduces
caspase-3 activation, suppresses caspase-cleaved APP and increases in Abeta, and improved
histological outcome[168]. Administration of caspase-3 inhibitor z-DEVD-fmk (N-
benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone), a specific tetrapeptide inhibitor
of caspase-3, markedly reduces post-traumatic apoptosis and significantly improves
neurological recovery following TBI[143]. Intracerebral administration of z-DEVD-fmk after
TBI reduces caspase-3-like activity and DNA fragmentation in injured rat brain[169]. Given
that glutamate toxicity in TBI causes cortical neuron death and dysfunction, in a cellular
model of TBI, z-DEVD-fmk provides strong neuroprotection in PCNs following glutamate
exposure[120]. In addition, z-DEVD-fmk, like pan-caspase inhibitor BAF, significantly
attenuates apoptotic cell death in neuronal-glial cultures in vitro after mechanical injury by
combined 3-nitropropionic acid and glucose deprivation treatment, whereas the caspase-1
selective inhibitor z-YVAD-fmk has no effect[121]. Inhibition of caspase-1 activation
reduces trauma-mediated brain tissue injury, a conclusion is supported by several findings: 1)
reduction of tissue injury and free radical production following TBI in the brain tissue of a
transgenic mouse expressing a dominant negative inhibitor of caspase-1[142]; 2)
pharmacological inhibition of caspase-1 by intracerebroventricular administration of the
selective inhibitor of caspase-1, acetyl-Tyr-Val-Ala-Asp-chloromethyl ketone (cmk) or the
non-selective caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fmk confers
neuroprotection[142]; 3) the pan-caspase inhibitor z-VAD-fmk (carbobenzoxy-valyl-alanyl-
aspartyl-[O-methyl]-fmk) reduces cold injury-induced brain trauma in mice by preventing
caspase-1 activation and DNA fragmentation[170]; 4) antibody therapy using anti-ASC
(apoptosis-associated speck-like protein containing a caspase recruitment domain)
neutralizing antibodies administered immediately after FP injury to injured rats reduces
caspase-1 activation, IL-1| processing, and cleavage of X-linked inhibitor of apoptosis
protein, thus significantly decreasing contusion volume after TBI[144]. Taken together, the
available evidence indicates that pharmacological inhibition of caspases by caspase inhibitors
prevents cell death and improves functional outcome after TBI, offering an experimental
rationale for the evaluation of effective pharmacological agents in human trauma patients.
Hypothermia reduces the evolving secondary deterioration after TBI. Clinical trials have
been performed in patients and different durations and temperatures have been studied in
experimental animals[171]. Post-traumatic hypothermia significantly attenuates caspase-3
activity and cell death within the hippocampus following FP injury[172]. In another report,
hypothermia promotes a rapid acute activation of caspase-3 in rats after TBI but suppresses
the activation of caspase-3 at later time points[173]. In contrast, heat acclimation attenuates
the activation of caspase-3 and provides sustained improvement in functional recovery and
reduction in lesion volume in mice after TBI[174].
Intraperitoneal administration of minocycline, a derivative of the antibiotic tetracycline,
before or after TBI in mice, improves neurological function and reduces tissue damage. Given
that the activity of caspase-1 is increased in mice that underwent TBI, and this increase is
significantly diminished in minocycline-treated mice, the neuroprotection of minocycline in
TBI is suggested to take place through a caspase-1-dependent mechanism[141]. Minocycline
can also block nitric oxide (NO)-induced neurotoxicity[175, 176] and inducible NO synthase
up-regulation[176]. Lu et al. found that NO induces macrophage apoptosis, as shown by
positive TUNEL staining and caspase-3 immunostaining after TBI[177]. Furthermore, the
same group reported the neuroprotective effects of aminoguanidine, a selective inducible NO
synthase inhibitor, after lateral FP brain injury in rats. In rats receiving prophylactic or post-
injury treatment of aminoguanidine after TBI, the number of caspase-3 immunopositive
neurons is reduced in the cerebrum[178].
Antioxidants and free radical scavengers have been reported to be neuroprotective in
TBI. Selenium is an antioxidant with protective function in ROS-mediated apoptotic neural
precursor cell death in vitro and in vivo in an experimental mouse model of TBI through
attenuation of secondary pathological events. This action most likely results from its
comprehensive effects in blocking caspase-3 and -9 activation, resulting in the maintenance
of functional neurons and in inhibition of astrogliosis[179]. Cerebral contusions are one of the
most frequent traumatic lesions and the most common indication for secondary surgical
decompression. Hyperbaric oxygen therapy has been used in treatment of these lesions[180].

Table II. Treatments for TBI by Inhibition of the Caspases. The neuroprotective effects
of treatments for TBI with targeting indicated caspases in different species and cell lines
are tabulated.

It decreases not only the number of TUNEL-positive cells[180] but also apoptosis in TBI via
inhibition of the activation of caspase-9[181]. In addition, peroxynitrite can inhibit caspase-3-
mediated apoptosis in neurons following TBI in vitro and in vivo mostly because of its effect
on cysteinyl oxidation of caspase-3[112]. The free radical scavenger S-PBN and MEK
inhibitor U0126, both confer neuroprotection in rats after TBI. They attenuate the early
activation of ERK and reduce activation of caspase-3 and subsequent DNA
fragmentation[182].
The NMDA receptor plays an important role in the pathophysiological process of
TBI[183, 184]. The protective effect of NMDA receptor antagonist MK-801 against TBI
along with the inhibition of caspase-3 has been reported[183]. Macrophage colony
stimulating factor (M-CSF) and its receptor are upregulated in the brain in an experimental
model of TBI. NMDA induces caspase-3 activation and neuronal apoptosis in organotypic
hippocampal cultures, whereas treatment with M-CSF (like caspase inhibitor z-VAD-fmk)
protects hippocampal neurons from NMDA-induced caspase-3 activation and neuronal
apoptosis[118].
The cyclin-dependent kinase inhibitor flavopiridol significantly inhibits the activation of
caspase-3 in rats following TBI in vivo and in rat PCNs induced by etoposide in vitro[185].
Cyclooxygenase-2-specific inhibitor DFU (5,5-dimethyl-3(3-fluorophenyl)-4(4-
methylsulfonyl)phenyl-2(5)H)-furanone) enhances functional recovery and decreases the total
number of activated caspase-3-immunoreactive cells in injured cortex and hippocampus in a
lateral cortical contusion rat model[115].
Rosiglitazone is a potent agonist of peroxisome proliferator-activated receptor-gamma
that confers neuroprotection in experiments models of focal ischemia, spinal cord injury and
Parkinsons disease[186]. Furthermore, rosiglitazone-treated TBI mice also show
significantly fewer TUNEL-positive apoptotic neurons and curtailed induction of caspase-3
and Bax compared to vehicle-treated controls[187].
Simvastatin not only provides neuroprotection in H-IE but also confers beneficial effects
in TBI. These effects include activation of Akt, Forkhead transcription factor 1, and NFkB
signaling pathways, which suppress the activation of caspase-3 and apoptotic cell death,
thereby leading to neuron function recovery of rats after TBI[188]. In addition, a ketogenic
diet reduces cyto. c release and caspase-3 activation following TBI in juvenile rats[189].
A growing body of literature supports the benefit of hormone therapies for
neuroprotection and improved cognitive recovery after TBI. The neuroprotective effect of
estrogens, with significant reduction of active caspase-3, has been demonstrated in Sprague-
Dawley male rats after TBI[190]. Furthermore, premarin, an estrogen sulfate, protects against
cortical and hippocampal apoptosis after FP injury through mechanisms stimulating estrogen
receptor-alpha and preventing caspase-3 activation in male rats as well[191]. Moreover, the
neurosteroid progesterone and its metabolite allopregnanolone reduce the expression of the
pro-caspase-3 and the activity of caspase-3 and confer anti-astrogliotic effects after TBI in
adult male rats[192, 193]. In addition, the administration of progesterone improves
morphologic and functional outcome and the marked attenuation of caspase-3
immunoreactivity after diffuse TBI in rats[194]. Studies also suggest that allopregnanolone
appears to be more potent than progesterone in facilitating CNS repair after TBI[193]. EPO
belongs to the cytokine superfamily and has traditionally been viewed as a hematopoiesis-
regulating hormone. It improves functional recovery, and reduces caspase-3 activation and
neuronal apoptosis, as well as inflammation in a rodent model of TBI[195].
Genetic deletion or overexpression affects the expression or activation of caspase(s) and
functional outcome after TBI. Bid is a proapoptotic member of the Bcl-2 family that mediates
cell death. Compared with normal animals, mice genetically deficient in Bid (Bid-/-) in a CCI
model of TBI show the decreased early post-traumatic brain cell death and tissue damage, as
well as decreased numbers of cells expressing cleaved caspase-3[196]. However,
overexpression of Bcl-2, a major member of the Bcl-2 family, is only partially
neuroprotective; there is no significant difference in the cleavage of caspase-3 or -9 in
hippocampal samples from Bcl-2 transgenic or wild-type mice after TBI[197]. Transgenic
expression of interleukin-6 (IL-6) in the CNS under the control of the glial fibrillary acidic
protein (GFAP) gene promoter (GFAP-IL6 mice) affords neuroprotection against acute TBI
associated with reduced oxidative stress, neurodegeneration, and apoptosis including reduced
caspase-3 TUNEL staining[198]. In addition, neuronal survival has been found to correlate
with increased expression of uncoupling protein 2 (UCP-2). Transgenic mice overexpressing
UCP-2 show diminished brain damage after TBI while UCP-2 reduces cell death and inhibits
the oxygen and glucose deprivation-induced caspase-3 activation in rat PCNs[199]. 1321N1
astrocytic cells expressing recombinant P2Y2 nucleotide receptors (P2Y2R-1321N1) are a
well-established in vitro model of TBI. Activated caspase-9, but not caspase-8, is inhibited in
P2Y2R-1321N1 astrocytic cells, suggesting the direct involvement of this nucleotide receptor
in modulating cleaved caspase-9 after traumatic injury towards cell survival; while PD1693, a
MKK3/6 inhibitor, inhibits trauma-induced death and abolishes the expression of cleaved
caspase-9 in P2Y2R-1321N1 astrocytic cells[122]. Additionally, sAPPalpha (soluble amyloid
precursor protein alpha), is a product of the non-amyloidogenic cleavage of an amyloid
precursor protein. sAPPalpha reduces neuronal injury and improves functional outcome as
well as significantly reduces the number of caspase-3 apoptotic cells in the hippocampus and
cortex of rat brains following diffuse TBI[200].
We and other researchers have worked on caspase related stem cell therapy for
neurological disorders[201-203], and there is increasing interest in the use of stem cells as a
therapeutic tool in TBI. Direct intrathecal implantation of mesenchymal stromal cells in rat
brain with TBI leads to enhanced neuroprotection via an NFkB-mediated increase in IL-6
production and inhibition of caspase-3[201]. Moreover, treatment of TBI with human
mesenchymal stem cells during the acute phase of rat brain injury can enhance neurological
functional outcome, with increased levels of neurotrophic factors and decreased caspase-
3[203].
Interestingly, the pan-caspase inhibitor BAF reduces acute cell death in rats after TBI by
inhibiting mitochondrial release of cyto. c and initiator caspases-2 and effector caspase-3, but
not through caspase-8[167]. Moreover, even though caspase-8 is activated in a palmitic- or
stearic acid-induced nerve growth factor differentiated PC12 cell model of TBI, blockade of
caspase activity with the pan-caspase inhibitor z-VAD-fmk does not prevent cell death[123].
The evidence may imply that targeting caspase-8 in the cell death receptor pathway of TBI is
not crucial. To date, there is no treatment for TBI that targets caspase-10 and -12.

4. Perspective

Because they are significant clinical problems, effective treatment strategies are urgently
needed to cure H-IE and TBI based on their pathophysiological process. The synergistic
mitochondrial death pathway, cell death receptor pathway, and ER death pathway could be
coactivated to mediate neonatal HIBI and TBI. In future years, combination therapies
targeting multiple caspases may become a viable strategy to enhance the beneficial effects of
each. Such strategies will be employed to explore multimodal and neuroprotective therapies
for neonatal HIBI and TBI using currently available compounds and treatment combinations
(e.g. selective caspase-3 inhibitor as a magic bullet in combination with hypothermia). In
addition, the development of more specific caspase inhibitor(s) should elucidate the action
mechanism of caspases in the pathophysiology of neonatal HIBI and TBI, thus providing a
new perspective in our understanding of the regulation of neuronal apoptotic cell death in the
two neurological disorders. Mesenchymal stem cell therapy has provided beneficial effects
for TBI[201, 203]. Furthermore, trials of stem cell therapies are ongoing, offering great
promise as new treatment modalities for neonatal HIBI and H-IE[204-207]. Hopefully, stem
cell therapy focused on caspase-mediated apoptosis will culminate in treatment stategies to
reduce the risk of death or disability in infants with H-I encephalopathy and offer hope to
those suffering from TBI.
Acknowledgments
This work is supported by grants from the National Institutes of Health/National Institute
of Neurological Disorders and Stroke (to X.W.) and the Muscular Dystrophy Association (to
X.W.).
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Chapter 7
Alterations in N-Methyl-D-Aspartate
(NMDA) Receptor Function and
Potential Involvement in Anesthetic-
Induced Neurodegeneration
Cheng Wang
*
, Xuan Zhang, Fang Liu,
Merle G. Paule and William Slikker Jr.
National Center for Toxicological Research,
U.S. Food & Drug Administration, Jefferson, Arkansas
Abstract
Advances in pediatric and obstetric surgery have resulted in an increase in the
duration and complexity of anesthetic procedures. It is known that the most frequently
used general anesthetics have either NMDA receptor blocking or -aminobutyric acid
(GABA) receptor activating properties. It is also known that anesthetic agents can cause
widespread and dose-dependent apoptotic neurodegeneration in the developing brain.
Exposure of developing mammals to NMDA-type glutamate receptor antagonists
affects the endogenous NMDA receptor system and enhances neuronal cell death. The
NMDA receptor regulates a calcium channel and calcium influx that overwhelms the
mitochondrial buffering capacity can result in increased production of reactive oxygen
species (ROS) and cell death. Meanwhile, stimulation of immature GABA receptors is
thought to be excitatory early in development but inhibitory in mature neurons.
Stimulation of immature neurons by GABA agonists is thus thought to increase overall
nervous system excitability and may contribute to NMDA receptor-associated increased
excitability during early development. This increased excitability may contribute to
abnormal neuronal cell death during development.

*
Corresponding author: The Division of Neurotoxicology, HFT-132; National Center for Toxicological
Research/FDA; 3900 NCTR Road.; Jefferson, AR 72079-0502 USA., Phone: (870) 543-7259, Fax: (870) 543-
7745, Email: cheng.wang@fda.hhs.gov
Cheng Wang, Xuan Zhang, Fang Liu et al. 156
The type of excitotoxic insults that lead to neuronal apoptosis or necrosis are not
adequately understood but surely depend upon animal species, the concentration of
stressors, durations of exposures, the receptor subtypes activated and the stage of
development or maturity of a particular cell type at the time of exposure. It has been
proposed that prolonged blockade of the NMDA receptor in the developing brain by
NMDA receptor antagonists such as the dissociative anesthetics ketamine or
phencyclidine (PCP) causes a compensatory up-regulation of NMDA receptors. Neurons
bearing these up-regulated receptors are subsequently more vulnerable to the excitotoxic
effects of endogenous glutamate, because this up-regulation of NMDA receptors allows
for the influx of toxic levels of intracellular Ca
2+
under normal physiological conditions.
Although many more studies will be necessary in order to develop adequate
quantitative models to explain the relationships between altered NMDA receptor function
and anesthetic-induced neurodegeneration, a general hypothesis has been constructed and
tested in an interactive manner using carefully selected agents as defined by their
pharmacological and physiological properties. The integrative and iterative evaluation of
these kinds of models will lead to a better understanding of the potential neurotoxicity of
NMDA antagonists and GABA agonists in the developing human.
Introduction
Advances in pediatric and obstetric surgery have resulted in an increase in the duration
and complexity of anesthetic procedures. It is known that the most frequently used general
anesthetics have either N-methyl-D-aspartate receptors (NMDA) receptor blocking or -
aminobutyric acid (GABA) receptor activating properties. It is also known that anesthetic
agents can cause widespread and dose-dependent apoptotic neurodegeneration in the
developing brain. Since the first report [1] that NMDA-receptor antagonist administration
caused neurotoxicity in rats during early stage of central nerve system (CNS) development,
more attention has been paid to the pediatric population exposed to anesthetics. There is
evidence indicating that anesthetic drugs caused widespread and dose-dependent apoptosis in
the developing rat brain [2-4], and the vulnerability of brains to neuronal effects of pediatric
anesthetics is restricted to the period of rapid synaptogenesis, also known as the brain growth
spurt [1]. The brain growth spurt occurs at different times relative to birth in different species.
In rats and mice it happens in postnatal period. In human the brain growth spurt time extends
from the sixth month of gestation to several years after birth. Thus, the brain growth spurt
period lasts for several years, from pre to postnatal human development, during which
immature CNS neurons are susceptible to drugs with N-methyl-D-aspartic acid (NMDA)
antagonist or gamma amino butyric acid (GABA) mimetic properties [5]. Therefore, it is of
interest that most of the currently used general anesthetic drugs have either NMDA receptor
blocking property, such as ketamine or GABA receptor enhancing property, exemplified by
midazolam and benzodiazepines.
Activation of NMDA receptors mediates most of the excitatory neurotransmission in the
central nervous system (CNS). Normal neuronal development, differentiation and outgrowth
depend on stimulation of NMDA receptors. Since ketamine is widely used in clinical
anesthesia, the reports by different groups on the neurodegenerative effect of NMDA receptor
blockade in the developing rat brain raise concerns about the safety of ketamine and other
anesthetics used for neonates. Exposure of developing mammals to NMDA-type glutamate
Alterations in N-Methyl-D-Aspartate (NMDA) Receptor Function and Potential 157
receptor antagonists affects the endogenous NMDA receptor system and enhances neuronal
cell death. The NMDA receptor regulates a calcium channel and calcium influx that
overwhelms the mitochondrial buffering capacity can result in increased production of
reactive oxygen species (ROS) and cell death. Meanwhile, stimulation of immature GABA
receptors is thought to be excitatory early in development but inhibitory in mature neurons.
Stimulation of immature neurons by GABA agonists is thus thought to increase overall
nervous system excitability and may contribute to NMDA receptor-associated increased
excitability during early development. This increased excitability may contribute to abnormal
neuronal cell death during development.
One of the intracellular transduction pathways that may contribute to anesthetic agent
induced neurotoxicity involves N-methyl-D-aspartate receptors. NMDA receptors and the
consequent Ca
2+
influx caused by their activation are major events that may contribute to
potential neuronal cell death. Therefore, long-term blockade of NMDA receptors by
anesthetic agents during the synaptogenesis stage in developing brain can cause apoptotic
degeneration of neurons.
Neurotransmission and Anesthetic Agents
Glutamatergic Transmission and Potential Anesthetic-Induced
Neurodegeneration during the Development
The amino acid L-glutamate is generally recognized as the major excitatory
neurotransmitter of the mammalian central nervous system (CNS) and glutamate receptors
play a major role in fast excitatory synaptic transmission. Glutamate promotes neuronal
migration, differentiation and plasticity during development and throughout life [6].
Malfunctions of the glutamate system can affect neuroplasticity and cause neuronal toxicity.
In the case of anesthetic-induced neurodegeneration, many glutamate-regulated processes
seem to be perturbed. Abnormal neuronal development, abnormal synaptic plasticity and
neurodegeneration have been proposed as mechanisms that underlie anesthetic-induced
neuronal cell loss. It is becoming clear that some of the most important functions of the
nervous system, such as synaptic plasticity and synaptic formation, critically depend on the
behavior of NMDA receptors, and that neurological damage caused by a variety of
pathological states can result from exaggerated or inappropriate activation of NMDA
receptors [7, 8].
The general anesthetics, such as ketamine, block subtypes of glutamate receptors: the N-
methyl-D-aspartate (NMDA) receptors. As a dissociative anesthetic, ketamine is commonly
used to produce analgesia in children in emergency departments [9]. Clinically, ketamines
role in pediatric anesthesia is well established. However, recent studies have indicated that
ketamine may cause dose-dependent, widespread apoptotic neurodegeneration in immature
rat and monkey brains [1, 10, 11]. The window of vulnerability appears restricted to the phase
of rapid synaptogenesis, also known as the brain growth spurt.
Glutamatergic transmission is mediated by receptor families that are classed as ionotropic
(iGluRs) or metabotropic (mGluRs). iGluRs are ligand-gated ion channels which can be sub-
classified into the following groups based upon their ligand binding properties: N-methyl-D-
aspartate receptors (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate
receptors (AMPA), kainate receptors (KA) and, more recently, delta receptors. NMDA
receptors appear to be heteromeric complexes [12]. NMDA-R1 (Grin1) subunits can form
homo-oligomeric receptors that are functional, and the presence of this subunit is required to
produce detectable NMDA-activated channel currents in vitro [13]. NMDA-R2 (Grin2A-D)
subunits produce functional receptors only when co-expressed with NMDA-R1 [12], and co-
expression of NMDA-R1 with NMDA-R2 subunits increases their responsiveness to NMDA
and yields different functional properties [13].
Proposed mechanisms for the developmental neurotoxicity caused by anesthetic agents
such as ketamine include a compensatory up-regulation of NMDA receptors and subsequent
over-stimulation of the glutamatergic system by endogenous glutamate via this receptor up-
regulation [4, 10, 14]. This results in dysregulation of calcium signaling, oxidative stress [15],
and activation of the NF-kB signaling pathway [14]. However, the exact molecular
mechanisms underlying ketamine-induced apoptotic neuronal cell death remain elusive.
To better understand the molecular pathogenesis of anesthetic-induced developmental
neurotoxicity, systems biology approaches should be carried out to examine the changes in
gene expression profiles in developing brains, which have been shown to display a high
susceptibility to ketamine-induced apoptotic neuronal cell death.
Changes in Gene Expression Profiling and Anesthetic-Induced
Consistent with previous morphological and biochemical findings on anesthetic agents,
such as ketamine, our microarray study [16] identified disruptions in glutamate receptor
signaling, synaptic long-term potentiation, PTEN (phosphatase and tensin homolog deleted
on chromosome 10) signaling, pyrimidine metabolism, circadian rhythm signaling, etc. In
addition, we also found remarkable changes in genes related to apoptosis. More specific and
sharper differences did exist in that the Troponin T1 (Tnnt1) gene was significantly induced
in the adult mouse brain [17], but it was not observed in our system. Conversely, while a
significant up-regulation of the Grin1 (NR1) and Grin2 (NR2) genes was observed in our
study, the previous report did not show similar results [17]. These similarities and differences
most likely reflect the unique response patterns of adult and developing brains toward
ketamine administration. Importantly, these data indicate that potential anesthetic-induced
neurotoxicity may depend on the dose of anesthetic, the duration of exposure, the route of
administration, the receptor subtype activated, and the stage of development or maturity at the
time of exposure.
The gene expression of the NMDA receptor subunit gene, Grin1 (NR1), was significantly
up-regulated in ketamine-treated rat pups as detected in microarray experiments and
subsequently confirmed with TaqMan analyses. The NMDA receptor NR1 subunit is widely
distributed throughout the brain and is the fundamental subunit necessary for NMDA channel
function. In the study [16], by using in situ hybridization techniques to detect the relative
densities of NMDA receptor NR1 subunits, a potential parallel relationship between enhanced
apoptosis and NMDA receptor expression levels was examined (Figure 1). Our in situ data
provided direct evidence that repeated ketamine exposure results in a substantial increase in
autoradiographic density (labeling) of NR1 subunit mRNA in the frontal cortex and
hippocampus. These data indicate that ketamine-induced pathological change is closely
associated with a remarkable up-regulation of NMDA NR1 subunit mRNA.
It is possible that increased expression of Grin1 (NR1) was accompanied by an altered
expression of other glutamate receptor subunits. Our microarray analyses and Q-PCR data
[16] revealed an increase in Grin2a (NR2A; 1.5 fold) and Grin2c (NR2C; 1.7 fold), but no
significant effects were observed in Grin2b (NR2B) or Grin2d (NR2D). Our findings are
consistent with those of previous in situ hybridization and immuno-blotting data that
demonstrated a compensatory up-regulation of NMDA-R1 and NMDA-R2 receptors
following prolonged exposure to NMDA receptor antagonists [10, 18, 19].

Figure 1. NMDA receptor NR1 subunit mRNA abundance in PND 7 rats. In situ hybridization was
performed on rat brain sections (coronal) using a
35
S-labeled oligonucleotide probe specific for the
NMDA receptor NR1 subunit. Panel I shows a general view of NR1 in situ hybridization signals in
frontal cortical areas from both control and ketamine-treated rats. Panel II illustrates that the
autoradiographic density (labeling) for NR1 subunit mRNA was higher in ketamine-treated (20 mg/kg
x 6 injections) rat brain frontal cortex (B) compared to control (A). Scale bar = 90 m
In Situ Hybridization (Frontal Cortex)
(NMDA Receptor NR1 Subunit mRNA)
Control Ketamine
I
Control
Ketamine (20 mg/kg x 6)
II
A B
Shi et al., 2010
In Situ Hybridization (Frontal Cortex)
(NMDA Receptor NR1 Subunit mRNA)
Control Ketamine
I
Control
Ketamine (20 mg/kg x 6)
II
A B
Shi et al., 2010
Table 1. Selective validation of the microarray results by Q-PCR
Gene symbols Fold-change (Q-PCR) Fold-change (microarray)
Grin1 (NR1)
Grin2a (NR2A)
Grin2b (NR2B)
Grin2c (NR2C)
Grin2d (NR2D)
1.8*
1.5*
1.0
1.7*
1.2
1.5*
1.2
0.9
1.5*
1.1
* P<0.05, as compared to the control.

Figure 2. Ketamine-induced neurodegeneration in PND 7 rats assessed by TUNEL labeling.
Representative photographs indicate that TUNEL-positive cells are more numerous in layers II and III
of the frontal cortex in the ketamine treated rat brain (B). Only a few TUNEL-positive cells were
observed in the control (saline treated) rat brain (A). Scale bar = 60 m
NMDA receptor density has also been shown to increase in cultured cortical neurons
after exposure to the NMDA receptor antagonists D-AP5, CGS-19755, and MK-801, but not
after exposure to the AMPA/kainate receptor antagonist CNQX [20]. In our microarray study,
no significant changes were detected in the gene expression patterns of AMPA or kainate
receptors after repeated ketamine exposure. These findings support our previous
pharmacological data showing that application of the non-NMDA receptor antagonist,
CNQX, or nifedipine (an antagonist of the L-type voltage sensitive calcium channel) did not
produce a significant protective effect against ketamine-induced neuronal apoptosis [4]. We
hypothesize that continuous blockade of NMDA receptors by ketamine causes a
compensatory up-regulation of NMDA receptors and this up-regulation makes neurons
bearing these receptors more vulnerable, after ketamine withdrawal, to the excitotoxic effects
of endogenous glutamate, because this up-regulation of NMDA receptors allows for the
accumulation of toxic levels of intracellular calcium even under normal physiological
conditions.
In order to understand the underlying mechanism of anesthetic (e.g. ketamine)-induced
neurodegeneration, brain tissues from the frontal cortical levels, where the most severe
TUNEL-Assay
(PND-7 rat pups)
A B
Control (10 l saline/g, 6 injections) Ketamine (20 mg/kg, 6 injections)
TUNEL-Assay
(PND-7 rat pups)
A B
Control (10 l saline/g, 6 injections) Ketamine (20 mg/kg, 6 injections)
neuronal damage was expressed, were selected for RNA isolation and microarray analysis
[16]. Consistent with the TUNEL assay and previous in vivo data (Figure 2), a total of 32
genes were found to be involved in apoptosis, and among them, 15 genes were up-regulated
and 17 genes were down-regulated (Table 2) in animals exposed to 6 injections of 20 mg/kg
ketamine, compared with the controls [16] . The apoptosis-related genes are a group of genes
that has two distinct modes of operation: pro-apoptosis or anti-apoptosis. In response to
various inducers such as stressful stimuli or sustained elevation of intracellular calcium
levels, the ultimate fate of the brain cell is determined by the roles of these apoptosis-related
genes in regulating the life/death cell balance.
Table 2. Apoptosis-related genes identified by GOFFA
Gene symbols Gene names
1 Acvr1c activin A receptor, type IC
2 Ahr aryl hydrocarbon receptor
3 Alms1 Alstrom syndrome 1
4 Amigo2 adhesion molecule with Ig like domain 2
5 Atp7a ATPase, Cu++ transporting, alpha polypeptide
6 Bnip3 BCL-2/adenovirus E1B 19 kDa-interacting protein 3
7 Bub1b budding uninhibited by benzimidazoles 1 homolog, beta
8 Cd24 CD24 antigen
9 Cdc2a cell division cycle 2 homolog A (S. pombe)
10 Inhba inhibin beta-A
11 Myc myelocytomatosis oncogene
12 Ntf3 neurotrophin 3
13 Pak7_predicted p21 (CDKN1A)-activated kinase 7 (predicted)
14 Pdia2_predicted protein disulfide isomerase associated 2 (predicted)
15 Rasa1 RAS p21 protein activator 1
16 Tnfrsf11b tumor necrosis factor receptor superfamily, member 11b
17 Unc5c unc-5 homolog C (C. elegans)
18 Agt angiotensinogen (serpin peptidase inhibitor, clade A, member 8)
19 Alb Albumin
20 Apoe apolipoprotein E
21 Bag3 Bcl-2-associated athanogene 3
22 Cebpb CCAAT/enhancer binding protein (C/EBP), beta
23 Clu Clusterin
24 Cryab crystallin, alpha B
25 Gjb6 gap junction membrane channel protein beta 6
26 Hrk harakiri, BCL-2 interacting protein (contains only BH3 domain)
27 Igfbp3 insulin-like growth factor binding protein 3
28 Inpp5d inositol polyphosphate-5-phosphatase D
29 Jun Jun oncogene
30 Mal myelin and lymphocyte protein, T-cell differentiation protein
31 Rassf5 Ras association (RalGDS/AF-6) domain family 5
32 Txnip thioredoxin interacting protein
Genes 1-17 were down-regulated and genes 18-32 were up-regulated.

The mechanism(s) underlying the anesthetic (e.g. ketamine)-induced neuronal cell death
have not been fully elucidated. However, our microarray data indicate that the majority
(approximately two-thirds) of up-regulated genes were pro-apoptotic in nature including Agt,
Clu, Gjb6, Hrk, Igfbp3, Inpp5d, Jun, Mal, Rassf5 and Txnip. Meanwhile, from these up-
regulated genes, expression of Alb, Apoe, and Cryab may provide inhibition of apoptosis [21].
It has been reported that silencing the expression of Bag3 (Bcl-2-associated athanogene 3)
leads to reduced protein levels of Bcl-XL, Mcl-1 and Bcl-2 in colon cancer cells and
increased apoptosis [22]. As a critical gene, Cebpb [CCAAT/enhancer binding protein
(C/EBP)] acts as a major regulator of metabolic homeostasis and is involved in many cellular
processes, such as differentiation, growth, immune responses, neoplastic growth,
development of the reproductive system, and pro- and anti-growth pathways [23].
On the other hand, in genes that have been down-regulated (17 genes), about one-half are
anti-apoptotic genes. The over-expression of Acvr1c has been shown to suppress the apoptotic
effects and Amigo2 acts as an anti-apoptotic factor [24, 25]. Bnip3 encodes cellular proteins
that interact with Bcl-2. In cortical cells, cyanide induces a rapid up-regulation of Bnip3
expression, followed by a caspase-dependent cell death [26]. Down-regulation of Cd24,
Cdc2a, and disruption of the Rasa1 gene in early embryonic mice induce apoptosis of
neuronal cells [27, 28]. In neocortical and hippocampal tissues, apoptotic effects can be
demonstrated following Ahr activation [29] and this gene was found to be up-regulated in our
studies. These observations may imply that the frontal cortex is the brain region most
vulnerable to ketamine-induced neurotoxicity during development, and the neuronal survival
in the early phases of the apoptotic cascades mostly depends on the balance between the pro-
and anti-apoptotic factors of the apoptosis-related genes.
Gabaergic Neurotransmission and Potential Anesthetic-Induced
Anesthetics that block the NMDA receptor (glutamate subtype) and/or positively
modulate or gate the GABA
(A)
receptor have been associated with apoptotic
neurodegeneration in the developing neonate [4, 11, 30, 31]. To induce or maintain a surgical
plane of anesthesia, it is common practice in pediatric or obstetrical medicine to use a
combination of agents from these two classes.
The anesthetic gas nitrous oxide (N
2
O), an NMDA receptor antagonist, and isoflurane
(ISO), a volatile anesthetic that acts on multiple receptors, including the postsynaptic GABA
receptor, are most commonly used in surgical procedures for human infants. Therefore, this
practice raises the concern that there is a risk of deleting immature neurons by increasing
apoptotic cell death in the developing brain. In our previous study [32], to further clarify the
brain damaging potential of N
2
O and ISO, PND-7 rat pups were exposed to clinically relevant
doses of these anesthetics, either individually or in combination for 2, 4, 6 or 8 hours, and the
neurotoxic effects were evaluated 6 hours after

cessation of anesthesia. No significant
increase in apoptotic neurodegeneration in layers II and III of the frontal cortex was detected,
as indicated by caspase-3 immunostaining, silver staining and Fluoro-Jade C staining, in
animals exposed to N
2
O (75 vol%) alone for up to 8 hours [32]. This was similar to findings
from previous studies performed using the same model [11]. Meanwhile, it has also been
reported that animals treated with isoflurane (0.75. 1.0, or 1.5%), which acts on multiple
receptors including postsynaptic GABA receptors, exhibited dose-dependent neurod-
egeneration in the thalamic nuclei and parietal cortex [11]. However, our previous study
demonstrated that application of isoflurane in a low dose (0.55 %; for 2, 4, 6 and 8 hours)
showed no significant enhancement of apoptotic cell death in the frontal cortex compared
with the corresponding control animals. This finding is very important because this
concentration somewhat reflects the threshold dose of the neurotoxic effect of isoflurane [32].
In order to determine if a combination of NMDA antagonists and GABA agonists will
prevent or enhance each others effects, the combination of N
2
O (75%) with ISO (0.55%) has
been applied. Co-administration of N
2
O and ISO is a frequently-used inhaled anesthetic
combination to facilitate surgery in the neonate. Co-administration of N
2
O and ISO is a
frequently-used inhaled anesthetic combination to facilitate surgery in the neonate. A striking
enhancement in brain damage was noted [32] when N
2
O (75%) was combined with ISO
(0.55%). Maximal neurodegeneration was observed after 6-8 hours, however, only a small
amount of damage (not significant) was detected at 4 hours, and no significant effects at 2
hours. These findings were also consistent with previous studies performed using the same
animal model [11]. The concordance between increased caspase-3 immunostaining, silver
staining and Fluoro-Jade C staining, as well as a decreased BCL-X
L
/Bax ratio suggests this
regimen produces cell death primarily through an apoptotic mechanism. In recent years there
has been a great deal of interest in mechanisms of cell death, especially those that may be
associated with the potential neurotoxic effects of anesthetics. Olneys group [33, 34]
conducted a series of studies that led to the observation that during the developmental period
of synaptogenesis, brief exposure to ethanol, which has both NMDA antagonist and
GABAmimetic properties, can trigger enhanced apoptosis in the mammalian brain. The
ability of ethanol to induce widespread apoptotic neurodegeneration throughout the forebrain
during synaptogenesis provides a more likely explanation than has heretofore been available
for the reduced brain mass and lifelong neurobehavioral disturbances associated with fetal
alcohol spectrum disorder [35].
There is no doubt that the developing central nervous system is exquisitely sensitive to
derangements in the internal milieu [36]. Accentuated neurodegenerative mechanisms in the
immature brain increase neuronal susceptibility to various metabolic events or exposure to
anesthetic agents [36]. Anesthetics and anticonvulsant drugs that block NMDA receptors or
activate GABA
(A)
receptors consistently increase neuronal apoptosis in the neonatal brain [1,
5, 11, 34], suggesting that the physiological stimulation of the NMDA receptor is necessary
for neuronal synaptogenesis differentiation and survival during development. Lack of NMDA
receptor activation by glutamate decreases synaptogenesis and cell-cell interactions [37, 38].
Anesthetic agents directly suppress neuronal activation and also reduce extracellular
concentrations of excitatory neurotransmitters [39], thereby reducing developmental inputs to
immature neurons. Anesthetic agents suppress neuronal activity by as-yet unknown
mechanisms. Similar to the proapoptotic effect of NMDA antagonists, vulnerability of the
developing rat brain to GABA mimetics also changes with age [34], again raising the
possibility that, depending upon the time of exposure [33], different patterns of
neurodegeneration and potentially different neurobehavioral disturbances might occur.
Meanwhile, immature GABA receptors are excitatory early in development and convert to an
inhibitory role in mature neurons [40]. It can be postulated that prolonged supra-physiological
stimulation of immature neurons by GABA agonists enhances the excitatory component in
the action of GABA and may contribute to increased excitability during early development
[41]. This increased excitability, along with NMDA antagonist-induced alteration of NMDA
receptors, could lead to neuronal cell death. Consistent with previous reports [11], it appears
that

more profound neurodegeneration is induced if both NMDA and GABA
(A)

receptors are
simultaneously altered, in that a more robust neurodegenerative response is seen after
exposure to

ethanol than to either an NMDA antagonist or GABAmimetic drug

alone.
Although this principle is corroborated by the present

demonstration that combining a
nontoxic concentration of the NMDA

antagonist N
2
O with a GABAmimetic agent induced a
pattern of neurodegeneration more pronounced than was induced by

either drug category
alone, more supportive data from additional agent combinations, such as ketamine and
midazolam, and experimental models (in vitro and in vivo studies of nonhuman primate and
rodent models) will be necessary to confirm this conclusion. In addition, the timing of the
switch in the chloride reversal potential in various brain regions should be considered. At
PND 7, many regions have either completed the switch or are well along in the process of
switching. Thus, the matter of GABAergic excitation as a contributor to damage is complex
and most likely region dependent.
Anesthetic-Induced Toxicity
and Potential Protection
Lines of evidence have indicated that activation of NMDA receptors caused neuronal cell
death [1, 5, 34, 42-44]. Although neuronal apoptosis can be the final result of anesthetic-
induced toxicity, the pathways leading from mitochondrial dysfunction are not completely
understood. It is increasingly apparent that mitochondria lie at the center of the cell death
regulation process. Our previous data suggest the possible link between the formation of
reactive oxygen species (ROS) and the regulation of either BCL-2 related genes or
transcription factors [10, 14, 15, 45]. It was postulated that the continuous exposure of
developing brains to general anesthetics may cause selective cell death by a mechanism that
involves a dysregulation of NMDA receptor subunits [4, 14, 18, 31] accompanied by loss of
mitochondrial membrane potential, alterations of calcium homeostasis and subsequent free
radical formation. In addition, several factors may contribute to the topographic differences in
anesthetic-induced neurodegeneration, such as animal species, developmental stage at the
time of drug exposure, doses and duration of anesthesia exposure, etc.
It is proposed that the administration of general anesthetics during critical developmental
periods will result in a dose-related increase in neurotoxicity and loss of neurons [4, 10, 14,
30]. Polymers of -2,8-linked sialic acid neural cell adhesion molecule (PSA-NCAM), a
neuronal specific marker, is formed by the enzymatic transfer of large, negatively charged
carbohydrate polymers of -2,8-linked sialic acid to the fifth immunoglobulin domain of the
NCAM molecule. To determine whether the expression levels of PSA-NCAM correlate with
inhaled anesthetic-induced cell death, Western blot analysis for PSA-NCAM was performed
[32]. An anesthetic combination (75% N
2
O + 0.55% ISO for 6 hours) caused a substantial
increase in caspase-3, silver and Fluoro-Jade C staining, along with a concomitant decrease in
PSA-NCAM staining. The decrease in PSA-NCAM corresponded to an approximately 45%
decrease in PSA-NCAM immunoreactivity as assessed using an immunoblot assay. This
decrease could be the direct result of neuronal loss induced by anesthetics, because this
reduction of PSA-NCAM expression is consistent with the enhanced neurodegeneration as
indicated by increased caspase-3, silver and Fluoro-Jade C staining positive neuronal cells.
L-carnitine is a dietary supplement and has been reported to prevent apoptotic death [46].
L-carnitine is well known to exhibit membrane modulatory effects and thereby preserve
cellular membrane integrity. The fact that co-administration of L-carnitine blocked cell death,
as well as the loss of PSA-NCAM immunoreactivity, further indicates that the neuronal cell
death induced by an anesthetic combination in developing rats is most likely apoptosis.
Several reactive oxygen species, including nitric oxide and superoxide anion (O
2
), have
been implicated in glutamate-induced neuronal death. However, little is known about the
signaling pathway that mediates the postulated roles of peroxynitrite (ONOO). In previous in
vitro studies, general anesthetics such as ketamine administration caused a significant up-
regulation of nitrotyrosine expression accompanied by enhanced neuronal apoptosis as
indicated by cell death detection ELISA and decreased PSA-NCAM expression. Protein
tyrosine nitration occurs during many neurodegenerative states and is an important marker of
oxidative stress induced by peroxynitrite, and possibly other nitric oxide (NO
-
)-derived
oxidants. The toxicity of NO
-
is linked to its ability to combine with superoxide anions (
-
O
-
2
)
to form peroxynitrite, an oxidizing free radical that can cause DNA fragmentation and lipid
oxidation [47, 48]. Recent findings show that peroxynitrite may act as a signaling molecule
capable of up-regulating protein tyrosine phosphorylation, which plays an important role in
the regulation of cell communication, proliferation, migration, differentiation and survival
[49, 50]. In the brain, NO
-
is produced by nNOS and is believed to be controlled by activation
of NMDA receptors [51]. The finding that nNOS is connected to the NMDA receptor via a
postsynaptic density protein (PSD95) indicates a direct link between NMDA receptor
activation and nNOS stimulation [52]. The interaction of superoxide and NO
-
, which results
in the formation of peroxynitrite, can be prevented by targeting either superoxide or NO
-
. The
previous study [53] demonstrated that pharmacological manipulation of NOS by 7-
nitroindazole provides protection against ketamine-induced neuronal loss. Importantly, no
protective effect was observed with cultures co-treated with ketamine and 7-nitroindazole,
when 7-nitroindazole was omitted during ketamine washout. Primary neuronal cell cultures
are thought to mainly contain neuronal NOS (nNOS). Therefore, 7-nitroindazole was used in
this study since it has been reported to be a selective nNOS inhibitor. It has been
demonstrated that pretreatment with 7-nitroindazole blocks methamphetamine (METH)-
induced dopaminergic neurotoxicity in mice [54] and selective nNOS knock-out mice are
resistant to METH-induced dopaminergic neurotoxicity [55]. In addition to 7-nitroindazole,
several other NOS inhibitors and iron porphyrin compounds have been utilized, such as
FeTPPS and FeTMPyP, which efficiently degrade peroxynitrite to nitrate under physiological
conditions. It appears, however, that 7-nitroindazole is the most selective nNOS inhibitor [56-
58].
The precise mechanisms by which NMDA regulates Bax (a proapoptotic protein) is
unknown, but the prevention of this effect in vitro by the addition of catalase and superoxide
dismutase [15], or in vivo by M40403 (superoxide dismutase mimetic) suggest the possible
involvement of reactive oxygen species (ROS). Among the potential downstream regulators,
the effect of ketamine on Bax and BCL-X
L
(an anti-apoptotic protein) was measured. Bax is a
pore-forming cytoplasmic protein which translocates to the outer mitochondrial membrane,
influencing its permeability and inducing cytochrome c release from the intermembrane space
of the mitochondria into the cytosol, subsequently leading to cell death [59].
It is important to indicate that up-regulation of the NR1 subunit of the NMDA receptor is
an important first step in the pathway to anesthetic-induced neurotoxicity. These up-regulated
calcium channel receptors are vulnerable to endogenous glutamate concentrations within the
tissues after ketamine washout. In our previous studies [4, 14] , to test whether the
administration of antisense oligonucleotides (ODNs) targeted to the NR1 NMDA receptor
subunit blocks steady state protein levels, an antisense ODN-targeting exon was used. Co-
administration of antisense ODN specifically prevented NR1 up-regulation and blocked the
reduction of PSA-NCAM expression induced by ketamine.
Of particular interest to the data at hand are the possible mechanisms by which
anesthetics such as ketamine could up-regulate NMDA receptors. Surprisingly, there is not an
abundance of literature concerning this issue, but recently it has been demonstrated that the
distal region of the NR1 promoter contains an active NF-kB site, which is developmentally
regulated and appears to bind Sp3/Sp1 somewhat better than the NF-kB subunits [60]. The
NMDA receptor NR1 subunit is widely distributed throughout the brain and is the
fundamental subunit necessary for NMDA channel function. Our previous study [14]
demonstrated that ketamine produced a significant up-regulation in NR1 protein expression.
This result was consistent with literature demonstrating that treatment with NMDA
antagonists produces an up-regulation of the NMDA receptor complex as measured by an
increase in Bmax of NMDA receptor binding sites [20, 61].
The transcription factor NF-kB is known to respond to changes in the redox state of the
cytoplasm and has been shown to translocate in response to NMDA-induced cellular stress
[62]. NF-kB is normally sequestered in the cytoplasm, bound to the regulatory protein IkB
kinase. The net result is the release of the NF-kB dimmer, which is then free to translocate
into the nucleus.
NF-kB translocation appears to be a necessary step in cell death induced by PCP [45],
cyanide, and excitotoxic stimuli [63]. In our previous studies, ketamine produced a
remarkable increase in translocation of NF-kB into the nucleus. The protection against
cortical neuronal cell death and decreased PSA-NCAM by a peptide inhibitor of NF-kB
translocation, SN-50, suggested that there was a causal relationship between these effects.
There is evidence in the literature suggesting that the transcriptional regulation of target genes
by NF-kB is tissue specific and possibly gene specific within a given cell type. The ability of
SN-50 to prevent ketamine-induced cell death demonstrated that NF-kB is crucial to those
processes. However, whether anesthetic-induced NF-kB translocation is specifically
responsible for apoptotic or necrotic pathways observed in anesthetic studies is still unknown.
Resolution of this question will require additional experiments.
Conclusion
The integrative and iterative evaluation of the anesthetic models (in vivo & in vitro) lead
to a better understanding of the potential neurotoxicity of NMDA antagonists and GABA
agonists in the developing brains.
For example, a total of 462 down-regulated genes and 357 up-regulated genes were
identified after ketamine exposure in developing rat brain. Among them, 32 genes are
associated with apoptotic pathways. Perturbations of NMDA-type glutamate and other
receptor signaling were identified in our microarray analysis. Q-PCR assay confirmed that
NMDA receptor genes including Grin1 (NR1), Grin2a (NR2A) and Grin2c (NR2C) were
significantly up-regulated. The ketamine-induced up-regulation of NMDA receptor Grin1
(NR1) mRNA signaling was further confirmed by in situ hybridization. These observations,
together with morphological evidence suggest that repeated anesthetic (e.g. ketamine)
exposure causes compensatory up-regulation of NMDA receptors and subsequent over-
stimulation of the glutamatergic system triggering enhanced apoptosis in developing neurons,
and that neuronal survival in the early phases of apoptotic cascades depends mostly on the
balance between pro- and anti-apoptotic factors.

Figure 3. The cartoon illustrating the working model of anesthetic (e.g. ketamine)-induced neuronal cell
death and potential protection mechanisms
In summary, Figure 3 illustrates a potential model of anesthetic (i.e. ketamine)-induced
neuronal cell death in the developing brain. Excessive activation of up-regulated NMDA
receptors results in a calcium overload that exceeds the buffering capacity of the
mitochondria and interferes with electron transport in a manner that results in an elevated
production of reactive oxygen species, and the dissociation of some transcription proteins,
such as NF-kB, and their transport into the nucleus. In the nucleus these transcription factors
bind to several DNA sequences of several known genes. The consequence of this binding is
Ca
C
a
+
+
o
v
e
r
l
o
a
d
O
2
-
O
2
-
I
K
K
Ca
++
Ca
++ Ca
++
glutamate
NR1
NR2
c
a
s
p
a
s
e
s
c
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Figure 3.
UP-regulated
NMDA Receptor
Modified from McInnis et al., 2002
L-carnitine
not completely understood, but the loss of the balance of pro- and anti-apoptotic genes is
apparent; the diminished formation of anti-apoptotic heterodimers in favor of pro-apoptotic
homodimers. These homodimers are thought to create mitochondrial membrane pores through
which cytochrome c can leak into the cytoplasm where it can activate caspases that play a
critical role in the ultimate demise of the neuron. As shown in above (text), several recent
studies using antisense ODNs targeting specific NMDA receptor subunits, or blockers of
oxidative stress such as L-carnitine, the superoxide dismutase mimetic, M40403, and the
NOS inhibitor, 7-nitroindazole have indicated that reduction of oxidative stress may protect
the developing animal from anesthetic-induced brain cell death.
Disclaimer
This document has been reviewed in accordance with United States Food and Drug
Administration (FDA) policy and approved for publication. Approval does not signify that the
contents necessarily reflect the position or opinions of the FDA nor does mention of trade
names or commercial products constitute endorsement or recommendation for use. The
findings and conclusions in this report are those of the author(s) and do not necessarily
represent the views of the FDA.
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Chapter 8
Genetics and Molecular Biology
of Alzheimer's Disease and
Frontotemporal Lobar Degeneration:
Analogies and Differences
Daniela Galimberti
*
, Chiara Fenoglio
and Elio Scarpini
Elio Scarpini, Dept. of Neurological Sciences,
"Dino Ferrari" Center, University of Milan, Fondazione C Granda, IRCCS Ospedale
Maggiore Policlinico, Milan, Italy
Abstract
Alzheimers disease (AD) is the most common cause of dementia in the elderly,
whereas Frontotemporal Lobar Degeneration (FTLD) is the most frequent
neurodegenerative disorder with a presenile onset. The two major neuropathologic
hallmarks of AD are extracellular Amyloid beta (A|) plaques and intracellular
neurofibrillary tangles (NFTs). Conversely, in FTLD the deposition of tau has been
observed in a number of cases, but in several brains there is no deposition of tau but
instead a positivity for ubiquitin.
In some families these diseases are inherited in an autosomal dominant fashion.
Genes responsible for familial AD include the Amyloid Precursor Protein (APP),
Presenilin 1 (PS1) and Presenilin 2 (PS2). The majority of mutations in these genes are
often associated with a very early onset (40-50 years of age).
Regarding FTLD, the first mutations described are located in the Microtubule
Associated Protein Tau gene (MAPT). Tau is a component of microtubules, which
represent the internal support structures for the transport of nutrients, vesicles,
mitochondria and chromosomes within the cell. Mutations in MAPT are associated with
an early onset of the disease (40-50 years), and the clinical phenotype is consistent with

*
Corresponding author: phone ++ 39.2.55033847, FAX ++ 39.2.50320430, E-MAIL: daniela.galimberti@unimi.it
Daniela Galimberti, Chiara Fenoglio and Elio Scarpini 174
Frontotemporal Dementia (FTD). Recently, mutations in a second gene, named
progranulin (GRN), have been identified in some families with FTLD. Progranulin is
expressed in neurons and microglia and displays anti-inflammatory properties.
Nevertheless, it can be cleaved into granulins which, conversely, show inflammatory
properties. The pathology associated with these mutations is most frequently
characterized by the immunostaining of TAR DNA Binding Protein 43 (TDP-43), which
is a transcription factor. The clinical phenotype associated with GRN mutations is highly
heterogeneous, including FTD, Progressive Aphasia, Corticobasal Syndrome, and AD.
Age at disease onset is variable, ranging from 45 to 85 years of age.
The majority of cases of AD and FTLD are however sporadic, and likely several
genetic and environmental factors contribute to their development. Concerning AD, it is
known that the presence of the c4 allele of the Apolipoprotein E gene is a susceptibility
factor, increasing the risk of about 4 fold. A number of additional genetic factors,
including cytokines, chemokines, Nitric Oxide Synthases, contribute to the susceptibility
for the disease. Some of them also influence the risk to develop FTLD.
In this chapter, current knowledge on molecular mechanisms at the basis of AD and
FTLD, as well as the role of genetics, will be presented and discussed.
1. Alzheimers Disease and Frontotemporaal
Lobar Degeneration
Alzheimers disease (AD) is the most common cause of dementia in the elderly, with a
prevalence of 5% after 65 years of age. The disease was originally described by Alois
Alzheimer and Gaetano Perusini in 1906, and it is clinically characterized by a progressive
cognitive impairment, including impaired judgment, decision-making and orientation, often
accompanied, in later stages, by psychobehavioural disturbances as well as language
impairment. The two major neuropathologic hallmarks of AD are extracellular beta-amyloid
(A|) plaques and intracellular neurofibrillary tangles (NFTs). The production of A|, which
represents a crucial step in AD pathogenesis, is the result of the cleavage of a bigger
precursor, named Amyloid precursor protein (APP), which is over-expressed in AD [1]. A|
forms highly insoluble and proteolysis resistant fibrils known as senile plaques.
Neurofibrillary tangles are composed of the tau protein. In healthy controls, tau is a
component of microtubules, which are the internal support structures for the transport of
nutrients, vesicles, mitochondria and chromosomes within the cell. Microtubules also
stabilize the growing axons, which are necessary for the development and growth of neurites
[1]. In AD, tau protein is abnormally hyperphosphorylated and forms insoluble fibrils, which
originate deposits within the cell.
Frontotemporal Lobar Degeneration (FTLD) occurs most often in the presenile period,
and age at onset is typically 45-65 years, with a mean in the 50s. Distinctive features in FTLD
concern behaviour, including disinhibition, loss of social awareness, overeating and
impulsiveness. Despite profound behavioural changes, memory is relatively spared [2].
Conversely to AD, which is more frequent in women, FTLD has an equal distribution among
men and women. The current consensus criteria [3] identify three clinical syndromes:
Frontotemporal Dementia (FTD), Progressive nonfluent Aphasia (PA) and Semantic
Dementia (SD), which reflect the clinical heterogeneity of FTLD. Frontotemporal dementia is
characterized by behavioural abnormalities, whereas PA is associated with progressive loss of
Genetics and Molecular Biology of Alzheimer's Disease and Frontotemporal Lobar 175
speech, with hesitant, nonfluent speech output [4], and SD is associated with loss of
knowledge about words and objects. This variability is determined by the relative
involvement of the frontal and temporal lobes, as well as by the involvement of right and left
hemispheres [5].
Despite the majority of AD and FTLD are sporadic and likely caused by the interaction
between genetic and environmental factors, so far it was observed that clinically typical AD
and FTLD can cluster in families and be inherited in an autosomal dominant fashion,
suggesting a genetic cause.
2. Familial AD
Autosomal dominant AD forms are characterized by mutations in three genes: |APP
[6], PSEN1 [7] and PSEN2 [8].
In 1987, a region of linkage with AD was reported on the long arm of chromosome 21,
which encompassed a region harboring the |APP gene, a compelling candidate for AD [9].
The gene is located at chromosome 21q21.22 and encodes for a transmembrane protein that is
normally processed into amyloid fragments. In 1991, the first missense mutation in APP was
reported [6]. Since then, 32 different mutations have been described in the |APP gene in 89
families (http://molgen-www.uia.ac.be). All these mutations cause amino acid changes in
putative sites for the cleavage of the protein, thus altering the APP processing, such that more
pathological A|42 is produced [10]. Interestingly, the chromosome 21, in which |APP
resides, is triplicated in Down syndrome and most of the cases manifest also AD by the age of
50. Post-mortem analyses of Downs patients who die young show diffuse intra-neuronal
deposits of A|, suggesting that its deposition is an early event in cognitive decline. The recent
discovery of an extra copy of the |APP gene in familial AD [11] provides further support
that increased A| production can cause the disease.
The other two genes causing familial AD are Presenilin (PSEN)1 (14q24.3) and PSEN2
(1q31-q42). Presenilins represent a central component of -secretase, the enzyme responsible
for originating A| from the C-terminal fragment of the APP protein. Mutations in presenilins
also alter APP cleavage, leading to an increased production of A|42. So far, more than 178
mutations in PSEN1 have been identified and 14 additional mutations have been found in the
homologous gene PSEN2 (http://molgen-www.uia.ac.be).
Most variants in PSEN1 are missense mutations resulting in single amino-acid
substitutions. Some are more complex, for example, small deletions or splice mutations. The
most severe mutation in PSEN1 is a donor-acceptor splice mutation that causes a two-
aminoacid substitution and an in-frame deletion of exon 9. However, the biochemical
consequences of these mutations for -secretase assembly seem to be limited [12,13]. All
these clinical mutations are likely to cause a specific gain of toxic function for PSEN1,
determined by an increase of the ratio between A42 and A40 amyloid peptides, thus
indicating that presenilins might modify the way in which -secretase cuts APP.
Mutations in presenilins occur in the catalytic subunit of the protease responsible for
determining the length of A| peptides therefore generating toxic A| fragments. However,
presenilins have also non-proteolytic functions [14,15], the disruption of which might also
contribute to familial AD pathogenesis.
Despite several carriers develop the disease early (40-50 years of age) with a typical AD
phenotype, in some cases patients carrying the same mutation develop signs and symptoms
resembling FTD instead of AD [16]. In addition, other mutations are associated with
myoclonus, seizures, bilateral spasticity, parkinsonian features or ataxia [17].
3. Familial FTLD
Frontotemporal Lobar Degeneration is a heterogeneous disease characterized by a
strong genetic component in its aetiology as up to 40% of patients report a family history of
the disease in at least one extra family member [18]. In 1994 an autosomal dominantly
inherited form of FTD with parkinsonism was linked to chromosome 17q21.2 [19].
Subsequently, other familial forms of FTD were found to be linked to the same region,
resulting in the denomination frontotemporal dementia and parkinsonism linked to
chromosome 17 (FTDP-17) for this class of diseases.
In 1998, MAPT gene on chromosome 17q21, which encodes the microtubule associated
protein tau was described as the cause of the disease in these families [20-22]
Currently, 44 different mutations in the MAPT gene have been described in totally 132
families (http://molgen-www.uia.ac.be). MAPT mutations are either non-synonimous or
deletion, or silent mutations in the coding region, or intronic mutations located close to the
splice-donor site of the intron after the alternatively spliced exon 10 [23]. Mutations are
mainly clustered in exons 9-13, except for two recently identified mutations in exon 1 [24].
As regards possible effects on MAPT mutations, different mechanisms are involved,
depending on the type and location of the mutation. Many of them disturb the normal splicing
balance, producing altered ratios of the different isoforms. A number of mutations promote
the aggregation of tau protein, whereas others enhance tau phosphorylation [25].
However, after the discovery for MAPT as causal gene for FTDP-17, there were still
numerous families with autosomal dominant FTLD genetically linked to the same region of
chr17q21 that contains MAPT but in which no pathogenic mutations had been identified,
despite extensive analysis of this gene [26-28]. The neuropathological phenotype in these
families was similar to the microvacuolar-type observed in a large proportion of idiopathic
FTD cases with ubiquitin immunoreactive neuronal inclusions. Moreover, clinically, the
disease in these families was consistent with diagnostic criteria for FTLD [3]. Sequence
analysis of the whole MAPT region failed to find a mutation and tau protein appeared normal
in these families [9] Moreover the minimal region containing the disease gene for this group
of families was approximately 6.2 Mb in physical distance. This region defined by markers
D17S1787 and D17S806 is particularly gene rich, containing around 180 genes. Collectively,
these data strongly argued against MAPT and pointed to another gene. Systematic candidate
gene sequencing of all remaining genes within the minimal candidate region was performed
and after sequencing 80 genes, including those prioritized on known function, the first
mutation in progranulin gene (GRN) was identified. It consists in a 4-bp insertion of CTGC
between coding nucleotides 90 and 91, causing a frameshift and premature termination in
progranulin (C31LfsX34) [30]. Tese results have been contemporarily replicated by Cruts et
al. [31], who analyzed other families with a FTLD-U disease without MAPT pathology,
finding a mutation five base pairs into the intron following the first non coding exon of the
GRN gene (IVS0+5G-C). This is predicted to prevent splicing out of the intron 0, leading the
mRNA to be retained within the nucleus and subjected to nuclear degradation [31]. At present
there is no obvious mechanistic link between the mutations in MAPT and GRN, currently
assuming that their proximity on chromosome 17 is simply a coincidence. Progranulin is
known by several different names including granulin, acrogranin, epithelin precursor,
proepithelin and prostate cancer (PC) cell derived growth factor [32]. The protein is encoded
by a single gene on chromosome 17q21, which produces a 593 amino acid, cysteine rich
protein with a predicted molecular weight of 68.5 kDa. The full-length protein is subjected to
proteolysis by elastase and this process is regulated by a secretory leukocyte protease
inhibitor (SLPI) [33]. Progranulin and the various granulin peptides are implicated in a range
of biological functions including development, wound repair and inflammation by activating
signaling cascades that control cell cycle progression and cell motility [32]. Excess
progranulin appears to promote tumour formation and hence can act as a cell survival signal.
Despite the increasing literature on the function of progranulin, its role in neuronal function
and survival remains unclear. In the human brain, GRN is expressed in neurons but
significantly is also highly expressed in activated microglia [30], with the result that GRN
expression is increased in many neurodegenerative diseases.
Since the original identification of null-mutations in FTLD in 2006, numerous novel
mutations have been reported, spanning most exons, and to date more than 68 GRN mutations
have been described (http://www.molgen.ua.ac.be/)
Interestingly, the majority of mutations identified create functional null alleles, causing
premature termination of the GRN coding sequence. This leads to the degradation of the
mutant RNA by nonsense mediated decay, creating a null allele [30,31]. The presence of a
null mutation causes a partial loss of functional progranulin protein, which in turn leads
eventually to neurodegeneration (haploinsufficency mechanism), although how loss of GRN
causes neuronal cell death remains unclear. Estimates of the frequency of GRN mutations in
typical FTD patient populations suggests that they account for about 5-10% of all FTD cases,
although numbers vary markedly depending on the nature of the populations considered
[31,34,35].
Neuropathology analysis revealed that ubiquitin immunoreactive neuronal cytoplasmatic
and intranuclear inclusions were present in all cases with FTDP-17, where pathological
findings were available [36]. Furthermore, soon after the identification of mutations in GRN,
biochemical analyses demonstrated that truncated and hyperphosphorylated isoforms of the
TAR-DNA binding protein (TDP-43) are major components of the ubiquitin-positive
inclusions in families with GRN mutations as well as in idiopathic FTD and a proportion of
Amyotrophic Lateral sclerosis (ALS) cases [37]. TDP43 is a ubiquitously expressed and
highly conserved nuclear protein that can act as a transcription repressor, an activator of exon
skipping or a scaffold for nuclear bodies through interactions with survival motor neuron
protein. Under pathological conditions, TDP-43 has been shown to relocate from the neuronal
nucleus to the cytoplasm, a consequence of which may be the loss of TDP-43 nuclear
functions [37]. The mechanism by which loss of progranulin leads to TDP-43 accumulation
and whether this is necessary for neurodegeneration in this group of diseases is still to be
clarified.
In conclusion, the function of progranulin in the brain is currently unclear and why loss
of this protein leads to a neurodegenerative diseases in mid-life remains to be established, and
its possible role as regulator of a repair activity in the central nervous system, as it is well
known to happen in periphery, remains a challenge for science. The gene encoding for TDP-
43, named TARDBP, has been extensively studied and a number of mutations found in its C-
terminal glycine rich region. Unexpectedly, the clinical phenotype of carries was ALS, and
aggregates made of TDP-43 have been described in brain and spinal cord of such patients (see
[38] for review).
A recently published collaborative study [39] analyzed GRN in a population of 434
patients with FTLD, including FTD, PA, SD, FTD/ALS, FTD/MND, CBD, PSP. Fifty eight
variants were identified, including 24 pathogenic variants. The frequency of GRN mutations
was 6.9% of all FTLD-spectrum cases, 21,4% of cases with a pathological diagnosis of
FTLD-U, 16% of FILD-spectrum cases with a family history of a similar neurodegenerative
disease, and 56.2 of cases of FTLD-U with a family history. Clinical information were
available for 31 GRN mutation-positive patients from 28 different families. The most
common clinical diagnosis was FTD (n=24); 3 patients were diagnosed with PA, 3 with AD
and 1 with CBD. The majority of GRN mutations Introduced a premature termination codon,
suggesting that their corresponding mRNA will be degraded through nonsense mediated
decay, supporting the hypothesis that most GRN mutations create functional null allele [39].
Two additional genes have been shown to cause FTLD. In 1995 Brown et al. [40]
reported linkage to the pericentromeric region of chromosome 3 in a large multigenerational
family with FTLD from Denmark. Nevertheless, the aberrant gene in this family has only
recently been identified [41]. It consists in a mutation of the splice acceptor site of exon 6 of
CHMP2B (charged multivescicular body protein 2B), which is part of the endosomal
ESCRTIII-complex. The change from G to C results in an alteration of the splice acceptor site
of exon 6, causing aberrant mRNA splicing of this transcript, which leads to the insertion of
201 base pairs of the intron between exons 5 and 6. In addition, a further transcript was
identified, resulting from the use of a cryptic splice site consisting of 10 base pairs from the 5
end of exon 6. Anyway, mutations in CHMP2B appear as a rare genetic cause of FTLD
mainly due to their rare frequency of occurrence, showing moreover that the CHMP2B locus
does not increase the risk for FTLD [42]
lastly, the first evidence of linkage with chromosome 9q21-22 comes from a study carried
out in families with Motor Neuron Disease (MND) and FTD [43]. Despite the evidence of
linkage to chr9q21-22 in several additional FTD-MND families, the gene responsible for the
disease in this locus has yet to be identified [44-46].
4. Sporadic AD
Risk genes are likely to be numerous, displaying intricate patterns of interaction with
each other as well as with non-genetic variables, and-unlike classical Mendelian (simplex)
disorders- exhibit no simple mode of inheritance. Mainly due to this reason, the genetics of
sporadic AD has been labeled complex [47]. The gene mainly related to the sporadic forms
of AD is the Apolipoprotein E (APOE) [48], which is located at chromosome 19q13.32 and
was initially identified by linkage analysis [49]. The relationship between APOE and AD has
been confirmed in more than 100 studies conducted in different populations. The gene has
three different alleles, APOE*2, APOE*3 and APOE*4. The APOE*4 allele is the variant
associated with AD. Longitudinal studies in Caucasian populations have shown that carriers
for one APOE*4 allele have a two-fold increase in the risk for AD [50]. The risk increases in
homozygous for the APOE*4 allele, and this allelic variant is also associated with an earlier
onset of the disease.
Several linkage studies have been performed, giving rise to additional candidate
susceptibility loci at chromosomes 1, 4, 6, 9, 10, 12 and 19. In particular, promising loci have
been found at chromosome 9 and 10 [51,52]. Recently, a wide genome analysis identified
variants at CLU (which encodes clusterin or ApoJ) on chromosome 8 and PICALM in
chromosome 11 associated with AD [53]. Data on CLU were replicated in an independent
study, which, in addition, demonstrated that CR1, encoding the complement component
(3b/4b) receptor 1and locate on chromosome 1, is associated with AD [54].
Also, a large number of candidate genes studies have been performed in order to search a
robust risk factor for the sporadic form of the disease. Several studies were mainly focused in
genes clearly involved in the pathogenesis of AD such as genes encoding for inflammatory
molecules or involved in the oxidative stress cascade, both considered major factors in AD
pathology . One of the strongest evidence of the role played by genetic variants in
inflammatory molecules to increase the risk of AD involves the Interleukin-1 (IL1) complex,
which is located at chromosome 2q14-21 and includes IL1-o, IL1-|, and IL1R antagonist
protein (IL-1Ra), all of which have significant polymorphisms found to be associated with
AD in several case- controls studies carried out in different populations [55-57]. Several
polymorphisms in IL-6, which is a potent inflammatory cytokine but has also regulatory
functions, have been investigated as well. The IL6 gene is located at chromosome 7p21 and
polymorphisms exist in the -174 promoter region and in the region of a variable number of
tandem repeats (VNTR), which is located in the 3untraslated region. Both of them have been
found associated with AD in case-controls studies [58,59]. Investigation of Tumor Necrosis
Factor-o (TNFo) polymorphisms was initiated because genome screening suggested a
putative association of AD with a region on chromosome 6p21.3, which lies within 20
centimorgans of the TNFo gene. Furthermore, other polymorphisms located in the promoter
region of TNFo have been associated with autoimmune and inflammatory diseases [60].
As with TNFo, investigations of the role of o-2macroglobulin (A2M) were initiated as a
result of screening studies of the genome. In this case, linkage was found in the region of
chromosome 1p, where A2M and its low-density lipoprotein receptor are found. Blacker et al.
[61] tested for association of polymorphisms with AD showing a strong involvement of this
gene in AD.
Moreover, polymorphisms in chemokines have been investigated with regard of
susceptibility of AD. In particular, Monocyte Chemoattractant Protein-1 (MCP-1) and
RANTES genes have been widely screened in different neurodegenerative diseases [62]. The
distribution of the A-2518G variant was determined in different AD populations with
concordant results [63,64] showing no evidence for association of this variant in AD
compared with controls. Moreover, Fenoglio et al. [63] found a significant increase of MCP-1
serum levels in AD carrying at least one G polymorphic allele. Therefore, the A-2518G
polymorphism does not seem to be a risk factor for the development of AD, but its presence
correlates with higher levels of serum MCP-1.
RANTES promoter polymorphism -403 A/G, found to be associated with several
autoimmune diseases, was examined in AD population, failing to find significant differences
between patients and controls [62]..
CCR2 and CCR5 genes, encoding for the receptors of MCP-1 and RANTES respectively,
have been also screened for association with AD. The most promising variants involve a
conservative change of a valine with an isoleucine at codon 64 of CCR2 (CCR2-64I) and a
32-bp deletion in the coding region of CCR5 (CCR5A32), which leads to the expression of a
non-functional receptor. A decreased frequency and an absence of homozygous for the
polymorphism CCR2-64I were found in AD, thus suggesting a protective effect of the
mutated allele on the occurrence of the disease [65]; conversely, no different distribution of
the CCR5A32 deletion in patients compared with controls were shown [65,66].
Another chemokine recently tested for susceptibility with AD is IP-10. A mutation
scanning of the gene coding region has been performed in AD patients searching for new
variants. The analysis demonstrated the presence of two previously reported polymorphisms
in exon 4 (G/C and T/C), which are in complete linkage disequilibrium, as well as a novel
rare one in exon 2 (C/T). Subsequently these SNPs have been tested in a wide case-control
study but no differences in haplotype frequencies were found [67].
Other genes under investigation are related to oxidative stress, a process closely involved
in AD pathogenesis. In this regard, genes coding for the nitric oxide synthase (NOS) complex
have been screened. The common polymorphism consisting in a T/C transition (T-786C) in
NOS3, previously reported to be associated with vascular pathologies, has been tested in AD,
but no significant differences with controls were found. Nevertheless, expression of NOS3 in
PBMC either from patients or controls seems to be influenced by the presence of the C
polymorphic allele, and is likely to be dose dependent, being mostly evident in homozygous
for the polymorphic variant. The influence of the polymorphism on NOS3 expression rate
supports the hypothesis of a beneficial effect exerted in AD by contributing to lower oxidative
damage [68].
An additional variant in NOS3 gene has been extensively investigated in AD patients,
although the results are still controversial. It is a common polymorphism consisting in a
single base change (G894T), which results in an aminoacidic substitution at position 298 of
NOS3 (Glu298Asp). Dahiyat et al. [69] determined the frequency of the Glu298Asp variant in
a two-stage case-control study, showing that homozygous for the wild-type allele were more
frequent in late onset AD. However, studies in other populations failed to replicate these
results [70-73]
More recently Guidi et al [74] correlated this variant with total plasma homocysteine
(tHcy) levels in 97 patients and 23 controls, demonstrating that the Glu/Glu genotype is
correlated with higher levels of tHcy, which represent a known risk factor for AD [75], and its
frequency was increased in AD patients [74]. Thus the mechanism by which this genotype
contributes to increase the risk in developing AD could be mediated by an increase of tHcy.
However, NOS-1 is the isoform most abundantly expressed in the brain. Recent genetic
analyses demonstrated that the double mutant genotype of the synonymous C276T
polymorphism in exon 29 of the NOS1 gene represents a risk factor for the development of
early onset AD [76], whereas the dinucleotide polymorphism in the 3UTR of NOS1 is not
associated with AD [77]. To date, the promoter region of NOS1, located approximately 200
kb upstream of these polymorphism, has not been investigated for susceptibility to AD. Due
to this reason and to further explore a possible association of NOS1 polymorphisms with AD,
the distribution of a functional polymorphisms and a variable number of tandem repeats
(VNTR) was analyzed very recently in a case-control study, which tested 184 AD patients as
well as 144 healthy subjects [78]. The functional variant considered is located in exon 1c,
which is one of the nine alternative first exons (named 1a-1i), resulting in NOS1 transcripts
with different 5-untraslated regions [79]. Three SNPs have been identified in exon 1c, but
only the G-84A variant displays a functional effect, as the A allele decreases the transcription
levels by 30% in in-vitro models [80]. Regarding exon 1f, a variable number of tandem
repeats (VNTR) polymorphism has been recently reported in its putative promoter region,
termed NOS1 Ex1f-VNTR. This VNTR is highly polymorphic and consists of different
numbers of dinucleotides (B-Q), which, according to their bimodal distribution, have been
dichotomized in short (B-J) and long (K-Q) alleles for association studies. Both Ex1c G-84A
and Ex1f-VNTR are associated with psychosis and prefrontal functioning in a population of
patients with schizophrenia [81]. Notably, both Ex1c and Ex1f transcripts are found in the
hippocampus and the frontal cerebral cortex, i.e. brain regions implicated in the pathogenesis
of schizophrenia as well as AD. The presence of the short (S) allele of NOS1 Ex1f-VNTR
represents a risk factor for the development of AD. The effect is cumulative, as in S/S carriers
the risk is doubled. Most interestingly, the effect of this allele is likely to be gender specific,
as it was found in females only. In addition, the S allele was shown to interact with the
APOE*4 allele both in males and females, increasing the risk to develop AD by more than 10
fold [78]. Thus, NOS1 seems to be a risk factor for AD, but only in female population. This
could be explained by a possible interaction with other genes or with additional
environmental factors present in females but not males. Epidemiological data indicate that the
prevalence of AD is increased in females compared with males. Therefore, it is conceivable
that different factors contribute to the development of the pathology in females rather than
males, including genetic ones.
5. Sporadic FTLD
The best well-known risk factor for late onset SAD, Apo E4, has also been considered as
a risk factor for sporadic FTLD. A number of studies suggested an association between FTLD
and APOE*4 allele [82-87]. Other Authors however,

did not replicate these data [88-90].
Recent findings demonstrated an association between the APOE*4 allele and FTLD in males,
but not females [91], possibly explaining the discrepancies previously reported. An increased
frequency of the APOE*4 allele was described in patients with SD compared to those with
FTD and PA [89].
Concerning the APOE*2 allele in the development of FTLD, heterogeneous data have
been obtained in different populations. Bernardi et al. [87] showed a protective effect of this
allele towards FTLD, whereas other Authors failed to do so [87]. Despite these results, a
recent meta-analysis comprising a total of 364 FTD patients and 2671 controls demonstrated
an increased susceptibility to FTD in APOE*2 carriers [93].
Besides pathogenic mutations, several polymorphisms have been reported to date, both in
MAPT and GRN. An association between Progressive Supranuclear Palsy (PSP) and a
dinucleotide repeat polymorphism in the intron between MAPT exons 9 and 10 was described
in 1997 [94]. The alleles at this locus carry 11 to 15 repeats. Subsequently, two common
MAPT haplotypes, named H1 and H2, were identified [95]. They differ in nucleotide
sequence and intron size, but are identical at the amino acid level. Homozygosity of the more
common allele H1 predisposes to PSP and Corticobasal Degeneration (CBD), but not to AD
or Pick Disease [95-96].
Regarding GRN, an association of a SNP located in the promoter and an increased risk to
develop FTLD in patients who did not carry causal mutations has recent been demonstrated
[97].
In addition, a known polymorphism in MCP-1 (A-2518G) has been shown to exert a
protective effects towards the development of FTLD [98], whereas NOS3 G894T
(Glu298Asp) and NOS1 C276T SNPs likely Increases the risk to develop FTLD [99-100].
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Chapter 9
The Cholinergic Neuron
in
Alzheimers Disease
Christian Humpel
*
and Celine Ullrich
Laboratory of Psychiatry and Exp. Alzheimers Research,
Department of Psychiatry and Psychotherapy,
Innsbruck Medical University, Austria
Abstract
Alzheimers disease (AD) is a chronic brain disorder characterized by cognitive
decline, neuronal and synaptic loss, beta-amyloid-containing plaques, neurofibrillary
tangles, inflammation and cerebrovascular damage. Numerous studies revealed that
cholinergic neurons in the basal forebrain (septum, diagonal band of Broca, basal nucleus
of Meynert) are affected in AD and a loss of acetylcholine directly correlates with
memory dysfunction. (1) We will give an overview on the cholinergic neurons in the
basal forebrain and discuss the role of the key enzyme choline acetyltransferase (ChAT).
(2) We review the protective role of nerve growth factor (NGF) to support the cholinergic
phenotype. (3) We demonstrate different in vitro and in vivo models, which are used to
study cholinergic CNS neurons. (4) We reconsider if cholinergic neurons degenerate in
AD or if cholinergic neurons only downregulate the key enzyme ChAT. (5) Finally, our
review will summarize recent therapeutic strategies on augmenting cholinergic
neurotransmission to improve or reverse cognitive deficits in AD. In summary our review
focuses on the cholinergic CNS neurons and their role in AD.
Alzheimers disease is a severe and chronic degenerative disorder characterized by a
progressive neurodegeneration, amyloid-containing plaques, neurofibrillary tangles, as
well as cognitive dysfunction. Cholinergic neurons in the basal forebrain are located in
six main central nuclei (Ch1-Ch6). The key enzymes for the cholinergic system, choline

*
Corresponding author: Dr. Christian Humpel, Department of Psychiatry and Psychotherapy, Anichstr. 35,
A-6020 Innsbruck, Austria, Phone: +43-512-504-23712; Fax: +43-512-504-23713, : christian.humpel
@i-med.ac.at
Christian Humpel and Celine Ullrich 190
acetyltransferase (ChAT) and acetylcholinesterase (AChE) can be used for
immunohistochemical staining and characterization of the system. Essential for the
development and survival of cholinergic neurons in the basal forebrain is the nerve
growth factor (NGF). The cholinergic neurotransmitter system in the basal forebrain is
severely affected in AD and loss of the neurotransmitter acetylcholine directly correlates
with cognitive dysfunction (Perry et al., 1981; Francis et al., 1985). Basic research of the
neuropathologic hallmarks and treatment strategies in AD is a fundamental goal, due to
immense costs of caring for patients with AD. The current review will highlight present
knowledge of the cholinergic dysfunction in AD and will demonstrate different models,
which are used to study AD, as well as possible therapeutic approaches.
The Cholinergic CNS Neuron
The cholinergic system plays a crucial role in learning, memory and cognition as well as
in the control of the cerebral blood flow, cortical plasticity, and sleep-wake cycle (Schliebs
and Arendt, 2006; Mufson et al., 2008). Choline acetyltransferase (ChAT) is the key enzyme
for the biosynthesis of acetylcholine (ACh) and is a good indicator for the functional activity
of cholinergic neurons (Oda, 1999). ChAT mediates the reaction involving the transfer of an
acetyl group from acetyl coenzyme A to choline at the synaptic endings of cholinergic
neurons (Figure 1A). Two receptors are responsible for the action of acetylcholine, the
nicotinic ACh receptors (nAChR) and the muscarinic ACh receptors (mAChR) (Pkski and
Klmn, 2008). The action of acetylcholine is determined by the activity of the enzyme
acetylcholinesterase (AChE), which is located presynaptically as well as postsynaptically and
cleaves acetylcholine into choline and acetate (Figure 1A) (Mesulam et al., 2002). In addition,
glial cells produce a second cholinesterase, butyrylcholinesterase (BChE), which has the
potency to substitute for acetylcholinesterase (Mesulam et al. 2002). After the enzymatical
cleavage of acetylcholine, choline is transported back from the synaptic cleft into the
presynaptic terminal and recycled.
The basal forebrain comprises six main central nuclei of cholinergic neurons (Ch1-Ch6),
which extend their cholinergic fibers in specific pathways (Figure 1B) (Mesulam and van
Hoesen, 1976; Mesulam et al., 1983; Mufson et al., 2003; Lucas-Meunier et al., 2003).
Cholinergic neurons from the septum (Ch1) and the vertical limb of the diagonal band of
Broca (Ch2) project only to the hippocampus, whereas pedunculopontinus nucleus (part of
Ch5) and laterodorsal tegmental nucleus (Ch6) from the brainstem project to the thalamus
(Mesulam et al., 1983; Mufson et al., 2003). Cholinergic nuclei from the lateral part of the
horizontal limb of the diagonal band of Broca (Ch3) project to the olfactory bulb (Mesulam et
al., 1983). The basal nucleus of Meynert (nBM) (Ch4) contains a population of large
cholinergic neurons and their fiber pathway innervates the entire cortex and amygdala (Wenk,
1997; Lucas-Meunier et al., 2003; Schliebs and Arendt, 2006). The homologous structure of
the basal nucleus of Meynert in human species conveys to the nucleus basalis magnocellularis
in the rat basal forebrain (Wenk, 1997; Lucas-Meunier et al., 2003). In addition, cholinergic
interneurons containing NADPH-diaphorase, GABA, calbindin and glutamate are found in
diverse brain areas, such as e.g. the striatum (Mufson et al., 2003; Schliebs and Arendt, 2006;
Lucas-Meunier et al., 2003).
The Cholinergic Neuron in Alzheimers Disease 191

Figure 1. Scheme of a cholinergic nerve terminal (A). Choline acetyltransferase (ChAT), enzyme for
the formation of acetylcholine (ACh), is anterogradely transported from the cell body to the presynaptic
nerve terminal. ChAT mediates the reaction of acetyl-coA and choline into ACh. The vesicular
acetylcholine transporter (VAChT) transports ACh into the synaptic vesicles. ACh is released into the
synaptic cleft and binds to postsynaptical ACh receptors. Acetylcholinesterase (AChE), located in the
presynaptic or postsynaptic membrane, terminates the reaction of ACh by enzymatically cleavage into
choline and acetate. Choline is recycled and transported back into the presynaptic terminal by choline
transporters. Metabolized acetate is taken up by glial cells. The postsynaptic cell (or adjacent glial cells)
secrete the nerve growth factor (NGF), which diffuses to presynaptically located NGF receptors, TrkA
or p75NTR. NGF stimulates the neuron as a "target-derived neurotrophic factor". Rat cholinergic
central pathways and nuclei (B). Cholinergic neurons are located in six main central central nuclei
(Ch1-Ch6), which project to different brain regions
Nerve Growth Factor and Cholinergic Neurons
Nerve growth factor (NGF) was the first discovered neurotrophin (Levi-Montalcini,
1987) and plays a key role in survival and phenotypic maintenance of cholinergic basal
forebrain neurons (Salehi et al., 2004). NGF activates various intracellular signaling pathways
via two membrane-bound receptors, the p75
NTR
(p75 neurotrophin receptor) and the TrkA
(tyrosine receptor kinase-A) (Yuen et al., 1996; Schindowski et al., 2008; Yuen et al., 1996;
Mufson et al., 2008; Schindowski et al., 2008). Binding of NGF to TrkA causes receptor
dimerization, which is endocytosed at the synapse and retrogradely transported to the cell
body (Rattray, 2001) (Figure 1A). NGF is synthesized as a precursor (proNGF), which is the
dominant form of NGF in the central nervous system (CNS) (Fahnestock et al., 2004; Allen
and Dawbarn, 2006). NGF is synthesized in the cortex and hippocampus and supports
cholinergic neurons as a target-derived neurotrophic factor (Salehi et al., 2004; Allen and
Dawbarn, 2006; Schindowski et al. 2008). The role of proNGF and mature NGF appears to be
contradictory: while mature NGF maintains survival and function to certain neuronal
populations, proNGF triggers cell death through p75
NTR
(Friedman, 2000). However, various
studies suggests that proNGF binds TrkA and promotes neuronal survival and neurite
outgrowth similar to mature NGF, but is probably less active than the mature NGF
(Fahnestock et al., 2004; Rattenholl et al., 2001). Thus, it is indicated that NGF may elicit cell
survival or cell death, depending on receptor and signaling pathways (Friedman, 2000).
Nevertheless, in the absence of NGF, cholinergic neurons show cell shrinkage, reduction in
fiber density and downregulation of transmitter-associated enzymes, e.g. ChAT or AChE,
resulting in a decrease of cholinergic transmission (Svendsen et al., 1991; Weis et al., 2001;
Humpel and Weis, 2002).
Various studies demonstrated increased levels of NGF in the cortex and decreased levels
of NGF in the basal forebrain in AD brains (Schindowski et al., 2008; Mufson et al., 2008;
Allen and Dawbarn, 2006). A marked reduction of TrkA has been shown in the basal
forebrain and in the cortex of AD brains (Schindowski et al., 2008; Mufson et al., 2003), thus
supporting that a dysfunctional retrograde transport could lead to an accumulation of NGF in
the cortex resulting in loss of trophic support for cholinergic neurons (Schindowski et al.,
2008).
In Vitro and in Vivo Models to Study
Cholinergic Neurons
The implication of the cholinergic system in the pathology of AD is an important issue in
AD research and the cholinergic phenotype can be studied in different in vitro and in vivo
models.
Dissociated primary cholinergic neurons, which were separated from diverse brain
regions, are widely used to study the cholinergic phenotype. The effects of trophic
interactions (Hefti et al., 1985; Wainer et al., 1991), toxic stimuli (Heaton et al., 1994) or
pharmacological substances (Bailey and Lahiri, 2010, Bennett et al., 2009) on cholinergic
neurons provide insights into the cholinergic system. The organotypic brain slice is an
outstanding in vitro model to study neurons in an almost intact brain environment. Brain
slices are able to maintain the survival of different cell types, the cytoarchitecture of the
tissue, the connections between cells and neuronal properties (Duff et al., 2002). The
organotypic brain slice model has been first introduced by Ghwiler and colleagues (Ghwiler
and Hefti, 1984, 1997) and was modified by Stoppini et al. (1991). In slices, individual cells
are in close contact and do not lose density-dependent regulatory mechanisms, three
dimensional architecture as well as tissue specific transport and diffusion probabilities. Thus,
slice cultures provide an easily accessible experimental model for studies of toxic,
degenerative and developmental changes in the brain (Zimmer et al., 2000). The
determination of cholinergic neurons is performed by immunohistochemical staining against
the enzyme ChAT (Figure 2C) and the addition of NGF supports the survival of cholinergic
neurons (Figure 2A,D) (Weis et al., 2001; Humpel and Weis, 2002). Single organotypic brain
slices of the nBM or co-cultures of the nBM together with cortex have been used to study
cholinergic neurons and the effects of aging (Marksteiner and Humpel, 2007), NGF
withdrawal (Weis et al., 2001; Humpel and Weis, 2002) and other neuroprotective (Zassler et
al., 2005) or neurotoxic (Zassler et al., 2003; Ullrich et al., 2009) substances.
Several in vivo models have been used to determine the mechanisms of cholinergic
dysfunction, cell death or recovery processes, such as injections of toxins (Smith, 1988; Waite
et al., 1995; Nilsson et al., 1992; Weinstock, 1997; Hanin, 1992), genetically modified
animals (Smeyne et al., 1994; Capsoni et al., 2006), fimbria-fornix transections (Hefti, 1986;
Widmer et al., 1993) or radiofrequency (Dubois et al., 1985; Hepler et al., 1985). The
development of cholinodeficient animal models is an important tool for studying the
dysfunction of cholinergic neurons in the basal forebrain. The injection of ethylcholine
aziridinium (AF64A), a toxin which has a structural similarity to choline, affects reference
and working memory in rats and specifically disrupts cholinergic nerve terminals (Weinstock,
1997; Hanin, 1992). A selective lesion of cholinergic nBM neurons can be performed by
injection of 192 IgG-saporin, which consists of the ribosome-inactivating protein saporin
conjugated to 192 IgG, a monoclonal antibody to the rat p75
NTR
. This immunotoxin produces
selective, dose-dependent and long-lasting cell death among p75
NTR
-positive cholinergic basal
forebrain neurons and leads to an impaired performance in learning and memory tasks (Waite
et al., 1995; Wenk, 1997; Schliebs and Arendt, 1998; Gu et al., 2000; Nilsson et al., 1992).
Additionally, fimbria-fornix transections represent a well characterized method to study the
effects of NGF on the survival of cholinergic neurons after axotomy (Hefti, 1986; Widmer et
al., 1993). On the other hand, radiofrequency current is used to destroy neuronal perikarya
and fibers and leads to lesions in the nBM (Dubois et al., 1985; Hepler et al., 1985).
In the past decades, the development of transgenic animal models of AD provides an
excellent opportunity to study the underlying neurobiological mechanisms of AD-related
cognitive deficits. The establishment of mice lacking NGF producing cells can be
characterized by severe loss of sensory and sympathetic neurons (Schliebs and Arendt, 1998;
Winkler et al., 1998). Mice lacking TrkA receptors demonstrate a substantial decrease in
AChE immunoreactive fibers in the cholinergic basal forebrain (Smeyne et al., 1994). In
AD11 anti-NGF mice, which express transgenic antibodies neutralizing NGF, a progressive
cholinergic neurodegeneration is seen, accompanied by intracellular neurofibrillary tangles,
deposition of amyloid peptide and an impaired spatial memory (Capsoni et al., 2006).

Figure 2. Cholinergic neurons in organotypic brain slices. Cholinergic neurons were incubated with (A,
C) or without (B) 10 ng/ml nerve growth factor (NGF) for two weeks and stained
immunohistochemically for the enzyme choline acetyltransferase (ChAT) using a chromogenic
substrate. Note a marked neurodegeneration of cholinergic neurons when slices where incubated
without NGF (B). Cholinergic ChAT-like immunoreactivity (green) is cytoplasmic as seen by nuclear
DAPI (blue) co-staining and these neurons are embedded in a glutamine synthase positive (red)
astroglial network (C). The number of cholinergic neurons remains stable when incubated for up to 6
weeks with NGF (filled squares), but declines markedly without NGF (filled circles). (D). The question
(?) was if NGF may enhance the cholinergic ChAT+ neurons when added after 2-weeks to slices
incubated without NGF. Our data show that the number of ChAT+ neurons did not increase, suggesting
cell death of cholinergic neurons and not only down-regulation of ChAT
The Impairment of the Cholinergic System in AD
A significant reduction of cholinergic basal forebrain neurons, as well as a substantial
loss of the cholinergic innervation in the cerebral cortex has been observed in AD brains
(Mufson et al., 2003; Mufson et al., 2008; Vogels et al., 1990; Mesulam, 2010). Furthermore,
a progressive memory impairment and other cognitive dysfunctions characterize the clinically
outcome of AD (McKhann et al., 1984). The concentrations of acetylcholine and ChAT are
markedly reduced in the frontal, parietal, temporal, and visual cortices (Davies and Maloney,
1976; Bowen et al., 1976; Ks et al., 1997) and in the hippocampus (Oda, 1999). Similarly,
the AChE-positive fibers are significantly declined, most severe in the temporal lobes (Geula
and Mesulam, 1989; Ks et al., 1997). It has been shown, that a loss of 55% of the cortical
cholinergic fibers was detected in AD brains (Geula and Mesulam, 1996). Furthermore,
various studies demonstrated a decreased number of nicotinic and muscarinic (M2) ACh
receptors in AD brains, in contrast to the maintenance of postsynaptic muscarinic (M1, M3)
receptors (Francis et al., 1999; Mesulam, 2010). This is in line with a previous study by
Drachman and Leavitt (1974), demonstrating that patients treated with the muscarinic
antagonist scolopamine exhibits similar features to that seen in AD brains. This indicates for
the first time that cortical cholinergic neurotransmission plays a crucial role in memory
function and in AD pathology. On the contrary, the numbers of ChAT-positive cells in the
putamen and the caudate nucleus are not affected in AD (Ks et al., 1997; Mesulam, 2010),
suggesting that this disorder does not affect the entire cholinergic basal forebrain system.
The causes for cholinergic degeneration in AD brains are not fully clear, whereas various
hypotheses exist. It has been demonstrated that cholinergic neurons in the Ch4 area are the
most sensitive neurons to age-related neurofibrillary degeneration (Sassin et al., 2000) and
that a decline in the number, size, or function of cholinergic neurons may be responsible for
the cognitive impairments associated with aging and AD (Wenk, 1997). A correlation of the
loss of cholinergic neurons of the basal forebrain and the extent of cognitive impairment
revealed, that at least 30% of the cholinergic basal forebrain neurons need to be degenerated
to see clinical symptoms (Schliebs and Arendt, 2006). Neuropathological studies described
morphological alterations in cerebral capillaries, dysfunction of the neurovascular unit and
ischemic infarcts in AD patients (Iadecola, 2004). Cholinergic neurons are closely associated
with cerebral blood vessels of the blood-brain barrier (BBB) and tend to degenerate due to
vascular dysregulation (Iadecola, 2004). Considering, Selkoe (2002) suggested that
cholinergic hypofunction in AD brains is caused by synaptic dysfunction initiated by
progressive accumulation of toxic beta-amyloid. Moreover, it is not known, if the cholinergic
depletion is an early or late event in AD. On the one hand, Perry and colleagues (1981)
demonstrated that ChAT activity is associated with early stages of AD pathology. Otherwise,
it was reported that the loss of cortical cholinergic function and cholinergic markers
correlated with severity of neuropathological lesions in AD (Davis et al., 1999).
Cholinergic neurons are detected by immunohistochemical analysis for the key enzyme
ChAT and a decline directly correlates with loss of cholinergic neurons (Perry et al., 1981;
Francis et al., 1985; Schliebs and Arendt, 2006). However, the question remains if cholinergic
neurons really die or only downregulate ChAT and cannot be visualized. It is well established
that a fimbria-fornix transection leads to a shrunken morphology and a reduced number of
ChAT-positive neurons (Naumann et al., 1992, 1994; Hefti, 1986). However, the number of
ChAT-positive neurons increases after in vivo axotomy within 6 months (Naumann et al.,
1994) suggesting the possibility of regeneration of transmitter synthesis within the cholinergic
neurons. In vitro (Hefti et al., 1985) and in vivo (Naumann et al., 1992) studies demonstrated
that NGF application results in reversal of ChAT expression and cell atrophy. Moreover,
Widmer et al. (1993) demonstrated that ChAT expression declines in transected cholinergic
neurons suggesting involvement of p75
NTR
after axotomy. Additionally, it was demonstrated
that dissociated cholinergic neurons cultured with or without NGF showed that NGF
enhanced cholinergic neuronal survival, but did not induce cellular ChAT activity (Hatanaka
et al., 1988). In organotypic bran slices, axotomized cholinergic neurons cultured without
exogenous NGF exhibited reduced and shrunken ChAT-positive neurons (Figure 2B). This
reduction can be prevented by application of recombinant NGF (Figure 2A) (Zassler et al.,
2005; Humpel and Weis, 2002). Furthermore, it has been demonstrated that NGF did not
stimulate the cholinergic phenotype in organotypic brain slices of the nBM, but rescued the
remaining cholinergic neurons from cell death (Humpel and Weis, 2002). Thus, this raises the
question if the cholinergic neurons in AD brains undergo cell death or downregulate their
ChAT activity, due to a disturbed NGF homeostasis.
Whereas, it is not known if the cholinergic dysfunction is the primary characteristic in
AD brains or if the deposition of beta-amyloid and neurofibrillary tangles promote the
cholinergic depletion. A link between cholinergic dysfunction and amyloid precursor protein
(APP) processing is demonstrated by co-localization studies of AChE with beta-amyloid
deposits in AD brains (Mesulam, 1986). This was supported by cell culture studies, showing
an upregulation of APP expression after NGF treatment (Villa et al., 2001). Moreover, only
little is known regarding the role of the impaired cholinergic system in the phosphorylation of
tau. Nonetheless, it has been shown that activation of mAChR decreased tau phosphorylation
in various studies (Sadot et al., 1996; Fisher, 1997; Rubio et al., 2006).
Therapeutic Strategies in AD
The cholinergic system has become an important target for therapeutic interventions, due
to its obvious role in the pathogenesis of AD. Diverse therapeutic strategies are in the focus of
intense research, such as the improvement of the cholinergic function in the basal forebrain,
as well as the maintenance of the decline in memory and cognitive function.
So far the most important therapeutic approach is to increase the availability of
acetylcholine by inhibiting AChE and thus to enhance cholinergic neurotransmission
(Blennow et al., 2006; Mufson et al., 2008). The efficacy of acetylcholinesterase inhibitors
donezepil, rivastigmine, and galantamine has been extensively studied (Darreh-Shori and
Soininen, 2010; Nordberg et al., 2009), but acetylcholinesterase inhibitors only temporarily
mitigate some of the symptoms in AD. Side effects implicating the gastrointestinal system
(e.g. nausea, vomiting, and diarrhoea, as well as fatigue or loss of appetite, limit their use
(Mufson et al., 2008). Whereas, cholinesterase inhibitors only counteract AD-related
deficiencies in cortical acetylcholine loss, but are not capable of slowing or reversing
cholinergic neuronal loss. Alternatively, the cholinergic neurotransmission can be enhanced
by activation of presynaptic nicotinic cholinergic receptors through appropriate nicotinic
agonists (Oddo and LaFerla, 2006; Buckingham et al., 2009) or by the stimulation of
muscarinic ACh receptors through the use of M1 agonists (Avery et al., 1997). Furthermore,
recent studies reported a relationship between smoking, nicotinic cholinergic signaling and
AD, suggesting that nicotinic treatments improve cognitive functions (Sabbagh et al., 2002;
Levin et al., 2006).
On the other hand, NGF delivery to the brain of patients appears to be an emerging
potential therapeutic approach in AD. It is important to note, that NGF does not cross the
BBB and must be delivered directly into the CNS. A continuous intracerebroventricular NGF
injection has been tested in clinical trials with AD patients (Olson, 1993; Seiger, 1993;
Eriksdotter Jnhagen et al., 1998; Winkler et al., 1998; Mufson et al., 2008). However, the
NGF application was stopped because undesirable side-effects occurred, such as weight loss,
back pain, pain from stimulation of dorsal root ganglion nociceptive neurons, sympathetic
axon sprouting in the cerebral vasculature and Schwann cell hyperplasia (Eriksdotter
Jnhagen et al., 1998; Tuszynski, 2007). An advanced method for NGF delivery into the AD
brain could be gene therapy, where genetically modified cells which express e.g. NGF or
ChAT are grafted into the brain (Tuszynski et al., 2005; Smith et al., 1999) or in combination
with e.g. adeno-associated virus (AAV) vectors systems (Klein et al., 2000; Blesch et al.,
2005; Mandel et al., 2006). Studies performed in aged monkeys using fibroblasts as vehicles
of ex vivo NGF gene delivery demonstrated reversal of neuronal atrophy and restoration of
cortical cholinergic inputs (Smith et al., 1999). In addition, studies were performed to deliver
NGF, coupled to a transport protein across the BBB, such as e.g. transferrin-NGF coupled
systems (Granholm et al., 1993; Begley, 2003) or the use of cell-based BBB-carrier systems,
such as blood cells containing incorporated drugs (Begley, 2003). The intranasal route of
administration of NGF could provide an alternative to ICV infusion and gene therapy (de
Rosa et al., 2005; Covaceuszach et al., 2009). Furthermore, a recent in vitro study has shown
that NGF loaded monocytes may migrate through a simple BBB and deliver NGF into the
brain (Bttger et al., 2010). There will be a need to develop non-invasive delivery methods
due to the large number of AD patients. In the case of NGF, this protein must be delivered
early before degeneration of cholinergic neurons occurs and must be delivered continuously
through life.
In summary, cholinergic dysfunction plays an important role in AD and loss of
acetylcholine in cortex and hippocampus correlates to cognitive decline. Therapeutic
strategies are explored to enhance acetylcholine levels in the brain or to counteract cell death
of cholinergic neurons.
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Chapter 10
Retinal Neurodegeneration Is an Early
Event in the Pathogenesis of Diabetic
Retinopathy: Therapeutic Implications
Rafael Sim* and Cristina Hernndez
Diabetes and Metabolism Reseach Unit, Institut de Recerca Hospital Universitari
Vall DHebron and CIBER for Diabetes and Associated Metabolic Diseases
(CIBERDEM, Instituto de Salud Carlos III), Barcelona, Spain
Abstract
Diabetic retinopathy (DR) remains the leading cause of blindness among working-
age individuals in developed countries. Although tight control of both blood glucose
levels and hypertension are essential to prevent or arrest progression of the disease, the
recommended goals are difficult to achieve in many patients and, consequently, DR
develops during the evolution of the disease. Therefore, new therapeutic strategies based
on the understanding of the pathophysiological mechanisms of DR are needed.
DR has been classically considered to be a microcirculatory disease of the retina due
to the deleterious metabolic effects of hyperglycemia per se, and the metabolic pathways
triggered by hyperglycemia. However, before any microcirculatory abnormalities can be
detected in ophthalmolscopic examination, retinal neurodegeneration is already present.
The two main features of retinal neurodegeneration are apoptosis and glial activation.
Most of the information regarding retinal neurodegeneration has been obtained from rats
with streptozotocin-induced diabetes (STZ-DM). Streptozotocin (STZ) is a potent
neurotoxic agent and is able to produce neural degeneration. Therefore,
neurodegeneration observed in rats with STZ-DM could be due to STZ itself rather than
the metabolic pathways related to diabetes. However, the recent observation that both
apoptosis and glial activation also occur in the retina of diabetic patients, even before any

*
Corresponding author: Diabetes and Metabolism Research Unit., Institut de Recerca Hospital Universitari Vall
dHebron., Pg. Vall dHebron 119-129. 08035 Barcelona. Spain, Telephone: 34 934894172. FAX: 34
934894032, E-mail: rsimo@ir.vhebron.net
Rafael Sim and Cristina Hernndez 204
microvascular abnormality could be detected in ophthalmologic examination, reinforces
the concept that neurodegeneration is a crucial pathogenic factor of DR.
chromatic discrimination and contrast sensitivity. These alterations can be detected by
means of electrophysiological studies in diabetic patients with less than two years of
diabetes duration, that is before microvacular lesions can be detected in ophthalmologic
examination. In addition, neuroretinal degeneration subsequently initiates and/or activates
several metabolic and signaling pathways which participate in the microangiopathic
process, as well as in the disruption of the blood-retinal barrier (a crucial element in the
pathogenesis of DR). Therefore, the study of the mechanisms that lead to
neurodegeneration will be essential for identifying new therapeutic targets in the early
stages of DR.
Introduction
Diabetic retinopathy (DR) is the leading cause of blindness in working-age individuals in
developed countries [1, 2]. The tight control of blood glucose levels and blood pressure are
essential in preventing or arresting DR development. However, the therapeutic objectives are
difficult to achieve and, in consequence, DR appears in a high proportion of patients. When
DR appears, laser photocoagulation remains the main tool in the therapeutic armamentarium.
The objective of laser photocoagulation is not to improve visual acuity but to stabilize DR,
thus preventing severe visual loss. When laser photocoagulation is indicated in time, the risk
of blindness is reduced by 90% in the following 5 years, and the loss of visual acuity is
reduced by 50% in those patients with macular edema. However, timely indication is often
passed and, therefore, the effectiveness of laser photocoagulation in current clinical practice is
significantly lower. In addition, laser photocoagulation destroys a part of the healthy retina
and, in consequence, side effects such as loss in visual acuity, impairment of either dark
adaptation and colour vision, and visual field loss may appear. Vitreo-retinal surgery could be
indicated in advanced stages of DR (ie., hemovitreous, retinal detachment). However, this
therapeutic option requires a skilful team of ophthalmologists, is expensive, and fails in more
than 30% of cases. With this scenario, it seems clear that new treatments based on the
physiopathological knowledge of DR are needed.
DR has been classically considered to be a microcirculatory disease of the retina due to
the deletereous metabolic effects of hyperglycemia per se, and the metabolic pathways
triggered by hyperglycemia (polyol pathway, hexosamine pathway, DAG-PKC pathway,
advanced glycation end-products [AGEs] and oxidative stress). However, before any
microcirculatory abnormalities can be detected under ophthalmoscopic examination, retinal
neurodegeneration must be already present. In other words, retinal neurodegeneration is an
early event in the pathogenesis of DR which antedates and participates in the microcirculatory
abnormalities that occur in DR [3-6]. Therefore, the study of the mechanisms that lead to
neurodegeneration will be essential for identifying new therapeutic targets in the early stages
of DR. This is the main aim of the present project.
Retinal Neurodegeneration Is an Early Event in the Pathogenesis of Diabetic 205

Figure 1. Overexpression of GFAP in Mller cells (glial activation) in the retina from a representative
diabetic donor (right panel) in comparison with non-diabetic donor (left panel). ONL: outer nuclear
layer. INL: Inner nuclear layer GCL: Gnglionar cell layer
Neurodegeneration as an Early Event in the Pathogenesis of DR
This concept was first introduced by Barber et al. [3]. These authors observed that one
month after inducing diabetes in rats by using streptozotocin there was a high rate of
apoptosis (TUNEL positive cells) in the neuroretina without a significant apoptosis in
endothelial cells. In the same paper the authors compared the retinas from diabetic (n=2) vs.
nondiabetic (n=3) donors and found a higher rate of apoptosis in the neuroretina from diabetic
donors, even in the case of a diabetic donor without microvascular abnormalities. These
findings have been further confirmed in experimental models. In addition, it has been
demonstated that, apart from apoptosis, another of the features of retinal neurodegeneration is
glial activation [4-8]. Our research group has been able to demonstrate that both apoptosis
and glial activation also occur in the retina of diabetic patients (n=10) and precede
microvascular abnormalities [9, 10] (Figure 1). In addition, these changes are also present in
retinal explants cultured with a media with a high content of AGEs [11]. These findings
suggest not only that neurodegeneration is an early event in the natural history of DR but also
that it could play a crucial role in its pathogenesis.
chromatic discrimination and contrast sensitivity. These alterations can be detected by means
of electrophysiological studies in diabetic patients with less than two years of diabetes
duration, that is before microvacular lesions can be detected under ophthalmologic
examination [6, 12, 13]. In addition, neuroretinal degeneration will initiate and/or activate
several metabolic and signalling pathways that will participate in the microangiopathic
process, as well as in the disruption of the blood-retinal barrier (a crucial element in the
pathogenesis of DR). Nevertheless, these metabolic pathways remain to be characterized.
The mechanisms involved in DR neurodegeneration are poorly understood. In addition, it
is unknown which of the two primordial pathological elements (apoptotis or glial activation)
is the first to appear and is, in consequence, the primary event. Nevertheless, it seems that
these diabetes-induced changes occur in the early stages of DR and that they are closely
related.
P38 MAP kinase activation has been involved in the apoptotic retinal cell death that
occurs in DR [6, 12, 13]. Apoptosis can be secondary to the classic mechanism related to
caspase activation or can be due to a caspase-independent pathway. In this regard, it has been
shown that high glucose concentrations can induce neuroretinal apoptosis without an increase
in the caspase pathway. This caspase-independent pathway is at least in part mediated by the
translocation of the apoptosis inducing factor (AIF) from the mitochondria to the nuclei [14].
Glial degeneration consists of astrocyte degeneration and the hypertrophy and
hyperplasia of Mller cells. These activated or gliotic cells can be identified by means of
immunohistochemical procedures as cells with prolongations similar to end-feeds that cross
through all the retinal layers and reach the limiting inner membrane (Figure 1). However,
although hypertrophic these cells are dysfunctional. In normal conditions Mller cells act as
suppliers of retinal neurons and endothelial cells (which are the main constituents of the inner
blood-retinal barrier). One of the primary functions of Mller cells is the absorption of fluid
through potassium channels (mainly the Kir4.1), which allows transcellular water transport.
Moreover, they play a key role in the uptake of potential neurotoxic metabolites such as
glutamate and GABA [15-17]. Therefore, Mller cell dysfunction can precipitate neuroretinal
apoptosis due to the accumulation of water (retinal edema) and neurotoxic metabolites.
Moreover, glial cells are in close contact with endothelial cells of capillaries and, in
consequence, glial degeneration could contribute to inner BRB disruption [18]. However, this
possibility has still to be confirmed and the potential mechanisms involved are far from being
elucidated.
Experimental Models to Study Retinal
Neurodegeneration in the Setting of DR
The experimental model currently used to study retinal neurodegeneration in DR is the
rat with STZ-DM. In this model the presence of neural apoptosis and glial reaction has been
detected one month after starting diabetes [3, 6-8]. In addition, electroretinographic
abnormalities have been shown two weeks after inducing diabetes [19]. Retinal ganglion cells
(RGCs) are the earliest cells affected and with the highest rate of apoptosis [20]. However, an
elevated rate of apoptosis has also been observed in the outer nuclear layer (photoreceptors)
and in the retinal pigment epithelium (RPE) [21, 22].
The mouse has been much less frequently used than the rat as an experimental model for
the study of DR and retinal neurodegeneration. This is because it is more resistant to the STZ
effect (mice need 35 doses of STZ to induce diabetes whereas in rats one dose is enough), it
has a lower eye cup and presents higher resistance to the development of DR lesions. This
relative protection to developing pathological lesions related to diabetes can be partly
attributed to lower activity in the polyol pathway in comparison with rats [8, 23].
The interpretation of the results of retinal neurodegeneration in murine models with
diabetes induced by STZ is hampered by the neurotoxic effect of STZ. It is worthy of mention
that pathological changes to the brain after intraventricular injection of STZ are very similar
to the neurodegeneration reported in DR [24, 25]. Therefore, neurodegeneration (apoptosis +
glial activation) observed in rats with STZ-DM could be due to STZ itself rather than the
metabolic pathways related to diabetes. For this reason, it would be advisable to use murine
models with a spontaneous development of diabetes or at least experimental models in which
diabetes has not been induced by a neurotoxic drug.

Figure 2. Retinal neurodegeneration observed in a C57BL/KsJ-db/db diabetic mouse (right panel) at 4
weeks after starting diabetes in comparison with a C57BL/KsJ non-diabetic mouse (left panel)
Because of its great potential for genetic manipulation, the mouse offers a unique
opportunity to study the molecular pathways involved in disease development. Among mice,
C57BL/KsJ-db/db is the model that best reproduces the neurodegenerative features observed
in patients with DR. C57BL/KsJ-db/db mice carry a mutation in the leptin receptor gene, and
they are a model for obesity-induced type 2 diabetes. They develop hyperglycemia starting at
~8 weeks of age as a result of excessive food consumption. It is noteworthy that they present
an abundant expression of aldose-reductase in the retina (this is an important differential trait
from other mouse models). Therefore C57BL/KsJ-db/db is a good model for investigating the
underlying mechanisms of retinal neurodegeneration associated with diabetes and for testing
new drugs. We have recently had the opportunity to verify retinal neurodegeneration in this
model at 4 weeks after starting diabetes (Figure 2)
Therapeutic Candidates to be Explored
As mentioned in the introduction, it is essential to find new drugs for DR treatment. In
this regard, the results of two seminal studies: the FIELD study on DR [26] and the DIRECT
programme [27, 28] have been recently published. These studies have demonstrated that
fenofibrate (FIELD study) and candesartan (DIRECT programme) have beneficial effects in
non-advanced DR independently of their primary actions (lipid lowering in the case of
fenofibrate and lowering blood pressure in the case of candesartan). The mechanisms
involved in these beneficial effects remain to be explored.
Fenofibrate
It has been shown that fenofibrate exerts a neuroprotective action in a murine model of
cerebral ischemia [29, 30], Parkinsons disease [31] and cerebral trauma [32]. Antioxidant,
antiinflammatory and antiapoptotic mechanisms have been involved. However, there are no
studies in DR.
Angiotensin II receptor blockers (ARBs)
Angiotensin and its receptors are overexpressed in the retina of diabetic patients and
ARBs exert various pleiotropic effects within the retina (unrelated to blood pressure) that
explain their therapeutic effectiveness in DR. Regarding neuroprotection it has been recently
reported that candesartan (the ARB with the better diffusion across the blood-brain barrier)
has a neuroprotective effect after brain focal ischemia [33, 34]. In addition, telmisartan and
valsartan inhibit the synaptophysin degradation that exists in the retina of a murine model of
DR [35]. Furthermore, valsartan is able to prolong the survival of astrocytes and reduce glial
activation in the retina of rats with hypoxia-induced retinopathy [36]. Taken together it seems
that neuroprotection could be a relevant mechanism involved in the beneficial effects of
candesartan (and eventually other ARBs) in DR. In fact, it has been recently shown that
losartan has neuroprotective effects in the retina of rats by re-establishing oxidative redox and
the mitochondrial function [37]. Nevertheless, there are no specific studies on the
neuroprotective effects of ARBs in patients with DR.
Other Candidates
Apart from fenofibrate and ARBs there are other potential therapeutic candidates. Based
on our background we have selected the following:
Somatostatin
Both neuroretina and retinal pigment epithelium produce somatostatin (SST), a peptide
with antiangiogenic and neuromodulatory actions. These effects are mediated by its binding
to 5 receptors which are also expressed in the retina. We have detected a marked deficit of
SST (mainly due to SST-28) in the vitreous fluid of diabetic patients [38, 39]. In addition, we
have also demonstrated that this deficit also exists in the retina at the early stages of DR and
is associated with retinal neurodegeneration [9, 40]. Furthermore, intravitreal delivery of SST
has been proposed as a new therapeutic approach for DR [41]. Recently, it has been shown
that SST and SST analogues with selective high affinity to SST receptors 2 and 5 protect the
retina from excitotoxicity induced by (RS)-alpha-amino-3-hydroxy-5-methyl-4-
isoxazolepropionic acid hydrobromide in rats [42, 43]. However, to the best of our knowledge
there are no studies evaluating the neuroprotective effect of SST in DR.
Erythropoietin
In previous studies we have demonstrated that in DR there is an increase in erythropoietin
(Epo) production by the retina that is unrelated to hypoxia and occurs in the early stages of
the disease [44-46]. Since Epo has neuroprotective properties, we have suggested that this
increase detected in the retina of diabetic patients could be contemplated as a compensatory
mechanism. In this regard, there is growing evidence that intravitreous or direct delivery of
Epo to the retina can prevent retinal neurodegeneration and the breakdown of the blood-
retinal barrier (BRB) [47, 48]. In addition, Epo protects retinal pigment epithelial cells from
oxidative damage [49, 50]. Nevertheless, there are no data on this issue in murine models
with spontaneous diabetes.
Ascorbic acid
We have recently compared by metabonomic analysis the metabolite profiles of vitreous
humours of diabetic patients with proliferative diabetic retinopathy (PDR) and non-diabetic
subjects. In this study we have detected a marked reduction of ascorbic acid levels in the
vitreous fluid in patients with PDR (Invest Ophthalmol Vis Sci first revision-).
Acid ascorbic (AA), which is an essential substance in humans, acts as

a cofactor in the
enzymatic biosynthesis of collagen, catecholamines,

and peptide neurohormones [51].
However, one of its most important functions is to act as an antioxidant and/or free

radical
scavenger [51-53]. Although most animals can synthesize vitamin C from glucose, humans
can only acquire the vitamin from dietary sources because they lack gluconolactone oxidase,
the enzyme required for AA biosynthesis. Vitamin C exists in two major forms. The charged
form, ascorbic acid (AA), is taken into cells via sodium-dependent facilitated transport. The
uncharged form, dehydroascorbate (DHA), enters cells via glucose transporters (GLUT) and
is then converted back to AA within these cells. Retinal cells appear to be dependent on
GLUT-1 transport of DHA rather than sodium-dependent AA uptake [54]. DHA uptake
through facilitative glucose transport is competitively inhibited by D-glucose. In fact, the
molecule of AA is very similar to D-glucose. Therefore, it could be postulated that chronic
hyperglycemia of long-standing diabetes reduces DHA transport via GLUT1 at the blood
retinal barrier (BRB). Exclusion of DHA from cells by hyperglycemia deprives the cells of
the central antioxidant action of AA, thus favoring the accumulation of reactive oxygen
species [55].

It should be noted that the retina is the only neural tissue that has a direct and
frequent exposure to light, thus leading to free radical production due to photo-oxidation
which becomes extremely toxic to retinal cells [56]. AA is present in the retina at a high
concentration

compared with its presence in other humans organs, and it is able to protect the
retina against

oxidative damage [51, 56, 57]. In fact, we have found ~20 fold higher levels of
AA in the vitreous fluid than in serum (unpublished results). Given that oxidative stress is a
key factor in the pathogenesis of DR, the significant lower levels of AA detected in the
vitreous fluid of PDR patients, point to this deficit as a crucial factor in determining DR
development. AA is a required cofactor for several intracellular hydroxylases, including
proline hydroxylase and dopamine hydroxilase.

Therefore, AA deficit could also participate in
the impairment of neuropetide production and, therefore, in neurodegeneration. In this regard,
it has been reported that AA added to cultures of SH-SY5Y cells (cells derived from
neuroblastoma) dramatically reduces the apoptosis induced by oxidative stress and decreases
beta-amyloid protein production [58]. At present there are no published studies on the
effectiveness of ascorbic acid as a neuroprotective agent in DR. In addition, it has been
demonstrated that AA has antiangiogenic properties [59, 60] and, consequently, the lower
levels that exist in diabetic patients can contribute to neovascularization, the hallmark of
PDR.
There are two main mechanisms which account for the lower levels of AA detected in the
vitreous fluid of PDR patients: First, competitive inhibition mediated by hyperglycema in AA
transport from systemic circulation to the retina; Second, AA consumption that exists in the
diabetic retina in order to compensate the elevated degree of oxidative stress. Taken together,
AA can be contemplated as a new therapeutic target. Prospective trials using diet supplements
of vitamin C for preventing or arresting DR, and experimental studies addressed to increasing
AA transport across BRB in the presence of hyperglycemia are needed.
Conclusion
The two most common forms of diabetes (type I and type II) are increasing at alarming
rates in developed countries, and the complications associated with diabetes impose enormous
burdens on health care systems. It should be emphasized that DR remains a leading cause of
blindness in the working-age population and represents a major concern for patients with
diabetes and for those who treat them, from both a quality of life and an economic standpoint.
By identifying new targets for the detection and treatment of DR in its early stages, we will be
able to reduce the healthcare costs associated with DR and will contribute towards improving
the quality of life of diabetic patients.
At present, DR is diagnosed by ophthalmoscopic examination in which we are looking
for microvascular abnormalities (ie., mycroaneurisms, microhaemorrhages, hard exudades,
neovessels). However, before any microcirculatory abnormalities can be detected by
opthalmoscopic examination, retinal neurodegeneration is already present. Therefore, it is
reasonable to hypothesize that retinal neurodegeneration is an early event in the pathogenesis
of DR which precedes and mediates the microcirculatory abnormalities that occur in DR.
Hence, the development of methods to explore the consequences of retinal neurodegeneration
(i.e., electroretinograms, visual evoked potentials, optical coherence tomography [OCT]) will
be essential not only for detecting the early stages of DR but also for testing new therapeutic
agents.
The effectiveness of current treatments of DR is limited, and they are currently indicated
at too advanced stages of the disease. Therefore, novel strategies to detect, prevent and treat
DR in its earliest stages are needed. The study of factors involved in neurodegeneration
permit us to identify new therapeutic targets in the early stages of DR and, in consequence, to
bring about improvements in clinical practice in the medium term. The combination of basic
research focused on retinal neurodegeneration and clinical trials addressed to testing new
neuroprotective agents in DR will open a new scenario aimed at reducing the burden and at
improving the clinical outcome of this devastating complication of diabetes.
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Chapter 11
Molecular Imaging
and Parkinsons Disease
Valentina Berti, Cristina Polito,
Maria T. R. De Cristofaro and Alberto Pupi.
Clinical Pathophysiology Department, Nuclear Medicine Unit,
University of Florence, Florence, Italy
Abstract
Parkinsons disease (PD) is a neurodegenerative disorder characterized by the loss of
dopaminergic (DA) terminals in the striatum, resulting in functional changes in
frontostriatal circuits.
DA transporter imaging ([
123I
]FP-CIT SPECT imaging) and brain metabolic imaging
([
18F
]FDG PET imaging) have been broadly employed to explore the biological substrate
of PD, and together they could highlight the pathological processes occurring in early
stages of PD.
To evaluate the functional association between DA degeneration and cortical
metabolism we performed both [
123I
]FP-CIT SPECT and [
18F
]FDG PET in the same PD
sample; through a multiple regression analysis with SPM we explored the correlation
between putaminal DA degeneration and cortical metabolic rate of glucose.
In the putamen, which is the first and most affected striatal region in PD, the severity
of dopaminergic impairment is directly related to cortical hypometabolism in premotor,
[
123I
]FP-CIT SPECT and [
18F
]FDG PET allow to identify the early functional
alterations in the frontostriatal circuits involved in PD.
Introduction
Molecular imaging has been widely used for the assessment of several neurological
disorders, and it has been demonstrated to provide useful complementary functional
Valentina Berti, Cristina Polito, Maria T. R. De Cristofaro et al. 216
informations to anatomic imaging, such as specific receptor density, cerebral perfusion,
cerebral metabolic rate of oxygen or cerebral metabolic rate of glucose.
Functional neuroimaging, in particular with [
18F
]fluorodeoxyglucose ([
18F
]FDG) and
positron emission tomography (PET), has revolutionized scientists ability to accurately study
cerebral glucose consumption of both normal and disordered brain. Most important, because
of the intimate relationship between neural activity and brain metabolism, this technique can
reveal the changes within local circuits of the brain at rest, thus highlighting the cerebral
dysfunction closer to the pathology. This ability has provided an obvious opportunity to study
several neurological disorders, such as neurodegenerative diseases.
Parkinsons Disease
The need for accurate and early diagnosis of neurodegenerative diseases motivated the
use of molecular imaging techniques, which are able to show the presence of pathological
alterations even before the condition is recognized clinically. Parkinsons disease (PD), as
other neurodegenerative disorders, is characterized by a long preclinical period, during which
potential therapeutic and restorative treatments could have a strong effect. For this reason,
during last decades increasing attention has turned toward the application of neuroimaging
techniques in PD.
PD is a progressive neurodegenerative disorder, resulting from the progressive death of
dopaminergic neurons in the nigrostriatal pathway, which is the best recognized
neuropathological feature in PD and causes the alteration of cortico-striatopallido-
thalamocortical circuits [Wichmann T, et al. 2003]. The alterations in brain circuits occurring
in PD result in motor disturbances, which begin only after a loss of approximately 70-80% of
striatal dopamine- thus, there is a long latent period which precedes the development of
clinical symptoms [Huang WS, et al. 2001].
Through the study of PD typical pathophysiological alterations, and therefore through the
analysis of alterations occurring in striato-cortical circuits, it could be possible to obtain
important informations about the pathways themselves and their functioning.
N--fluoropropyl-2-carbomethoxy-3-{4-iodophenyl}-nortropane ([
123I
]FP-CIT) single
photon emission computed tomography (SPECT) has been used to estimate the loss of striatal
dopaminergic terminals and it is the most widely used diagnostic technique to assess the
integrity of the dopaminergic system in vivo [Booij J, et al. 2001; Spiegel J, et al. 2007]. The
striatal radioactivity measured after administration of [
123I
]FP-CIT is a function of the
quantity of dopaminergic terminals in the striatum. In patients with PD, decreased [
123I
]FP-
CIT uptake in the striatum has been reported by numerous studies, with the decrease being
much more severe in the putamen than in the caudate nucleus. Moreover, PD patients showed
an inverse correlation between the degree of motor deficit and [
123I
]FP-CIT uptake in the
striatum, especially in the putamen [Marshall V, et al. 2001], and this correlation has been
found even in early stages of disease [Berti V, et al. 2008].
Nigrostriatal dopaminergic neuronal loss in PD is, however, the cardinal but not the only
neuro-functional step in the pathologic progression of the disease [Bezard E, et al. 2003;
Braak H, et al. 2002; Obeso JA, et al. 2004]. PET imaging with FDG as been widely
employed as a measure of local synaptic activity in the resting state and it can provide
Molecular Imaging and Parkinsons Disease 217
inferences regarding the status of neural pathways in PD patients. Indeed, the
neuropathological processes can alter the functional connectivity across the entire brain in a
disease-specific manner [Eckert T, et al. 2005] and [
18F
]FDG PET can highlight local changes
in brain metabolism accompanying local changes in neural activity.
[
18F
]FDG PET can improve diagnostic accuracy in the evaluation of patients with
movement disorders, and recently it has been demonstrated that the expression of specific
patterns of cortical hypometabolism could help in the differential diagnosis of parkinsonisms
[Spetsieris PG, et al. 2009].
A number of reports described the expression of an abnormal PD related metabolic
pattern, characterized by increased pallido-thalamic and pontine activity, associated with
reductions in lateral frontal, paracentral, inferior parietal and parieto-occipital regions
[Fukuda M, et al. 2001; Huang C, et al. 2007]. Besides, several studies showed the correlation
between regional cortical hypometabolism and severity of motor impairment in PD,
demonstrating an inverse correlation involving in particular anterior cingulate gyrus,
orbitofrontal and occipitotemporal regions [Nagano-Saito A, et al. 2004].
However, even though both [
123I
]FP-CIT SPECT and [
18F
]FDG PET are able to identify
the presence of PD pathophysiological alterations, taken apart they do not provide specific
information about the neural circuits involved in the disorder. Only from the combination of
the two neuroimaging techniques and thus from the relation of the impairment of nigrostriatal
dopaminergic system with cerebral metabolic reductions, it could be possible to indirectly
show the striato-cortical circuits in PD.
Recently, our group evaluated the functional association between dopaminergic
degeneration and cortical metabolism performing both [
123I
]FP-CIT SPECT and [
18F
]FDG
PET in the same group of PD de novo patients; through a multiple regression analysis with
Statistical Parametric Mapping (SPM) we explored the correlation between striatal
dopaminergic degeneration and cortical metabolic rate of glucose (CMRglc), thus indirectly
highlighting the alteration of striato-cortical circuits in PD [Berti V, et al. 2009].

Figure 1. Statistical parametric maps (SPMs) showing cortical regions with a direct association with
putaminal dopaminergic impairment. In PD patients, the putaminal dopaminergic neuronal loss
correlates with hypometabolism in premotor, dorsolateral prefrontal, anterior prefrontal and
orbitofrontal cortices. From left to right side of figure: SPMs are displayed on the right and left lateral
views of a 3D rendered standardized MRI
In the putamen, which is the first and most affected striatal region in PD, the severity of
dopaminergic impairment is directly related to cortical hypometabolism in premotor,
Consistently with what is known about the organization of basal ganglia circuits [Albin
R, et al. 1989; Obeso JA, et al. 2000], striatal dopaminergic impairment correlates with
hypometabolism in several frontal areas. These findings are consistent with the
pathophysiological changes occurring in the functional organization of the basal ganglia in
PD, since the increased neuronal firing activity in the output nuclei of the basal ganglia
characterizing PD leads to excessive inhibition of thalamocortical projections, which may be
at the basis of reduced CMRglc in specific regions [Obeso JA, et al. 2008].
Besides, the correlation between striatal dopaminergic impairment and hypometabolism
in frontal cortex emphasizes the functional inter-relationships between the neocortex and the
striatum, consistently with the concept of fronto-striatal loops, which has been widely used to
explain the functional organization of frontostriatal circuits. According to this model, this
neural system includes five parallel but functionally distinct loops, including a motor loops
and complex non-motor loops, which are involved in cognitive functions. At the striatum
level, the motor loop is mostly centered on the putamen and the associative loop mostly on
the caudate [Parent A, et al. 1995; Alexander GE, et al. 1986].
The functional correlation between dopaminergic loss in the putamen and cortical
hypometabolism in the premotor cortex indirectly reflects the presence of the classical fronto-
striatal motor loop. However, the association between putaminal dopaminergic impairment
and cortical hypometabolism is not confined to the frontal regions belonging to the motor
loop, but it involves also dorsolateral, anterior prefrontal, and orbitofrontal regions, which are
typically part of the associative and limbic loops [Alexander GE, et al. 1986]. These results
can be explained by the parallel loss of dopaminergic terminals in putamen and in the caudate
nucleus (even if to a minor extent in the latter), which is demonstrated by the correlation
between putaminal and caudate nucleus dopaminergic impairment. Indeed, even in the early
stages of the disease, the loss of dopaminergic projections in PD is not confined to the
putamen but at the same time also occurs in the caudate nucleus and in ventral striatum (even
if they are involved lately and to a lesser extent), which are the striatal relais of associative
and limbic frontostriatal loops, therefore connected with dorsolateral prefrontal, anterior
prefrontal and orbitofrontal cortices [Alexander GE, et al. 1986].
Conclusion
In conclusion, through the study of the pathophysiological alterations occurring during a
disease condition, it is possible to obtain important informations about specific brain circuits
and their functioning.
As an example, the double-tracer study of patients affected by PD, which, through the
nigrostriatal neuronal loss affects striato-cortical brain circuits, indirectly highlithed the
presence of specific brain circuits. Indeed, the correlation of dopaminergic impairment in
specific striatal regions with cortical metabolism has shown pathways connecting the
putamen and premotor and prefrontal cortex.
Molecular Imaging and Parkinsons Disease 219
Interestingly, the functional informations provided by molecular imaging are able to
show and confirm several anatomical informations, such as anatomical connections between
brain regions.
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Index

A
Abraham, 185, 187
absorption, 206
acetylcholine, xi, 189, 190, 191, 194, 196, 197, 198,
200, 201
acetylcholinesterase, xi, 189, 190, 196, 199
acetylcholinesterase inhibitor, 196
acid, ix, 3, 30, 43, 68, 74, 82, 83, 111, 113, 120, 132,
136, 138, 141, 146, 147, 155, 156, 157, 164, 175,
177, 182, 202, 208, 209, 214
adamantane, 103
adaptation, 117, 204
adenine, 2, 4, 31, 38, 108
adenovirus, 161
adhesion, 161, 164
adipose, ix, 107, 108, 109, 119
adipose tissue, ix, 107, 108, 109, 119
ADP, 5, 6, 105, 108, 109, 127
advantages, ix, 87, 99
aetiology, 176
aggregation, 20, 46, 52, 54, 57, 60, 65, 82, 176
aging population, 80
agonist, 88, 95, 96, 97, 100, 140, 153
AIDS, 15, 18, 19, 21, 24, 28, 34, 36, 38, 39, 41, 42
albumin, 92, 113
alcohol use, 170
alcoholism, 83
alcohols, 55
allele, xi, 170, 174, 177, 178, 179, 180, 181, 182,
185, 187
ALS, viii, 3, 7, 8, 20, 45, 46, 53, 54, 55, 56, 57, 58,
60, 114, 116, 177, 178, 184
alters, 36, 82, 84, 134, 147, 148, 150, 186
amines, 76, 78, 83
amino acids, 109, 131
ammonium, 69
amygdala, 190
amyloid beta, 38, 39, 122
amyloid deposits, 196
amyotrophic lateral sclerosis, viii, 3, 8, 27, 29, 40,
45, 46, 56, 57, 58, 60, 114, 123, 124, 145, 154,
184
anatomy, 219, 220
anesthetics, ix, x, 155, 156, 157, 162, 163, 164, 165,
166
angiogenesis, 82, 213, 214
ANOVA, 70, 71, 72, 73, 74, 75, 76, 77, 92, 97
antibiotic, 46, 131, 138
antibody, 92, 138, 193, 199
anticonvulsant, 163
antidepressant, 65, 81
antigen, 161, 171
anti-inflammatory drugs, 55, 56
antioxidant, 2, 3, 7, 9, 11, 17, 19, 22, 23, 24, 27, 30,
33, 34, 37, 38, 42, 43, 47, 49, 60, 65, 112, 114,
117, 132, 138, 209
antisense, 58, 166, 168
antisense oligonucleotides, 166
apoptosis pathways, 89, 147
apoptotic mechanisms, 100, 133, 134, 144
appetite, 70, 196
Arabidopsis thaliana, 48, 58
architecture, 183, 193
argon, 91
arrest, xi, 9, 13, 33, 170, 203
artery, 83, 127
aryl hydrocarbon receptor, 161
ascorbic acid, 68, 209, 214
aseptic, 90
aspartate, ix, 87, 88, 102, 103, 109, 126, 131, 156,
157, 158, 168, 169, 171, 172
aspartic acid, 156
asphyxia, 127, 143, 145, 146
assessment, 80, 82, 99, 101, 215
Index 222
astrocytes, vii, 1, 7, 8, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 27, 29, 30, 31, 36, 38,
39, 40, 41, 42, 43, 50, 83, 88, 105, 136, 208
astrocytoma, 36
astrogliosis, 50, 57, 138
asymmetry, 132
asymptomatic, 36
ataxia, vii, 1, 8, 12, 30, 31, 43, 176
atherosclerosis, 24, 41, 43
ATP, ix, 5, 6, 78, 107, 108, 109, 110, 112, 113, 117,
123, 131
atrophy, 23, 135, 148, 195, 197
Austria, 189
autoimmune diseases, 180
autosomal dominant, x, 173, 175, 176, 182
autosomal recessive, 8
axons, 123, 150, 174
B
back pain, 196
background noise, 55
bacteria, 6
barriers, 214
basal forebrain, xi, 189, 190, 192, 193, 194, 195,
196, 197, 199, 200, 202
basal ganglia, 113, 218, 219, 220
base pair, 177, 178
basic research, 210
BBB, 22, 23, 24, 195, 196
Bcl-2 proteins, 170
behaviors, 198
beneficial effect, 54, 125, 140, 142, 180, 207, 208
benzodiazepine, 152
biochemistry, 142
biological sciences, 46
biosynthesis, 30, 190, 209
biosynthetic pathways, 108
blindness, xi, 203, 204, 210
blood flow, 88, 190
blood pressure, 204, 207, 208
blood supply, 117
blood vessels, 195
blood-brain barrier, 22, 42, 154, 195, 197, 208
BMI, 13
body composition, 111
body weight, 70, 71, 111
bonds, 4, 5, 8, 18
brain contusion, 151, 152
brain damage, 128, 132, 133, 141, 143, 144, 145,
146, 147, 152, 154, 163
brain growth, 156, 157
brainstem, 111, 190
breakdown, 136, 149, 151, 208
budding, 161
Burkina Faso, 81
Butcher, 104
C
Ca
2+
, x, 2, 4, 5, 6, 10, 19, 29, 88, 89, 100, 101, 103,
111, 112, 114, 116, 117, 132, 156, 157, 172
cadmium, 32
calcium, ix, 7, 16, 19, 21, 30, 38, 40, 48, 59, 60, 88,
103, 107, 108, 111, 113, 116, 121, 127, 155, 157,
158, 160, 161, 164, 166, 167
caloric restriction, 83, 122
calorie, 70
cancer, 8, 24, 30, 33, 42, 162, 170, 177
cancer cells, 162, 170
candidates, 208
capillary, 69
cardiovascular disease, 41
cartoon, 167
Caspase-8, 126, 136, 151
caspases, 89, 90, 100, 103, 117, 125, 126, 128, 129,
130, 131, 132, 133, 134, 137, 138, 139, 141, 142,
143, 150, 152, 168
catalysis, 4
cataract, 29, 50
catecholamines, 78, 209
category a, 164
CCR, 186
cDNA, 119, 182
cell body, 191, 192
cell culture, 55, 66, 95, 103, 105, 123, 135, 149, 165,
172, 196, 202
cell cycle, 9, 10, 13, 33, 84, 177
cell fusion, 37
cell line, 20, 36, 41, 48, 55, 59, 103, 113, 114, 116,
139, 202
cell lines, 113, 139
cell membranes, 22
cell metabolism, 8
cell signaling, 3, 27, 84
cell surface, 17
cellular signaling pathway, 112
central nervous system, vii, ix, 1, 35, 36, 39, 40, 87,
88, 107, 109, 118, 119, 120, 133, 145, 153, 156,
157, 163, 171, 178, 192, 200
cerebellar development, 33
cerebellum, 12, 25, 80, 111, 113
cerebral amyloid angiopathy, 182
cerebral blood flow, 88, 190
cerebral contusion, 151
Index 223
cerebral cortex, 27, 42, 104, 130, 133, 135, 136, 181,
194
cerebral hypoxia, 144, 145
cerebrospinal fluid, 116, 123, 136, 137, 144, 149,
151, 187, 188, 198
cerebrum, 138
chaperones, 3, 4, 24, 43, 46, 48, 49, 54, 57, 58, 59
chemokine receptor, 186
chemokines, xi, 34, 151, 174, 179
chemoprevention, 43
child abuse, 151
cholinesterase, 82, 190, 196, 198, 200
cholinesterase inhibitors, 196, 198
choroid, 111
chromatography, 69
chromosome, 158, 175, 176, 177, 178, 179, 182,
183, 184, 185
chronic fatigue syndrome, 14, 34, 43
circadian rhythm, 132, 158
circulation, 209
class, 176
cleavage, 10, 16, 18, 89, 126, 127, 128, 133, 134,
136, 137, 138, 141, 144, 150, 151, 152, 174, 175,
190, 191
climate, 18
clinical diagnosis, 178
clinical symptoms, 195, 216
clinical syndrome, 174
clinical trials, 24, 79, 132, 196, 210
CNS, vii, viii, xi, 1, 2, 13, 15, 16, 18, 20, 21, 22, 23,
25, 26, 35, 50, 57, 80, 88, 99, 118, 132, 134, 135,
140, 141, 146, 156, 157, 189, 190, 192, 196, 199
CO2, 90
coding, 176, 177, 180
codon, 178, 180, 186
coenzyme, 109, 110, 113, 190
cognition, 190
cognitive ability, 64
cognitive deficit, xi, 153, 189, 193
cognitive deficits, xi, 153, 189, 193
cognitive dysfunction, xi, 189, 194
cognitive function, viii, ix, 63, 73, 77, 78, 80, 87,
196, 200, 218
cognitive impairment, 64, 174, 195, 212
cognitive performance, 71, 79
cognitive tasks, 64
coherence, 210
collagen, 209
colon, 150, 162, 170
colon cancer, 150, 162, 170
complexity, ix, 49, 52, 80, 149, 155, 156
complications, 42, 121, 210
composition, 83, 88, 89, 91, 111, 153, 169
compounds, 91, 131, 142, 165
computed tomography, 135, 216
computer software, 67
condensation, 144
conductance, 42, 101, 108, 121
connectivity, 217
consensus, 48, 174, 182
consumption, viii, 9, 63, 109, 207, 209, 216
contrast sensitivity, xii, 204, 205
control condition, 94, 97, 98
control group, 67, 131
controlled trials, 212
contusion, 135, 138, 140, 152, 153
convergence, 14, 34
conviction, 108
cooling, 131
coordination, 8, 131
corpus callosum, 130, 149, 150
correlation, xii, 34, 39, 91, 92, 93, 195, 215, 216,
217, 218, 219
cortex, 27, 42, 90, 104, 110, 111, 113, 115, 130, 131,
134, 136, 140, 141, 158, 159, 160, 162, 163, 170,
181, 187, 190, 192, 193, 194, 197, 218, 219
cortical neurons, 38, 93, 100, 101, 102, 103, 105,
117, 135, 136, 149, 150, 160, 169, 172
cost, 94
criticism, 55
crystallization, 66
CSF, 140, 200
cues, 25, 64
culture, 13, 17, 19, 22, 23, 33, 36, 50, 53, 55, 56, 66,
88, 93, 95, 99, 103, 104, 105, 123, 168, 169, 171,
196, 201, 211
cyanide, 65, 162, 166, 170
cycling, 8, 9, 13, 65
cyclooxygenase, 39
cystic fibrosis, 24
cystine, 17, 24, 38
cytoarchitecture, 37, 193
cytochrome, viii, 63, 65, 68, 74, 78, 83, 84, 105, 109,
110, 112, 113, 117, 126, 132, 145, 151, 153, 166,
168
cytokines, xi, 151, 174
cytoplasm, 5, 6, 7, 47, 89, 90, 98, 101, 108, 166, 168,
177
cytoskeleton, 30
cytotoxicity, 15, 116, 123, 143, 146
D
damages, iv, ix, 5, 6, 7, 20, 87, 97, 100
decay, 177, 178
decomposition, 172
Index 224
defects, 14, 83, 116
deficiency, 10, 11, 12, 16, 21, 25, 31, 32, 65, 79, 83,
117, 123
deficit, 38, 72, 208, 209, 216
degenerate, xi, 189, 195
degradation, 4, 25, 40, 43, 101, 127, 177, 208
dementia, vii, x, 1, 2, 3, 19, 22, 28, 36, 37, 38, 39,
40, 41, 42, 65, 76, 83, 114, 173, 174, 176, 183,
184, 185, 186, 187, 197, 198
demyelination, 28
depolarization, 88, 116, 123
deposition, x, 173, 175, 193, 196, 200
deposits, 174, 175, 196
deprivation, 2, 101, 117, 138, 141
destruction, 88, 89, 94
detection, 69, 123, 165, 210
detoxification, 7, 8, 76
developed countries, xi, 203, 204, 210
developing brain, ix, x, 143, 155, 156, 157, 158, 162,
164, 166, 167, 168, 169, 170
developmental change, 193
diabetes, xii, 24, 43, 80, 81, 108, 113, 118, 121, 203,
204, 205, 206, 207, 208, 209, 210, 211, 212, 213,
214
diabetic patients, xii, 203, 204, 205, 208, 209, 210,
213
diabetic retinopathy, vii, 209, 211, 212, 213
diagnosis, 54, 178, 200, 216, 217, 220
diagnostic criteria, 176, 182
diet, 67, 140, 153, 209
dietary fat, 113
differential diagnosis, 217
diffusion, 128, 135, 193, 208
dimerization, 192
direct action, ix, 88, 89
disability, 65, 127, 135, 142
disadvantages, 99
discrimination, xii, 204, 205
disease model, vii, 2, 198, 202
disease progression, 7, 15, 57
disequilibrium, 180
disorder, vii, x, xi, xii, 1, 8, 54, 114, 115, 163, 173,
189, 195, 215, 216, 217, 220
displacement, 99
disposition, 42
dissociation, 31, 167
disturbances, 163, 174, 198, 216
diversity, ix, 48, 87, 90, 169, 186
DNA, vii, x, 1, 3, 6, 8, 9, 10, 27, 29, 31, 32, 33, 36,
41, 50, 58, 59, 65, 82, 89, 90, 97, 100, 101, 103,
104, 115, 123, 127, 132, 133, 135, 137, 140, 143,
144, 147, 149, 152, 165, 167, 171, 174, 177
DNA damage, 10, 31, 32, 33, 97, 104, 115, 171
DNA lesions, 32
DNA repair, 8, 9, 10, 27, 127
DNA strand breaks, 31
donors, 14, 43, 108, 205
dopamine, 76, 115, 122, 123, 209, 216, 219
dopaminergic, xii, 29, 42, 115, 116, 123, 165, 172,
215, 216, 217, 218, 219
dosage, 19
Down syndrome, 175
down-regulation, 105, 114, 122, 194
drinking water, viii, 24, 63, 66, 67, 71, 72, 73, 74,
75, 76, 77
Drosophila, 48, 56, 57, 59, 61, 99, 102, 105
drug addict, 115
drug discovery, 125
drug resistance, 24, 43
drug treatment, 3
drugs, vii, 2, 22, 23, 24, 25, 26, 55, 56, 64, 92, 132,
156, 163, 197, 207, 212
E
edema, 133, 149, 150, 204, 206, 211, 213
electroencephalogram, 151
electron, ix, 2, 4, 5, 6, 7, 9, 28, 65, 68, 76, 78, 83,
107, 108, 109, 110, 115, 121, 167
electrons, 3, 4, 5, 6, 7, 23, 78, 108, 110, 112, 113
ELISA, 165
elucidation, viii, 2, 125
embryogenesis, 102
emission, 92, 99, 216
encephalitis, 14, 34, 38, 41
encephalomyelitis, 42
encephalopathy, 15, 35, 81, 125, 126, 127, 142, 143,
154
encoding, 52, 178, 179, 180
endonuclease, 136
endothelial cells, vii, 2, 7, 15, 18, 20, 36, 37, 83, 132,
205, 206
endothelium, 19, 40, 211
energy consumption, 9
environmental conditions, 2
environmental factors, xi, 114, 174, 175, 181
enzymes, xi, 7, 81, 114, 117, 126, 189, 192, 198
epidemiology, 184
epithelia, 126
epithelial cells, 19, 208, 214
epithelium, 38, 206, 208, 211
erythropoietin, 147, 208, 213, 214
ESI, 69, 73
estrogen, 140, 153, 170
ethanol, 163, 164, 199
ethylene, 64, 81
Index 225
eukaryotic cell, 4, 108
evil, 27
evoked potential, 210
excitability, x, 155, 157, 163
excitation, 68, 69, 91, 164
excitotoxicity, vii, ix, 21, 87, 88, 89, 90, 95, 96, 98,
100, 102, 117, 123, 152, 208
execution, 48, 53, 58, 125
exercise, 80
exons, 176, 177, 178, 181
experimental autoimmune encephalomyelitis, 42
exposure, x, 19, 20, 21, 46, 53, 65, 79, 90, 93, 94, 95,
96, 97, 98, 99, 111, 113, 137, 156, 158, 159, 160,
163, 164, 167, 168, 169, 209
extinction, 68
F
FAD, 4, 28, 183
falciparum malaria, 81
family history, 176, 178
family members, 103
FAS, 128
fat, 69, 70, 108, 113
fatty acids, 65, 109, 112, 114, 149
FDA, 55, 79, 131, 155, 168
FDA approval, 79
fetal alcohol syndrome, 170
fever, 65, 82
fiber, 135, 190, 192, 199
fibers, 190, 193, 194, 199
fibroblast growth factor, 39
fibroblasts, viii, 10, 52, 63, 197
flavopiridol, 140
fluid, 116, 123, 135, 137, 148, 152, 187, 188, 198,
206, 208, 209, 213
fluorescence, 69, 91, 92, 93, 94, 96, 97, 99, 100, 101,
104
folate, 122
food intake, 70
forebrain, xi, 144, 163, 168, 169, 170, 171, 189, 190,
192, 193, 195, 196, 197, 199, 200, 202
fragments, 137, 175
free radicals, ix, 3, 23, 27, 78, 107, 108, 123, 132
frequencies, 180, 187
frontal cortex, 153, 158, 159, 160, 162, 170, 218
frontal lobe, 183, 187
functional changes, xii, 22, 215
functional imaging, 220
fusion, 18, 37, 99, 133
G
gene expression, 21, 25, 41, 50, 51, 60, 61, 115, 158,
160, 186
gene promoter, 141
gene therapy, 197, 201
gene transfer, 197, 199
genes, x, 7, 8, 17, 29, 30, 34, 36, 48, 51, 52, 56, 58,
59, 60, 142, 149, 150, 158, 161, 162, 164, 166,
167, 173, 175, 176, 178, 179, 180, 181
genetic disease, 2, 8, 25
genetic factors, xi, 174
genetic linkage, 182
genetic mutations, 2
genetics, vii, xi, 174, 178
genome, 27, 100, 179
genomic instability, 9
genotype, 144, 180, 185, 187
gestation, 127, 156
glia, 15, 28, 30, 39
glial cells, 19, 36, 58, 88, 105, 115, 190, 191, 206
glioma, 33
glucose, xi, xii, 7, 10, 24, 39, 43, 54, 90, 112, 114,
117, 121, 138, 141, 203, 204, 205, 209, 211, 215,
216, 217
GLUT, 209
glutamate, viii, ix, x, 7, 17, 21, 38, 42, 56, 87, 93, 94,
97, 98, 102, 103, 104, 109, 116, 123, 134, 137,
143, 149, 155, 156, 157, 158, 159, 160, 162, 163,
165, 166, 167, 190, 202, 206
glutamate receptor antagonists, ix, 155, 157
glutathione, 3, 4, 17, 28, 29, 30, 39, 41, 42, 112, 117,
124, 132, 134
glycine, 91, 96, 103, 178, 184
glycogen, 49, 57, 147
glycolysis, 112
grades, 115
gray matter, 81, 148
growth factor, xi, 12, 18, 24, 32, 33, 39, 114, 122,
133, 161, 177, 184, 189, 190, 191, 192, 194, 198,
199, 200, 201, 202
growth hormone, 133
growth spurt, 156, 157
H
HAART, 19, 21, 22
haplotypes, 182
head injury, 135, 148, 151, 152, 153
health care system, 210
heat shock protein, viii, 31, 45, 47, 56, 57, 58, 59, 60
heavy metals, 46
Index 226
hematopoietic stem cells, 10, 27
heme, viii, 63, 78, 79
hemisphere, 133
hemorrhage, 127
hepatic failure, 27
hepatitis, 34
hepatoma, 33
heterogeneity, 174
hippocampus, 79, 85, 103, 115, 117, 124, 128, 130,
131, 132, 134, 135, 136, 138, 140, 141, 150, 153,
159, 171, 181, 190, 192, 194, 197
histidine, 27
histochemistry, 119
histogram, 92, 98
HIV, vii, 1, 2, 3, 14, 15, 16, 18, 19, 20, 21, 22, 25,
28, 34, 35, 36, 37, 38, 39, 40, 41, 42
HIV/AIDS, 42
HIV-1, 14, 19, 21, 22, 34, 36, 37, 38, 39, 41, 42
HIV-1 proteins, 19, 38
HO-1, 8
homeostasis, ix, 2, 6, 9, 10, 11, 16, 19, 24, 25, 26,
32, 38, 43, 51, 107, 108, 111, 117, 121, 162, 164,
196
homocysteine, 180, 186
host, 6, 18, 19, 21, 25, 128, 184
human brain, 36, 74, 76, 77, 83, 120, 124, 137, 151,
177
human development, 156
human immunodeficiency virus, 28, 35, 36, 37, 38,
39, 40, 41, 43
hybridization, 50, 119, 158, 159, 167
hybridoma, 170
hydrogen, 3, 4, 78, 83, 112, 117, 118, 132, 134, 146
hydrogen gas, 132
hydrogen peroxide, 3, 4, 78, 83, 112, 117, 118
hydroperoxides, 3
hydroxyl, ix, 3, 4, 55, 87, 88, 112
hyperglycemia, xi, 113, 122, 203, 204, 207, 209
hyperplasia, 196, 206
hypersensitivity, 9
hypertension, xi, 137, 203, 213
hyperthermia, 50, 58, 172
hypertrophy, 206
hypoglycemia, 101
hypothermia, 131, 138, 142, 144, 145, 148, 152
hypothesis, x, 121, 156, 178, 180, 182, 198, 202
hypoxia, 104, 117, 125, 127, 128, 143, 144, 145,
146, 147, 148, 198, 208
I
ICAM, 134
ice, 50, 68, 69
idiopathic, 176, 177, 219
image, 80, 92, 93, 94
image analysis, 80
images, 91, 93, 94, 95, 98, 128
imaging modalities, 127
imbalances, 52
immune reaction, 97
immune response, 150, 162, 185
immune system, 15
immunodeficiency, 8, 14, 24, 28, 35, 36, 37, 38, 39,
40, 41, 43
immunoglobulin, 164, 171, 202
immunomodulatory, 132
immunoreactivity, 116, 140, 164, 165, 194, 200, 213
impairments, viii, 13, 33, 64, 101, 115, 195, 199, 200
impulsiveness, 174
in situ hybridization, 50, 119, 148, 158, 159, 167
in vivo, xi, 13, 28, 33, 42, 50, 53, 58, 59, 60, 65, 66,
74, 79, 125, 128, 132, 133, 136, 137, 138, 140,
145, 152, 161, 164, 165, 166, 170, 172, 189, 192,
193, 195, 216
incidence, 20, 41, 127, 135, 210
induction, 18, 28, 30, 38, 39, 52, 53, 56, 58, 75, 78,
79, 84, 96, 100, 101, 114, 117, 136, 140, 148, 171
industrialized countries, 117
infants, 142, 145, 151, 162
infarction, 117, 120, 132
inferences, 217
inflammation, xi, 20, 38, 132, 140, 148, 153, 177,
189
inflammatory disease, 179
inflammatory responses, 60
inheritance, 178
inhibition, 12, 13, 18, 29, 32, 68, 76, 82, 83, 84, 101,
108, 126, 128, 130, 131, 134, 138, 140, 141, 142,
143, 147, 150, 152, 153, 154, 162, 209, 213, 218
inhibitor, 11, 12, 13, 17, 33, 49, 53, 81, 84, 100, 131,
133, 136, 137, 138, 140, 141, 142, 143, 145, 148,
161, 165, 166, 168, 171, 172, 177, 200
initiation, 6, 9, 10, 16, 39, 125, 131
injections, 159, 161, 193
inositol, 6, 161
insertion, 176, 178
insulin, 12, 33, 114, 119, 122, 161, 210
integration, 28
interference, 112
interneurons, 89, 190
intervention, viii, 43, 45, 55, 108, 128, 131
ion channels, 102, 149, 157
ionizing radiation, 9, 10, 32, 144
ions, 6, 38, 88, 102
IP-10, 180, 186
ipsilateral, 128, 133
Index 227
Ireland, 146
iron, 4, 23, 27, 79, 165
irradiation, 81
ischemia, 81, 85, 108, 117, 124, 125, 126, 127, 128,
131, 132, 140, 143, 144, 145, 146, 147, 148, 207,
208
isolation, 161
isoleucine, 180
isomerization, 4, 5
Italy, 173, 215
J
Jordan, 30, 35
K
K
+
, 88, 89, 100, 104
kidney, 65, 81, 112, 121
kinase activity, 59
kinetics, 48
Krebs cycle, 109
L
language impairment, 174
latency, 19, 43
lateral sclerosis, viii, 3, 8, 27, 29, 40, 45, 46, 54, 56,
57, 58, 59, 60, 114, 123, 124, 184
learning, ix, 79, 80, 87, 132, 152, 169, 190, 193, 200
left hemisphere, 175
leptin, 81, 207
lesions, xii, 32, 117, 124, 139, 150, 193, 195, 198,
204, 205, 206, 214
leukemia, vii, 1, 14, 15, 34, 35, 36, 37
lice, 23, 117, 201, 202
ligand, 5, 36, 126, 149, 157
lipid oxidation, 165
lipid peroxidation, 20, 21, 23, 101, 112, 117, 131,
132
lipids, 3, 132
liquid chromatography, 69
liver, 109, 111
localization, 30, 58, 116, 196, 200
locomotor, 115
locus, 178, 182, 184, 185
loss of appetite, 196
low-density lipoprotein, 179
LSD, 71, 72, 73
lumen, 2, 4, 5, 7
Luo, 29, 32, 84, 145, 213
lymphocytes, 15, 40, 56, 58
lymphoid, 8, 22
lymphoma, 32, 34
lysine, 49, 91
M
machinery, 53
macrophages, 15, 21, 41, 132
macular degeneration, 29
magnetic resonance, 120, 127, 135, 149, 150, 152
magnetic resonance imaging, 127, 135, 149, 152
magnetic resonance spectroscopy, 120
majority, x, xi, 21, 25, 114, 162, 173, 174, 175, 177,
178, 185
malaria, 81, 83
mammalian brain, 163
manic-depressive psychosis, 81
manipulation, 49, 84, 120, 165, 207
MAP kinases, 39
markers, 10, 13, 23, 24, 37, 148, 176, 195, 198
Marx, 33
matrix, 108, 109, 110, 111, 112, 113, 117, 118, 121,
127
MCP, 179, 180, 182, 185, 188
MCP-1, 179, 180, 182, 185, 188
media, 96, 100, 205
mediation, 131, 132
MEK, 12, 13, 140
melatonin, 131, 132, 144, 146
membrane permeability, 101
membranes, 5, 6, 18, 22, 99, 112
memory, viii, ix, xi, 63, 64, 67, 71, 72, 77, 79, 80,
82, 84, 87, 152, 174, 189, 190, 193, 194, 196,
198, 199, 212
menadione, 83
mental health, 84
mental illness, 84
mental retardation, 127
mesenchymal stem cells, 141, 154
mesoderm, 183
messengers, 112
meta-analysis, 181, 187
metabolic pathways, xi, 203, 204, 205, 206
metabolic syndrome, 11, 32
metabolism, xii, 6, 8, 40, 64, 70, 79, 80, 82, 88, 101,
108, 109, 111, 113, 120, 121, 158, 215, 216, 217,
218
meter, 67
methamphetamine, 165, 172
methanol, 69
methemoglobinemia, 65
Mg
2+
, 91, 102
Index 228
microscope, 91, 92
microscopy, ix, 87, 91, 93, 95, 96, 104, 105
migration, 33, 154, 157, 165, 168, 171
mitochondria, viii, ix, x, 2, 5, 6, 7, 9, 10, 15, 16, 17,
19, 27, 28, 29, 63, 64, 65, 66, 70, 73, 78, 79, 83,
87, 103, 104, 107, 108, 109, 111, 112, 113, 114,
115, 117, 119, 120, 121, 132, 134, 136, 137, 164,
166, 167, 170, 173, 174, 206
mitochondrial DNA, 6, 29, 115
mitogen, 49
mitosis, 104
model system, 198
modeling, 22, 41
moderates, 81
modification, 11, 12, 49, 69
modifier gene, 29
molecular biology, vii
molecular oxygen, 4, 5, 6
molecular weight, 42, 78, 110, 177
molecules, 3, 7, 24, 26, 47, 48, 49, 55, 81, 98, 102,
126, 127, 179
MOM, 214
monoclonal antibody, 193
monocyte chemoattractant protein, 185
morbidity, 117, 127, 134, 145
morphogenesis, 89
morphology, 148, 150, 195
morphometric, 150, 168, 199
mortality rate, 127
Moses, 149
motor neuron disease, 34, 35, 36, 116, 184
motor neurons, 56, 59, 60, 89, 116, 123
movement disorders, 217, 219
MRI, 127, 135, 150, 217
mRNA, 58, 105, 110, 111, 116, 117, 119, 128, 131,
134, 136, 137, 148, 149, 150, 158, 159, 167, 177,
178, 182
mtDNA, 5, 6, 10
multicellular organisms, 126
multiple regression, xii, 215, 217
multiple regression analysis, xii, 215, 217
multiple sclerosis, 3, 34, 150
multiplier, 69, 92
muscle strength, 64, 73, 77, 78
mutagenesis, 171
mutant, vii, 1, 15, 25, 26, 35, 36, 37, 43, 50, 54, 56,
58, 59, 60, 116, 177, 180
mutation, viii, 2, 15, 18, 25, 54, 175, 176, 177, 178,
180, 182, 183, 186, 207
myelin, 50, 161
myoblasts, 56
myoclonus, 176
N
Na
+
, 88, 89, 100
NaCl, 91
NAD, 9, 76, 83, 84, 131, 145, 146
NADH, 78, 102, 108, 109, 110, 113
National Institutes of Health, 142
nausea, 196
necrosis, ix, x, 87, 88, 89, 90, 92, 93, 94, 95, 96, 99,
100, 104, 126, 128, 135, 145, 150, 156, 161, 171,
185, 202
nematode, 126
neocortex, 200, 218
neonates, 126, 127, 128, 131, 143, 156
neovascularization, 209
nerve, xi, 39, 53, 57, 59, 102, 135, 136, 141, 149,
156, 189, 190, 191, 193, 194, 198, 199, 200, 201,
202
nerve growth factor, xi, 39, 135, 136, 141, 149, 189,
190, 191, 194, 198, 199, 200, 201, 202
nervous system, vii, ix, x, 1, 2, 6, 34, 35, 36, 39, 40,
50, 87, 88, 102, 107, 109, 118, 119, 120, 155,
157, 163, 169, 171, 178, 192, 200
neural development, 127
neuroblastoma, 34, 39, 114, 116, 209, 214
neurodegenerative diseases, vii, ix, 2, 3, 7, 13, 22,
27, 30, 31, 40, 42, 55, 58, 59, 87, 88, 107, 112,
115, 121, 122, 128, 131, 142, 145, 146, 177, 178,
179, 182, 216
neurodegenerative disorders, 27, 56, 108, 114, 118,
187, 216
neurofibrillary tangles, x, xi, 114, 173, 174, 189,
193, 196
neurogenesis, 32, 33, 79, 84, 85
neuroimaging, 216, 217
neurological disease, vii, 1, 34, 126
neuromotor, 42
neuronal apoptosis, x, 38, 125, 128, 131, 132, 133,
140, 145, 147, 149, 150, 153, 156, 160, 163, 164,
165, 170
neuronal cells, 29, 59, 60, 133, 162, 165, 170, 213
neuropathy, 3, 20
neuroprotection, ix, 56, 88, 107, 108, 116, 122, 131,
132, 133, 134, 136, 137, 138, 140, 141, 143, 146,
147, 148, 153, 154, 198, 208, 212
neuroprotective agents, 89, 100, 210
neurotoxicity, x, 36, 40, 102, 104, 112, 122, 132,
138, 152, 156, 157, 158, 162, 164, 165, 166, 168,
169, 171, 172, 202, 213
neurotransmission, xi, 156, 189, 195, 196
neurotransmitter, viii, xi, 87, 96, 99, 157, 171, 190
neurotrophic factors, 88, 101, 133, 141, 154
neutrophils, 6, 103, 132
Index 229
niacin, 131
nicotinamide, 2, 38, 108, 131
nicotine, 31
nigrostriatal, 116, 123, 216, 217, 218
nitrate, 65, 165
nitric oxide, 36, 39, 41, 65, 82, 115, 132, 133, 138,
146, 147, 148, 152, 165, 171, 172, 180, 186, 187,
188
nitric oxide synthase, 65, 115, 133, 147, 148, 172,
180, 186, 187, 188
nitrogen, 68, 132
nitrous oxide, 162
nitroxide, 43
NMDA receptors, ix, x, 87, 100, 102, 156, 157, 158,
160, 163, 164, 165, 166, 167, 168, 169, 171
N-methyl-D-aspartic acid, 156
noise, 55, 92
normal aging, 75, 83
Nrf2, 7, 8, 16, 17, 19, 22, 23, 27, 30, 32, 34, 39, 42
nuclei, xi, 89, 91, 92, 93, 94, 99, 101, 104, 130, 163,
189, 190, 191, 206, 218
nucleic acid, 83
O
obesity, 108, 207
occipital regions, 217
oedema, 213
oligodendrocytes, vii, 2, 11, 15, 16, 50, 57, 88, 136
oligomerization, 82
oligomers, 89, 114
oncogenes, 33, 104
opportunities, 118
organ, 84, 109, 126
organelles, 2, 4, 52, 108, 109
organism, 46, 48, 52, 56
oxidation, 4, 6, 7, 9, 22, 23, 28, 40, 60, 61, 65, 68,
69, 70, 78, 82, 84, 108, 109, 123, 140, 165, 209
oxidation rate, 68
oxidative damage, 7, 19, 65, 82, 108, 112, 114, 116,
121, 132, 180, 208, 209, 214
oxygen, viii, x, 2, 4, 5, 6, 7, 9, 12, 27, 28, 34, 48, 57,
63, 82, 84, 108, 109, 110, 117, 119, 120, 121,
123, 132, 139, 141, 146, 147, 152, 154, 155, 157,
164, 165, 167, 209, 213, 216
oxygen consumption, viii, 63
P
p53, 11, 16, 27, 32, 33, 36, 37, 115, 117
pain, 196
pancreas, 65
parallel, 17, 158, 218
paralysis, 43, 54, 116
parenchymal cell, 109
parietal cortex, 131, 163
parkinsonism, 115, 176, 183, 219
parvalbumin, 53
pathogenesis, ix, xii, 14, 20, 21, 27, 36, 54, 87, 88,
99, 125, 158, 174, 176, 179, 180, 181, 196, 204,
205, 209, 210
pathology, viii, x, 2, 13, 29, 45, 46, 54, 55, 97, 116,
118, 137, 174, 177, 179, 181, 185, 192, 195, 210,
216, 219
pathophysiology, 29, 142, 151
PBMC, 180
PCA, 220
PCP, x, 156, 166
PCR, 115, 159, 160, 167
PDGF, 133
peptidase, 161
peptides, 100, 114, 122, 175, 177
performance, 24, 42, 64, 71, 77, 79, 193
perfusion, 216
perinatal, 128, 143, 144, 145, 147, 149, 169
peripheral blood, 40
peripheral nervous system, 35
permeability, 5, 6, 10, 29, 90, 101, 112, 166, 211
permission, iv
permit, 210
peroxidation, 20, 21, 23, 101, 112, 117
peroxide, 3, 4, 78, 83, 112, 117, 118
peroxynitrite, 140, 148, 165, 171, 172
PET, xii, 80, 215, 216, 217, 219
pH, 27, 68, 91, 99
pharmacological treatment, 55
pharmacology, 199
phencyclidine, x, 156, 169
phenotype, x, xi, 9, 12, 43, 55, 173, 176, 178, 183,
185, 189, 192, 196, 202
phospholipids, 110, 123
phosphorylation, 6, 7, 10, 11, 12, 13, 32, 46, 47, 48,
49, 53, 108, 109, 112, 113, 117, 133, 147, 153,
165, 171, 176, 196, 201
photographs, 160
physical activity, 79, 114
physiology, ix, 29, 55, 60, 80, 107, 109
plasma membrane, 4, 5, 7, 15, 18, 19, 52, 88, 94, 99
plasminogen, 43
plasticity, 122, 157, 190
platform, 64, 67, 71, 72, 77
point mutation, 15, 35
Poland, 36
polarization, 116
polymerase, 5, 6, 105, 127
Index 230
polymorphism, 179, 180, 181, 182, 185, 186, 187,
188
polymorphisms, 179, 180, 181, 185
polypeptide, 161
polyphenols, 132, 146
polyunsaturated fat, 114, 147
polyunsaturated fatty acids, 114, 147
positron, 128, 143, 216
positron emission tomography, 128, 143, 216
potassium, 68, 122, 202, 206
prefrontal cortex, 187, 218
prevention, 27, 42, 81, 84, 105, 115, 165, 212
primary cells, 65
primary function, 108, 206
primate, 18, 122, 164, 220
probability, 89
probe, 67, 71, 72, 77, 159
progesterone, 140, 153
prognosis, 128
progressive neurodegenerative disorder, 216
progressive supranuclear palsy, 187
project, 190, 191, 204
proliferation, 3, 4, 11, 12, 13, 14, 25, 32, 33, 41, 85,
152, 153, 154, 165, 170
promoter, 7, 49, 55, 166, 172, 179, 180, 182, 186
prophylactic, 81, 138
prostate cancer, 177
protease inhibitors, 19, 21, 41
proteases, 68, 69, 89, 104, 125, 126, 152
protective mechanisms, 145
protective role, xi, 30, 115, 117, 189
protein family, 102
protein folding, vii, 1, 2, 4, 8, 20, 28, 40, 43
protein kinases, 60, 127
protein misfolding, 20, 47
protein oxidation, 4
protein sequence, 50
protein structure, 40
protein synthesis, vii, 1, 46
proteolysis, 36, 89, 101, 126, 137, 150, 151, 174, 177
proteome, 24
protons, ix, 7, 107, 108, 109, 110
proto-oncogene, 33
psychiatric disorders, 65
psychosis, 81, 181
pumps, 6
pyloric stenosis, 186
pyrimidine, 158
Q
quality of life, 65, 79, 210
quinolinic acid, 111, 120
quinone, 8, 76, 83
quinones, 76
R
radiation, 8, 9, 10, 31, 32, 33, 46, 65
radical formation, 82, 164
radicals, ix, 3, 4, 23, 27, 29, 34, 78, 107, 108, 119,
123, 132, 146
radiosensitization, 82
RANTES, 179, 180, 185
reactants, 28
reactions, 4, 6, 8, 22, 97
reactive oxygen, x, 2, 12, 48, 57, 84, 108, 119, 120,
123, 155, 157, 164, 165, 167, 209
reactivity, 211
recognition, 60, 93, 98, 99, 104, 126, 172, 198
recovery processes, 193
recruiting, 133
rectal temperature, 131
red blood cells, 42
regeneration, 85, 88, 99, 195
regression, xii, 70, 212, 215, 217
regression analysis, xii, 215, 217
repair, 8, 9, 10, 13, 27, 29, 57, 64, 84, 140, 143, 177,
178, 184
reparation, 97, 100
replacement, 25
repression, 12, 13, 48
repressor, 11, 12, 177
reserves, 57
residues, 7, 8, 9, 27, 49, 102, 126
resistance, viii, 16, 24, 36, 43, 63, 113, 117, 120, 206
respiration, 6, 9, 11, 32, 108, 109, 112, 113, 114,
116, 120
respiratory syncytial virus, 14, 34
responsiveness, 158
retardation, 13
reticulum, vii, 1, 4, 27, 28, 29, 31, 32, 35, 38, 40, 41,
43, 54, 57, 101, 126, 143
retina, xi, 82, 203, 204, 205, 207, 208, 209, 210, 211,
213, 214
retinal detachment, 204
retinitis, 214
retinitis pigmentosa, 214
retinoblastoma, 58
retinopathy, vii, xi, 203, 204, 208, 209, 211, 212, 213
retirement, 80
retrovirus, vii, 1, 2, 14, 15, 19, 20, 21, 22, 23, 26, 28,
29, 34, 35, 37, 38, 39, 42, 43
retroviruses, vii, 1, 3, 14, 15, 18, 20, 40
rhythm, 158
ribose, 5, 6, 105, 127
Index 231
RNA, 34, 50, 112, 161, 177, 186
RNAi, 112
rodents, 50, 56, 64, 67, 77, 111, 120
room temperature, 68, 90, 92
rosiglitazone, 140, 153
S
sarcopenia, 65, 79
SARS, 37
SARS-CoV, 37
scavengers, 132, 138, 146
schizophrenia, 181, 187
sclerosis, viii, 3, 8, 27, 29, 34, 40, 45, 46, 54, 56, 57,
58, 59, 60, 114, 123, 124, 177, 184
screening, viii, 63, 179, 184
secretion, 8, 15, 24, 88, 133, 154
self-assembly, 59
senescence, viii, 4, 63, 64, 65, 66, 73, 79, 81
senile dementia, 83, 198
sensitivity, xii, 8, 9, 31, 115, 121, 204, 205
septic shock, 65
septum, xi, 189, 190
sequencing, 176
serine, 49
serotonin, 78, 83
serum, 81, 92, 113, 179, 185, 209
serum albumin, 92, 113
shock, viii, 31, 45, 46, 47, 48, 50, 51, 56, 57, 58, 59,
60, 61, 65
shrinkage, 192, 202
sialic acid, 164
siblings, 185
side effects, 65, 79, 204
signal transduction, vii, 1, 6, 9, 30, 47, 52, 127
signaling pathway, xii, 9, 14, 38, 46, 112, 140, 158,
165, 192, 204
signalling, 118, 198, 201, 205
signals, 6, 22, 26, 49, 148, 159
signal-to-noise ratio, 92
signs, 13, 15, 19, 64, 176, 198
silver, 162, 163, 164
Sinai, 28
siRNA, 10, 13
skeletal muscle, 80, 83, 109, 116
skin, 126
SLPI, 177, 184
smoking, 196, 201
SNP, 182
sodium, 57, 209
software, 67, 69, 92
spasticity, 176
spatial learning, 80
spatial memory, viii, 63, 64, 67, 71, 72, 77, 80, 84,
148, 193, 199
species, x, 2, 12, 27, 28, 48, 57, 58, 76, 84, 108, 112,
119, 120, 123, 127, 130, 132, 139, 155, 156, 157,
164, 165, 167, 190, 209
spectroscopy, 120, 128
speech, 175
spinal cord, 50, 56, 58, 88, 105, 113, 116, 140, 154,
178
spinal cord injury, 117, 140
sprouting, 196
stabilization, 42, 170
stem cells, 10, 25, 27, 32, 33, 43, 84, 85, 141, 154
stenosis, 186
stimulus, 50
stomach, 109
stressors, x, 156
striatum, xii, 82, 116, 130, 131, 190, 215, 216, 218
stroke, vii, ix, 88, 107, 108, 114, 117, 118, 124, 127,
145, 153, 212
stromal cells, 141, 154
structural changes, 148
substitutes, 105
substitution, 175, 180
substitutions, 175
substrates, 4, 10, 19, 103, 109, 110, 126, 134
Sun, 89, 105, 148
suppression, 12, 32, 152, 153
surveillance, 4
survival, xi, 7, 11, 13, 17, 24, 25, 26, 30, 37, 40, 42,
46, 52, 53, 54, 59, 91, 102, 108, 112, 116, 118,
122, 131, 132, 133, 141, 162, 163, 165, 167, 170,
177, 190, 192, 193, 195, 199, 208
survival rate, 113
susceptibility, xi, 150, 158, 163, 171, 174, 179, 180,
181, 184, 188
suspensions, 82
swelling, 54, 89, 94
symptoms, vii, 2, 176, 195, 196, 216
synapse, 192
synaptic plasticity, 122, 147, 157
synaptic transmission, 157
synaptic vesicles, 191
syndrome, 11, 14, 19, 26, 32, 34, 43, 161, 170, 175
synergistic effect, 132
synthesis, vii, viii, 1, 31, 46, 63, 79, 82, 108, 110,
122, 146, 171, 195
T
T cell, 10, 15, 20, 22, 34, 35, 37
tandem repeats, 179, 181
tangles, x, xi, 114, 173, 174, 189, 193, 196
Index 232
tau, x, 13, 33, 114, 173, 174, 176, 183, 184, 187,
196, 201
TBI, 126, 129, 135, 136, 137, 138, 139, 140, 141,
142
technical assistance, 80
telangiectasia, vii, 1, 8, 30, 31, 32, 33, 43
temperature, 34, 35, 68, 69, 90, 92, 111, 118, 120
temporal lobe, 175, 194, 201
tension, 68, 73
terminals, xii, 193, 215, 216, 218
termination codon, 178
testing, viii, 63, 64, 67, 68, 207, 210
thalamus, 111, 130, 135, 136, 144, 190
therapeutic agents, 84, 200, 210
therapeutic approaches, xi, 22, 37, 190
therapeutic intervention, viii, 45, 108, 125, 196
therapeutic interventions, viii, 45, 196
therapeutic targets, vii, ix, xii, 107, 117, 204, 210
therapeutics, 127, 137, 182, 219
therapy, 21, 25, 26, 102, 115, 128, 138, 139, 141,
142, 147, 148, 152, 154, 197, 198, 201
thermoregulation, 111
tissue, ix, 4, 10, 41, 43, 64, 85, 88, 89, 107, 108, 109,
110, 111, 115, 116, 119, 130, 132, 133, 137, 138,
141, 144, 145, 150, 151, 166, 184, 193, 199, 201,
209
tissue plasminogen activator, 43
TNF, 17, 36, 179
toxicity, 41, 42, 54, 56, 58, 65, 83, 104, 123, 137,
157, 164, 165
toxicology, 146, 170
toxin, 193
training, 67, 71
transcription, x, 7, 17, 38, 47, 48, 50, 51, 55, 58, 59,
60, 61, 140, 164, 166, 167, 170, 174, 177, 181,
186
transcription factors, 50, 59, 164, 167
transcripts, 120, 181
transduction, vii, 1, 6, 9, 30, 47, 52, 56, 157
transection, 195, 200
transferrin, 197, 199
transformations, 91
translation, 4, 10, 52, 56, 186
translocation, 7, 17, 48, 49, 53, 58, 90, 98, 134, 143,
166, 169, 170, 206
transmission, 95, 157, 192
transplantation, 25
transport, ix, x, 2, 4, 5, 6, 9, 40, 65, 78, 83, 102, 107,
108, 110, 123, 167, 173, 174, 192, 193, 197, 201,
206, 209, 214
trauma, 117, 124, 135, 138, 141, 148, 149, 152, 153,
207
traumatic brain injury, 104, 125, 126, 148, 149, 150,
151, 152, 153, 154
tremor, 115
trial, 41, 67, 71, 72, 77, 81, 201, 212
triggers, 28, 48, 88, 150, 192, 202
tropism, 34
trypsin, 90
tumor, 32, 33, 43, 146, 148, 161, 185
tumor cells, 146
tumor necrosis factor, 148, 161, 185
tumorigenesis, 184
turnover, 4, 116
type 1 diabetes, 212
type 2 diabetes, 24, 43, 121, 207, 212
tyrosine, 18, 116, 165, 171, 192
tyrosine hydroxylase, 116
U
UK, 34
ultrasonography, 127
UN, 38
underlying mechanisms, 207
uniform, 88
UV, 46, 65, 102, 214
UV light, 65, 214
UV radiation, 46
V
vaccine, 34, 41
validation, 160
valine, 131, 180
vascular dementia, 187
vascular endothelial growth factor (VEGF), 24
vasculature, 196
vasodilation, 133
viral infection, viii, 2, 3, 27
virus infection, vii, 1, 37
viruses, 14, 15, 17, 37
vision, 71, 204
visual acuity, 204
visual field, 204
visualization, 103
vitamin B3, 131
vitamin C, 209
vomiting, 196
vulnerability, 30, 54, 60, 116, 123, 150, 156, 157,
163
Index 233
W
wealth, 54, 125
weight loss, 196
western blot, 50, 132
white matter, 135, 145
wild type, 10, 116
withdrawal, 160, 193
workers, 115, 117
working memory, 193
Y
yeast, 120
Z
zinc, 112

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