Apoptosis 1996; 1:5-10
Papers
Neuronal apoptosis v e r s u s necrosis induced by
glutamate or free radicals
P. N i c o t e r a , *w M. A n k a r c r o n a , * E. B o n f o c o , * S. O r r e n i u s *
a n d S. A. L i p t o n *
w of Biology, University of Konstanz, Konstanz, Germany; *institute of
Environmental Medicine, Division of Toxicology, Karolinska Institute, Stockholm,
Sweden; ~;Laboratory of Cellular and Molecular Neuroscience, Children's Hospital
and Program in Neuroscience, Harvard Medical School, Boston MA, USA
The type of cell death encountered in neuronal cell
cultures exposed to excitatory amino a c i d s - s u c h as
glutamate, the major excitatory neurotransmitter in the
central nervous system, or free radicals, such as nitric
oxide (NO') and superoxide anoin (O2"'), which react
to form peroxynitrite ( O N O O ' ) - appears to depend on
the intensity of the exposure and may involve two
temporarily distinct phases. Following relatively ful-
minant insults, an initial phase of necrosis - associated
with extreme energy depletion - may simply reflect the
failure of neurons to carry out the 'default' apoptotic
death program used to efficiently dispose of aged or
otherwise unwanted cells. Neurons recovering mito-
chondrial energy potential after an initial fulminant
insult or following a more subtle inciting injury may
subsequently undergo apoptosis, possibly associated
with a factor released from mitochondria that triggers
this death program. The maintenance of balanced energy
production may be a decisive factor in detemining the
degree, type, and progression of neuronal injury
caused by 'excitotoxins' and free radicals. Similar
events could possibly occur in vivo after ischemia or
other insults.
Key words: Apoptosis; excitotoxicity; glutamate; cytoskeleton;
nitric oxide; NMDA.
Received 9 J u n e 1996; accepted 2 4 J u n e 1996.
Apoptosis versus necrosis: two
distinct forms of neuronal cell death
Apoptosis and necrosis are different modes of cell death
with distinctive morphological and molecular features,
as well its with entirely different effects on the surrounding
Correspondence to Dr. Stuart A. Lipton, Laboratory of Cellular
and Molecular Neuroscience, Children's Hospital, 300
Longwood Avenue, Enders Bldg, Suite 361, Boston, MA 02115
USA. Tel: 617-355-6440; Fax: 617-730-0636; e-mail:
lipton_s@al .tch.harvard.edu
9 1996 Rapid Science Publishers
tissue. Apoptosis is an active process of cell destruction,
characterized by cell shrinkage, chromatin aggregation
with extensive genomic fragmentation, and nuclear
pyknosis. 1'2 In vivo, phagocytic cells normally sequester
antigenically modified apoptotic cells, preventing
inflammation and damage to the surrounding tissue) '4
In contrast, necrosis is characterized by passive cell
swelling, intense mitochondrial damage with rapid
energy loss, and generalized disruption of internal homeo-
stasis. This swiftly leads to membrane lysis, release of
intracellular constituents that evoke a local inflammatory
reaction, edema, and injury to the surrounding tissue)
Apoptosis plays an important role in both physiological
and pathological conditions. In the development of the
embryo, overproduction of cells ultimately requires that
surplus elements die as part of the process that balances
growth and differentiation with death. In the case of the
central and peripheral nervous systems, apoptosis, occurs
extensively during development. 6 However, dysregula-
tion of apoptosis may also underlie the etiology of several
diseases. For example, suppression of apoptosis may be
involved in the proliferation of preneoplastic or neoplastic
cells and in the development of autoimmune diseases, v
whereas increased apoptosis may be involved in neuro-
degenerative diseases, ~-1~ during retroviral infection
(HIV-1), n and perhaps in the development of diabetes
mellitus.~e'~3
Studies in vitro and in vivo have recently shown that
apoptosis could be the ultimate effect of the neuro-
degenerative processes underlying Alzheimer's disease TM
and Parkinson's diseases, as well as neuronal cell death
in the penumbra of a stroke. 15-Iv
Signaling in apoptosis
Although there are probably multiple signals leading to
apoptosis, recent work suggests that activation of a pro-
Apoptosis. Vol I . No 1 . 1996 5
 P. Nicotera et al.
tease, or family of proteases, related to the nematode
gene ced-3 and human interleukin-1B converting enzyme
(ICE) may be a common point of convergence) 8 Activation
or suppression of this protease family may be triggered
by intracellular messengers such as Ca 2+, cAMP, or by
free radicals, including nitric oxide and superoxide anion.
The biochemical machinery responsible for these events
appears to be present in all mammalian cells, except
blastomeres. Some of these proteolytic systems may be
Ca 2§ dependent, and direct evidence that an increase in
Ca 2§ can mediate apoptotic death has been provided by
several laboratories including ours. 12'19 Thus, elevation
of the intracellular Ca 2. level appears to represent a
relatively common trigger for apoptosis in cells of diverse
tissue origins. Proteins that mediate the effects of Ca 2§
are ubiquitously involved.
The accumulation of reactive oxygen species, resulting
in the perturbation of the cellular prooxidant-antioxidant
balance (oxidative stress), has also been implicated as an
important mechanism of cytotoxicity in a number of
biological systems. Both apoptosis and necrosis can be
induced by oxidative stress. For example, low prooxidant
concentrations induce apoptosis in RINm5F pancreatic
B cells, whereas higher concentrations promote necrosis.2~
The involvement of free radicals in the pathogenesis of
neurodegenerative disorders is suggested by a number of
observations. Behl et al. 21 have recently reported that the
amyloid B peptide kills neural and other cells by H202-
mediated mechanisms. Troy and Shelanski have shown
that suppression of copper/zinc superoxide simustase (Cu,
Zn-SOD) activity induces apoptosis in PC12 cells. 22
Analogously, antioxidants may protect cells from
apoptosis (reviewed by Buttke and Sandstrom23). For
example, apoptotic cell death induced by tumor necrosis
factor (TNF) can be prevented by antioxidants such as
N-acetylcysteine (NAC) and thioredoxin 24'25 or by over-
expression of mitochondrial Mn-SOD. 26There are several
possible roles for oxidation in apoptosis. Protein oxida-
tion may be an essential influence on gene expression
required for the signals leading to apoptosis. Oxidative
events, for example, may affect D N A binding of several
transcription factors. 27'28However, it is also possible that
oxidative events are involved in cytoskeletal alterations
in cells undergoing apoptosis or are required to initiate
either cell shrinkage or changes in higher-order chromatin
structure.
Nitric Oxide (NO) a multifunctional messenger
N O is normally generated from the terminal guanidine
residue of L-arginine by nitric oxide synthase (NOS), with
the consequent co-production ofcitrulline. Four different
forms of NOS have been identified, two of which are
6 Apoptosis. Vol 1 . No I . 1 9 9 6
constitutive in brain and in endothelium and are
activated by Ca 2§ via calmodulin. The third is an
inducible and Ca2§ enzyme expressed in
astrocytes or activated macrophages and in cytokine-
stimulated pancreatic B-cells, .3 and the last has been
cloned from human hepatocytes.
N O has a dual role as physiological messenger and as
a contributor to lethal processes. Classically, N O
mediates endothelium-dependent relaxation, takes part
in neurotransmission, and is a key player in the cellular
immune response. 29 Multiple reactions occur between
oxygen, superoxide, and transition metals with the
following products: N203 [equivalent to (NOa-)(NO*)],
peroxynitrite (OONO-), and metal-NO adducts, respec-
tively. These reactions determine the biological activity
of the N O group in its various redox-related forms. Other
reactions involving N O t transfer result in nitrosative
reactions at nucleophilic centers with the prevalence of
S-nitrosothiol formation) ~ Accordingly, thiol- and tran-
sition metal-containing proteins serve as major target
sites for NO-related species) ~ NO-target interaction
achieves both cGMP-dependent and cGMP-independent
transducing mechanisms. Cyclic GMP-independent NO-
induced responses account for the antimicrobial, the
cytostatic, and the cytotoxic capacity of NO. Excess
production of N O has been shown to underlie, at least
in part, glutamate-induced neuronal toxicity in cultures
of cortical and striatal neurons) 2 It has also been reported
that N O mediates apoptosis in murine peritoneal macro-
phages 33 and in the mouse pancreatic beta cell line
RINm5F. 13 Notably, the onset of cell killing was corre-
lated to increased NO, produced directly by NO-donors 3~
or induced by interleukin-l[3.13 In the latter study, the
lethal role of N O generation was confirmed by the
protective action of a medium containing an inhibitor
of NOS, N - m o n o m e t h y l a r g i n i n e . Finally, radicals
generated by the interaction of N O with oxygen species
can induce DNA damage) 4 Our studies, described in the
following sections, have recently focused on the
cytotoxicity of nitric oxide in conjunction with reactive
oxygen species in cortical neurons and cerebellar granule
cells.
NO-induced neuronal cell death: contribution
of apoptosis
Excessive activation of excitatory amino acid receptors
has been implicated as a mechanism for neurotoxicity in
both acute and chronic neurologic diseases)5-38This form
of neuronal cell death has been termed 'excitotoxicity.'
The underlying process responsible for neuronal cell
death after overactivation of glutamate receptor subtypes
- of which the N-methyl-D-aspartate (NMDA) receptor

is one of the most prominent because of its permeability
to Ca 2" - have only recently begun to be clarified. In a
variety of neurologic diseases, such as stroke and head
trauma, excitotoxicity may be related to excessive
glutamate release and/or lack of clearance, which results
in excessive stimulation of NMDA-receptors) 6 This can
result in either an acute or chronic process, possibly
dependent on the level of N M D A receptor stimulation.
O t h e r excitatory amino acid receptor subtypes also
contribute to these processes, but in many cases the
N M D A receptor has a prominent role.
The interaction of glutamate with excitatory amino
acids receptors initiates a cascade of events involving
excessive Ca 2§ entry and activation of several enzymes,
including phospholipases, proteases, and N O S ) 2'~5-~9
Phospholipase A2 activation leads to the generation of
arachidonic acid and other metabolites as well as to the
formation of oxygen free radicals. This can lead to an
oxidative stress and, together with the activation of Ca 2,-
dependent catabolic processes, 4~ can lead to either
necrosis or apoptosis. 4~ In addition, it has been shown
that N O - generated by activation of NOS - can react
with 02- (superoxide anion), forming O O N O - (peroxyni-
trite). 42 This substance, or one of its degradation
products, is neurotoxic 43 and can be present during
inflammation, sepsis and ischemia/reperfusion. In fact,
N O alone, even at high levels, is not toxic to cortical
neurons, but only becomes neurotoxic after reaction with
02- to form O N O O ) 3
We recently investigated whether apoptosis or necrosis
would be induced in cerebrocortical neurons in culture
by excessive glutamate receptor activation or by its down-
stream products, N O and 02-. We used high and low
concentrations of glutamate agonists (such as NMDA),
NO-donors [such as 3-morpholinosydnonimide (SIN-l)
and S - n i t r o s o c y s t e i n e ( S N O C ) ] , or p e r o x y n i t r i t e
(OONO-). 44 We found that exposure of cortical cultures
to relatively short durations or low concentrations of
N M D A , SNOC, S I N - l , or peroxynitrite induced delayed
neuronal cell death characterized by apoptotic features.
In contrast, intense exposure to high concentrations of
N M D A or peroxynitrite induced relatively rapid necrotic
cell death in neurons. SOD and catalase attenuated neuronal
cell death, most likely by reducing the formation of
peroxynitrite since they were only effective if peroxyni-
trite had not yet formed. These findings suggest that the
intensity of the original insult may determine the ensuing
pathway to either necrotic or apoptotic neuronal cell
death. The nature of the original insult as well as the
decision to enter the necrotic versus the apoptotic pathway
might have therapeutic implications in terms of the
p o s s i b l e effectiveness o f S O D / c a t a l a s e or N M D A -
a n t a g o n i s t s , as well as the necessary timing of such
interventions.
Neuronal apoptosis in excitotoxicity
Possible mechanisms for NO-induced neuronal
apoptosis: role of cytoskeletal alterations
Alterations in the neuronal cytoskeletal architecture are
some of the most generalized neuropathological changes
in neurodegenerative disorders: abnormally phosphory-
lated forms of the microtubule-associated z protein are
typical histological features of Alzheimer's disease 45'46
and are found after exposure to agents that elicit Ca 2§
overload; 47 spherical inclusions ofneurofilaments are seen
in Parkinson's disease; and accumulation of abnormally
phosphorylated neurofilament proteins in motoneurons
is a feature of amyotrophic lateral sclerosis. In apoptosis,
important cytoskeletal changes precede the breakdown
of the nucleus and other typical cellular alterations. For
example, fragmentation of lamins (cytoskeletal elements
surrounding the nucleus) has been observed in relation-
ship to formation of large D N A fragments 48 and precedes
fragmentation of chromatin in neurons exposed to
glutamate (M. Ankarcrona and P. Nicotera, unpublished
observations). However, the role of cytoskeletal altera-
tions in the development of apoptosis remains unknown.
In particular, it is unclear whether such alterations are
an unavoidable side-effect of cell volume changes and
nuclear pyknosis, or, in contrast, these events are
inextricably linked to a chain of events leading to the
apoptotic program.
We have recently investigated the effects of primary
cytoskeletal damage on the survival of cerebellar granule
cells. 49 A m o n g several cytoskeletal agents, we used
colchicine, a microtubule disrupting drug which also
promotes x protein alterations. Colchicine binds to and
depolymerizes microtubules. Exposure to colchicine also
elicits alterations similar to those found in Alzheimer's
disease, including increased neuronal immunoreactivity
for the modified ~ protein found in neurofibrillary tan-
gles) ~ Additionally, it has been shown that in vivo
treatment with colchicine causes neurofibrillary degen-
eration 51 and neuronal cell loss) 2 Considering these
properties, colchicine has been used as an in vitro model
to mimic some cytoskeletal alterations found in neurode-
generative disorders, such as Alzheimer's disease. Our
results showed that both 0c and B-tubulins as well as
"c-protein were rapidly fragmented after treatment of
cerebellar granule cells with colchicine. Such alterations
are accompanied by Ca 2+ channel modification and over-
expression of FOS protein and NOS, the latter associated
with an increased production of NO. Following these
events, cerebellar granule cells u n d e r w e n t typical
apoptosis. To investigate which event was more relevant
for the induction of apoptosis in colchicine-treated cells,
we used Ca 2+ channel blockers, inhibitors of the NOS,
and taxol, a microtubule stabilizing agent. The results
showed that neither Ca 2. channel alterations nor the
Apoptosis. Vol 1 9 No 1 . 1 9 9 6 7
P. Nicotera et al.
increase in NOS activity per se were sufficient to cause
apoptosis in this system. In contrast, taxol prevented the
cytoskeletal alterations and rescued the neuronal cells
from apoptosis. 49
Disturbances of cytoskeletal organization can be
caused by several mechanisms including modifications
in protein phosphorylation/dephosphorylation, Ca 2~
overload, protease activation, or protein-protein cross
linking. Among these factors, Ca 2§ overload and oxidative
stress are known to produce cytoskeletal alterations prior
to cell death. 4~ A role for cytoskeletal alterations in the
onset of neuronal apoptosis associated with N O is
supported by our very recent findings that N O donors
induce microfilament and microtubule disruption and
that apoptosis is partially prevented by taxol. The mecha-
nism whereby N O contributes to damage of the
cytoskeleton and induces apoptosis i s currently under
investigation in our laboratories.
Glutamate elicits necrosis or apoptosis
depending on mitochondrial function
Recently our laboratories began to investigate the mecha-
nism(s) underlying the decision of neurons to undergo
apoptosis or necrosis following glutamate exposure. 41We
found that glutamate can induce either early necrosis or
delayed apoptosis in cerebellar granule cell cultures,
similar to the aforementioned results on cerebrocortical
neurons. During and shortly after exposure to glutamate,
a subpopulation of cerebellar granule cells died by
necrosis. In these neurons, mitochondrial membrane
potential collapsed, nuclei swelled, and intracellular
debris were scattered in the incubation medium. Neurons
surviving the early necrotic phase recovered mitochon-
drial potential and energy levels. Later, they underwent
apoptosis, as shown by the formation of apoptotic nuclei
and chromatin degradation into high and then low
molecular weight fragments. These results suggest that
mitochondrial function is a critical factor that helps
determine the mode of neuronal cell death in excitotoxicity.
During exposure to glutamate in our experiments, both
mitochondrial membrane potential and ATP levels
declined in many neurons. In those with irreversibly
dissipated mitochondrial membrane potentials, necrosis
rapidly ensued. The initial surviving neuronal population
recovered both mitochondrial membrane potential and
energy levels, and subsequently underwent delayed apop-
tosis. In fact, prior to the time that DNA fragmentation
and apoptotic nuclei were evident, mitochondrial
membrane potential, metabolism, and energy charge had
been restored to near normal levels. At that point,
repeated challenge with glutamate resulted in repeated
collapse of mitochondrial membrane potential, rapidly
8 Apoptosis. Vol 1. No 1. 1996
followed by necrosis of a second neuronal subpopulation.
According to one hypothesis, loss of energy and onset of
rapid necrosis simply prevent the activation of the
'default' apoptotic cell death program. This postulate is
supported by the observation that treatment of cerebellar
granule cells with the combination of a relatively low
concentration of glutamate (100 ~M) plus the irreversible
mitochondrial uncoupler carbonyl cyanide m-chlorophenyl-
hydrazone (CCCP, 10 ~tM), but not CPP alone resulted
in necrosis of 60% of the neuronal population rather than
in the delayed apoptosis observed with this low concen-
tration of glutamate alone.
The role of mitochondria in apoptosis is still a matter
of intense debate. The observation that apoptosis occurs
in respiration-deficient cells had cast doubts on the role
of mitochondrial energy production in apoptotic cell
death. 53 Similarly, in a n o t h e r p r e p a r a t i o n where
mitochondrial factors had been suggested to induce the
nuclear changes typical of apoptosis, inhibitors of
mitochondrial respiration had little effect in preventing
nuclear damage. 54 However, in both of these studies ATP
production was provided by non-mitochondrial sources.
Our more recent studies suggest that neurons do not die
by apoptosis when energy levels are very low. 4t
Since mitochondria are the main source of ATP in
neuronal cells, in the absence of substrate supplementa-
tion, collapse of mitochondrial membrane potential and
impaired respiration causes ATP depletion. Our experi-
ments suggest that under these conditions necrosis
intervenes before the apoptotic program has a chance to
develop. 41 Possibly, mitochondrial energy generation
may be needed to maintain the osmotic integrity of the
neuron via ion exchange mechanisms to avoid cell lysis.
Energy levels can easily be maintained by substrate
supplementation in experimental model systems in vitro.
Under these conditions, after an insult to the neurons
one would expect that necrosis could be avoided and
apoptosis would be able to develop with time. In contrast,
in vivo after a fulminant insult it would be very difficult
to envisage sufficient ATP generation under anaerobic
conditions to maintain active cellular processes for very
long in most neurons, and thus necrosis would be favored.
Nevertheless, a relatively minor loss of mitochondrial
membrane potential and energy production may still be
important as a signal for apoptosis, as long as adequate
energy production is maintained for the progression of
the apoptotic process. For example, a decrease in mito-
chondrial membrane potential - such as that observed
in our studies during the initial phases of glutamate
injury - may produce the release of signaling molecules
from the mitochondria that could trigger downstream
degradative processes (Figure 1). Ongoing work in our
laboratories appears to favor such an hypothesis.
In conclusion, the type of cell death encountered in

Figure 1. Model incorporating several features of apoptotic injury
in neurons. After a relatively mild insult by glutamate at the NMDA
receptor (generating NO and O2" to form ONOO) a drop in ATP
may result, but not a sufficient compromise in energy to severely
disrupt the pumps and result in necrosis. Nonetheless, increase
Ca2+ cycling into the mitochondrial matrix ensues, possibly
because of injury to the respiratory chain via ONOO. As ATP
synthesis recovers, an as yet unknown mitochondrial signal is
produced and released via the mitochondrial permeability transition
pore. This mitochondrial factor then contributes to the apoptotic
pathway, eventually triggering nuclear chromatin fragmentation.
X glucose
glutamate
NMDA Recep
.......... I
mitochondrial
H+ neuronal
yloplasrn
ATP resphratory mitrochondrial
synlhase chain matrix
neuronal cell cultures exposed to glutamate may depend
on the intensity of the exposure and may involve two
temporally distinct phases. Similar events could possibly
occur in vivo after ischemia or other insults. Necrosis -
associated with extreme energy depletion - may simply
reflect the failure of neurons to carry out the 'default'
apoptotic death program used to efficiently dispose of
aged or otherwise unwanted cells. The maintenance of
balanced energy production may be a decisive factor in
d e t e r m i n i n g the degree, type, and progression of neuro-
nal injury caused by excitotoxins and free radicals.
Acknowledgements
This manuscript is adapted from an oral and written
p r e s e n t a t i o n at the Asilomar Conference on Neural
Regeneration, held in December, 1995 in Pacific Grove,
California. The work was supported in part by grants
from the ILSI (International Life Science Institute), The
Swedish Natural Science Research Council (NFR) No.
B-AA/BU 101 73-301, The Swedish Medical Research
Council (MFR) No. 03 X- 2471 (to PN), and N I H grants
P01 H D 2 9 5 8 7 and R01 EY09024 (to SAL).
References
1. Kerr JFR, Wyllie AH, Currie AR. Apoptosis: a basic biological
phenomenon with wide ranging implications in tissue kinetics.
B r j Cancer 1972; 26: 239-257.
Neuronal apoptosis in excitotoxicity
2. Wyllie AH, Kerr, JFR Currie AR. Cell death: The significance
of apoptosis. Int Rev Cytol 1980; 68:251-306.
3. Duvall E, Wyllie AH, Morris RG. Macrophage recognition of
cells undergoing programmed cell death (apoptosis). Immunol-
ogy 1985; 56: 351-358.
4. SavillJS, Fadok V, Henson P, Haslett C. Phagocyte recognition
of cells undergoing apoptosis. Immunol Today 1993; 14:131-
136.
5. SchwartzLM, Smith SW, Jones MEE, Osborne BA. Do all pro-
grammed cell deaths occur via apoptosis? Proc Natl Acad Sci
USA 1993; 9O: 980-984.
6. Deckwerth TL, Johnson Jr EM. Neurotrophic factor depriva-
tion-induced death. Ann N Y Acad Sci 1993; 679:121-131
7. Thompson CB. Apoptosis in the pathogenesis and treatment
of disease. Science 1995; 267: 1456-1462.
8. Kure S, Tominaga T, Yoshimoto T, Tada K, Narisawa K. Glu-
tamate triggers internucleosomal DNA cleavage in neuronal
ceils. Biochem Biophys Res Commun 1991 ; 179: 39~45.
9. MacManusJp, Buchan AM, Hill IE, Rasquinha I, Preston E.
Global ischemia can cause DNA fragmentation indicative of
apoptosis in rat brain. NeuroscienceLett 1993; 164:89-92
10. Raft MC, Barres BA, Burne JF, Coles HS, Ishizaki Y, Jacob-
sonMD. Programmed cell death and the control of cell survival:
Lessons from the nervous system. Science 1993; 262: 695-700.
11. Ameisen JC, Capron A. Cells dysfunction and depletion in
AIDS: the programmed cell death hypothesis. Immunol Today
1991; 12: 102-105.
12. Juntti-Berggren L, Larsson O, Rorsman P, et al. Increased
activity of L-TypeCa2*channels exposed to serum from patients
with Type I diabetes. Science 1993; 261: 86-90.
13. Ankarcrona M, Dypbukt JM, Briine B, Nicotera P. Interleukin
1B-induced nitric oxide production activates apoptosis in pan-
creatic RINm5F cells. Experiment Cell Res 1994; I213: 172-177.
14. Games D, Adams D, Alessandrini R, et al. Alzheimer-type
neuropathology in transgenic mice overexpressing V717F 9-
amyloid precursor protein. Nature 1995; 373: 523-527.
15. Linnik MD, Zobrist RH, Hatfield MD. Evidence supporting
a role for programmed cell death in focal cerebral ischemia in
rats. Stroke 1993; 24: 2002-2009.
16. MitchellJ, LawsonS, Moser B, Laidaw SM, Cooper AJ, Walkin-
shaw G, Waters CM. Glutamate-induced apoptosis results in
a loss of striatal neurons in the Parkinsonian rat. Neuroscience
1994; 63: 1-6.
17. Su JH, Anderson AJ, Cummings BJ, Cotman CW. Immuno-
histochemical evidence for apoptosis in Alzheimer's disease.
NeuroReport 1994; 5: 2529-2533.
18. Yuan J, Shaham S, Ledoux S, Ellis HM, Horvitz HR. The c.
elegans cell death gene ced-3 encodes a protein similar to
mammalian interleukin-1B-convertingenzyme. Cell 1993; 75:
641-652.
19. Nicotera P, Zhivotovsky B, Bellomo G, Orrenius S. Ion
signalling in apoptosis. In: Schimke RT, Mihich E, eds. Apop-
tosis. New York: Plenum Press, 1994:97-115.
20. Dypbukt JM, Ankarcrona M, Burkitt M, Sj6holm ~l, Str/Sm
K, Orrenius S, Nicotera P. Different prooxidant levelsstimulate
growth, trigger apoptosis, or produce necrosis of insulin-
secreting RINm5F cells: The role of intracellular polyamines.
J B i d Chem 1994; 269: 30553-30560.
21. Behl C, Davis JB, Lesley R, Schubert D. Hydrogen peroxide
mediates amyloid B protein toxicity. Cell 1994; 77: 817-827.
22. Troy CM, Shelanski ML. Down-regulation of copper/zinc
superoxide dismutase causes apoptotic death in PC12 neuronal
cells. Proc NatIAcadSci USA 1994; 91: 63844387.
23. Buttke TM, Sandstrom PA. Oxidative stress as a mediator of
Ap@tosis. Vol 1 . No I . I996 9
P. Nicotera et al.
apoptosis. Immunol Today 1994; 15: 7-10.
24. Chang DJ, Ringold GM, Heller RA. Cell killing and induction
of manganous superoxide dismutase by tumor necrosis factor-
alpha is mediated by lipoxygenase metabolites of arachidonic
acid. Biochem Biophys Res Commun 1992; 188: 538-546.
25. Matsuda M, Masutani H, Nakamura H,etal. Protective activity
of adult T cell leukemia-derived factor (ADF) against tumor
necrosis factor dependent cytotoxicity on U937 cells.J Immunol
1991; 147: 3837-3841.
26. Hirose K, Longo DL, Oppenheim JJ, Matsushima K. Over-
expression of mitochondrial manganese superoxide dismu~_ase
promotes the survival of tumor cells exposed to interleukin-1,
tumor necrosis factor, selected anticancer drugs, and ionizing
radiation. FASEBJ 1993; 7: 361-368.
27. Abate C, Patel N, Rauscher III FJ, Curran T. Redox regulation
of Fos and Jun DNA-binding activity in vitro. Science 1990;
29: 1157-1161.
28. Meyer M, Schreck R, Baeuerle PA. H202 and antioxidants
have opposite effects on activation of NF-KB and AP-1 in
intact cells: AP-1 as secondary antioxidant-responsive factor.
EMBOJ 1993; 12: 2005196;2015.
29. Nathan C. Nitric oxide as a secretory product of mammalian
cells. FASEBJ 1992; 6: 3051-3064.
30. Mohr S, Stamler JS, Briine B. Mechanism of covalent modi-
fication of glyceraldehyde-3-phosphate dehydrogenase at its
active site thiol by nitric oxide, peroxynitrite and related
nitrosating agents. FEBS Letters 1994; 348: 223-227.
31. Stamler JS. Redox signaling: nitrosylation and related target
interactions of nitric oxide. Cell 1994; 78: 931-936.
32. Dawson VL, Dawson TM, London ED, Bredt DS, Snyder SH.
Nitric oxide mediates glutamate neurotoxicity in primary cor-
tical cultures. Proc Natl Acad Sci USA 1991; 88: 6368-6371.
33. Albina JE, Cui S, Mateo RB, Reichner JS. Nitric oxide-
mediated apoptosis in murine peritoneal macrophages. J
Immunol 1993; 150: 5080-5085.
34. Noronha-Dutra AA, Epperlein MM, Woof N. Reaction of
nitric oxide with hydrogen peroxide to produce potentially
cytotoxic singlet oxygen as a model for nitric oxide-mediated
killing. FEBS Lett 1993; 321: 59~62.
35. Choi DW. Glutamate neurotoxicity and diseases of the nervous
system. Neuron 1988; 1: 623-634.
36. Lipton SA, Rosenberg RA. Mechanisms of disease: Excitatory
amino acids as a final common pathway in neurologicdisorders.
N EngIJ Meal 1994; 330: 613-622.
37. Coyle JT, Puttfarcken P. Oxidative stress, glutamate, and
neurodegenerative disorders. Science 1993; 262: 689-695.
38. Meldrum B, Garthwaite J. Excitatory amino acid neurotoxicity
and neurodegenerative diseases. Trends Pharmacol Sci 1990; 11:
379-387.
39. Manev H, Favaron M, Guidotti A, Costa E. Delayed increase
of Ca2§ influx elicited by glutamate: role in neuronal death.
Mol Pharmacol 1989; 36: 106-112.
40. Nicotera P, Bellomo G, Orrenius S. Calcium-mediated mecha-
10 Apoptosis. Vol 1 9No 1 9 1996
nisms in chemically induced cell death. Annu Rev Pharnmcol
Toxicol 1992; 32: 449-470.
41. Ankarcrona M, Dypbukt JM, Bonfoco E, Zhivotovsky B,
Orrenius S, Lipton SA, Nicotera P. Glutamate-induced neuro-
nal death: a succession of necrosis or apoptosis depending on
mitochondrial function. Neuron 1995; 15: 961-973.
42. Beckman JS, Beckman TW, Chen J, Marshall PA, Freeman
BA. Apparent hydroxyl radical production by peroxynitrite:
implications for endothelial injury from nitric oxide and
superoxide. ProcNatl AcadSci USA 1990; 87: 1620-1624.
43. Lipton SA, Choi Y-B, Pan Z-H, etal. A redox-based mechanism
for the neuroprotective and neurodestructive effects of nitric
oxide and related nitroso-compounds. Nature 1993; 364: 626-
632.
44. Bonfoco E, Krainc D, Ankarcrona M, Nicotera P, Lipton SA.
Apoptosis and Necrosis: Two distinct events induced respec-
tively by mild and intense insults with NMDA or nitric
oxide/superoxide in cortical cell cultures. Proc Natl Acad Sci
USA 1995b; 91:7162-7166
45. Bancher C, Brunnen C, Lassman H, et al. Accumulation of
abnormallyphosphorylated I;precedes the formation ofneurofi-
brillary tangles in Alzheimer's disease. Brain Res 1989; 477:
90-99.
46. Shankar SK, Yanagihara R, Garruto, NR, Grunke-Iqbal I,
Kosik KS, Gajdusek DC. Ann Neurol 1989; 25: 146-151.
47. Mattson ME Antigenic changes similar to those seen in
neurofibrillarytangles are elicited by glutamate and Ca2§ influx
in cultured hippocampal neurons. Neuron 1990; 2: 105-117.
48. Oberhammer F, Hochegger A, Fr6schl K, Tiefenbacher G,
Pavelka M. Chromatin condensation during apoptosis is
accompaniedby degradation ofLamin A + B, without enhanced
activation of cdc2 kinase.J Cell Biol 1994; 126:827-8
49. Bonfoco E, Ceccatelli S, Manzo L, Nicotera P. Colchicine
induces apoptosis in cerebellar granular cells. Exp Cell Res
1995a; 218:189-200
50. Mattson MP. Effects of microtubule stabilization and destabi-
lization on tau immunoreactivity in cultured hippocampal
neurons. Brain Res 1992; 582: 107-118.
51. Wisniewski H, Terry LD. Experimental colchicine
encephalopathy: induction of neurofibrillary degeneration. Lab
Invest 1967; 17: 577-587.
52. Goldsmidt RB, Stewart O. Neurotoxic effects of colchicines.
Differential susceptibility of CNS neuronal populations. Neuro-
sci 1982; 7: 695-714.
53. Jacobson MD, Bume JF, King MP, Miyashita T, Reed JC, Raff
MC. Bcl-2 blocks apoptosis in cells lacking mitochondrial
DNA. Nature 1993; 361: 365-369.
54. Newmeyer DD, Farschon DM, Reed JC. Cell-free apoptosis
in Xenopus Egg extracts: Inhibition by Bcl-2 and requirement
for an organelle fraction enriched in mitochondria. Cell 1994;
79: 353-364.