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Neurobiology of Disease 68 (2014) 126136

Contents lists available at ScienceDirect

Neurobiology of Disease
journal homepage: www.elsevier.com/locate/ynbdi

Angiotensin II type 1 receptor blocker losartan prevents and rescues


cerebrovascular, neuropathological and cognitive decits in an
Alzheimers disease model
Brice Ongali a, Nektaria Nicolakakis a, Xin-Kang Tong a, Tahar Aboulkassim a, Panayiota Papadopoulos a,
Pedro Rosa-Neto b,c, Clotilde Lecrux a, Hans Imboden d, Edith Hamel a,
a

Laboratory of Cerebrovascular Research, McGill University, Montral, QC H3A 2B4, Canada


Brain Imaging Centre, Montreal Neurological Institute, McGill University, Montral, QC H3A 2B4, Canada
c
Douglas Hospital Research Centre, McGill University, Montral, QC H3A 2B4, Canada
d
Institute of Cell Biology, University of Bern, Switzerland
b

a r t i c l e

i n f o

Article history:
Received 7 February 2014
Revised 17 April 2014
Accepted 27 April 2014
Available online 5 May 2014
Keywords:
AT1 receptor
AT4 receptor
Insulin-regulated aminopeptidase (IRAP)
Memory
Angiotensin receptor blocker (ARB)
Oxidative stress
Amyloid
FDG-PET

a b s t r a c t
Angiotensin II (AngII) receptor blockers that bind selectively AngII type 1 (AT1) receptors may protect from
Alzheimers disease (AD). We studied the ability of the AT1 receptor antagonist losartan to cure or prevent AD
hallmarks in aged (~18 months at endpoint, 3 months treatment) or adult (~12 months at endpoint, 10 months
treatment) human amyloid precursor protein (APP) transgenic mice. We tested learning and memory with the
Morris water maze, and evaluated neurometabolic and neurovascular coupling using [18F]uoro-2-deoxy-D-glucosePET and laser Doppler owmetry responses to whisker stimulation. Cerebrovascular reactivity was assessed
with on-line videomicroscopy. We measured protein levels of oxidative stress enzymes (superoxide dismutases
SOD1, SOD2 and NADPH oxidase subunit p67phox), and quantied soluble and deposited amyloid- (A)
peptide, glial brillary acidic protein (GFAP), AngII receptors AT1 and AT2, angiotensin IV receptor AT4, and
cortical cholinergic innervation. In aged APP mice, losartan did not improve learning but it consolidated memory
acquisition and recall, and rescued neurovascular and neurometabolic coupling and cerebrovascular dilatory
capacity. Losartan normalized cerebrovascular p67phox and SOD2 protein levels and up-regulated those of
SOD1. Losartan attenuated astrogliosis, normalized AT1 and AT4 receptor levels, but failed to rescue the cholinergic decit and the A pathology. Given preventively, losartan protected cognitive function, cerebrovascular
reactivity, and AT4 receptor levels. Like in aged APP mice, these benets occurred without a decrease in soluble
A species or plaque load. We conclude that losartan exerts potent preventive and restorative effects on AD hallmarks, possibly by mitigating AT1-initiated oxidative stress and normalizing memory-related AT4 receptors.
2014 Elsevier Inc. All rights reserved.

Angiotensin II (AngII), the main player of the brain reninangiotensin system (RAS), acts mainly through AngII type 1 (AT1) receptors. In cardiovascular diseases, AngII is present in abnormally high
quantities in the brain (Sweitzer, 2003) where it can up-regulate AT1
receptor expression (Zhu et al., 2011). Deleterious effects of brain

Abbreviations: A, amyloid peptide; ACh, acetylcholine; AD, Alzheimers disease;


AngII, angiotensin II; APP, amyloid precursor protein; AT1, AngII type 1 receptors; AT2,
AngII type 2 receptors; AngIV, angiotensin IV; AT4, AngIV receptor; ARBs, angiotensin
receptor blockers; CBF, cerebral blood ow; CGRP, calcitonin gene-related peptide;
CGU, cerebral glucose uptake; ET-1, endothelin-1; [18F]FDG-PET, [18F]uoro-2-deoxyD-glucose-PET; GFAP, glial brillary acidic protein; L-NNA, N-nitro-L-arginine; MAP,
mean arterial blood pressure; RAS, renin-angiotensin system; SOD1, superoxide dismutase1; SOD2, superoxide dismutase 2; WT, wild-type.
Corresponding author at: Montreal Neurological Institute, 3801 University Street,
Montral, QC, Canada H3A 2B4. Fax: +1 514 398 8106.
E-mail address: edith.hamel@mcgill.ca (E. Hamel).
Available online on ScienceDirect (www.sciencedirect.com).

http://dx.doi.org/10.1016/j.nbd.2014.04.018
0969-9961/ 2014 Elsevier Inc. All rights reserved.

AngII have been imputed primarily to reactive oxygen species (ROS)


generated by membrane-bound NADPH oxidase (Berry et al., 2001;
Cai et al., 2003). AngII may also impair cognitive performance by affecting memory acquisition, consolidation and recall (Braszko et al., 2003;
Tota et al., 2013), lowering cerebral blood ow (CBF) (Tota et al.,
2013; Washida et al., 2010) or brain acetylcholine levels (Tota et al.,
2013). Recent evidence suggests alterations in the brain RAS in
Alzheimers disease (AD) (Kehoe et al., 2009). Yet, benets of targeting
the RAS system for dementia warrant further clarication.
The angiotensin receptor blockers (ARBs), which selectively prevent
the actions of AngII on AT1 receptors, are thought to have positive effects on CBF and neuronal function (Maxwell and Hogan, 2010). Consistent with previously documented protective effects of ARBs on AD
(Fogari et al., 2003; Fournier et al., 2009), recent prospective cohort
(Li et al., 2010) and nested case-control (Davies et al., 2011) analyses reported lower incidence and rate of progression to dementia and AD in
patients prescribed ARBs. Similarly, studies in young amyloid- (A)-

B. Ongali et al. / Neurobiology of Disease 68 (2014) 126136

injected mice (Mogi et al., 2008; Takeda et al., 2009; Tsukuda et al.,
2009) or adult Tg2576 AD mice (Wang et al., 2007) have shown the capacity of ARBs to improve cognition. In contrast, AngII inhibition in
young or aged triple transgenic AD mice indicated no benet on the amyloid, tau or cognitive pathology (Ferrington et al., 2011, 2012). In view
of these contradictory ndings, it is timely and important to further investigate the capacity of brain penetrant ARBs in preventing or reversing cerebrovascular, neuronal and mnemonic impairments in young or
aged AD mice, the latter of highest relevance to AD patients.
Herein, we undertook a three-month therapy with the AT1 receptor
antagonist losartan in ~15 month-old human amyloid precursor protein
(APP) transgenic mice and investigated the impact of treatment on impaired spatial memory in the Morris water maze, CBF and cerebral glucose uptake (CGU) responses evoked by whisker stimulation, and
cerebrovascular reactivity. Molecular correlates of functional responses
were investigated with Western blot and immunohistochemistry. A
companion prophylactic study was conducted wherein losartan was administered in two month-old APP mice until ~12 months of age. Our results revealed that losartan did not restore learning in aged APP mice,
but it consolidated acquisition and recall memory capacities, and fully
rescued cerebrovascular function without reducing the A pathology.
Moreover, losartan downregulated AT1 receptor protein levels and normalized those of the memory-enhancing AT4 receptors. In the prophylactic cohort, losartan prevented the development of cerebrovascular
dysfunction and cognitive deterioration, and concomitantly normalized
AT1 and AT4 receptor levels. These unprecedented data highlight new
mechanisms of recovery in AD pathology, and support losartan for further testing in preventing or curing AD.
Material and methods

127

Table 1
Effects of losartan (L) treatment on body weight gain in aged and adult APP mice, and their
respective wild-type (WT) littermates.
Aged

WT (11)

WT-L (19)

APP (10)

APP-L (12)

Initial (g)
Final (g)
Gain (%)

40.8 3.1
42.5 2.9
4.6 1.2

47.8 2.1
47.8 2.5
0.0 2.9

34.7 3.9
36.4 3.9
5.0 2.8

39.5 3.0
42.4 3.0
6.9 2.1

Adult

WT (15)

WT-L (9)

APP (12)

APP-L (20)

Initial (g)
Final (g)
Gain (%)

27.4 1.0
38.3 1.9
27.0 2.9

25.0 1.2
40.4 2.5
37.2 2.6

24.3 1.4
32.8 2.5
24.2 3.1

25.3 1.0
36.2 1.5
29.8 1.6

Data represent mean SEM. Number of animals is in parentheses. p b 0.05 for


comparison between initial and nal body weights (repeated-measures two-way
ANOVA followed by Newman-Keuls). : different from WT (p b 0.05), APP (p b 0.01)
and APP-L (p b 0.05) (two-way ANOVA followed by Newman-Keuls post-hoc test).

2012). Mice were given three days of visible platform training for habituation purposes and for the assessment of any visual, locomotor or motivational decits. Wall cues were then switched, the platform moved to
a different pool sector (target quadrant) and submerged for ve days of
hidden-platform training. Daily escape latencies were recorded, and
used to plot a learning curve. To test memory, the platform was removed and mice were allowed a free swim (probe trial) during which
percent time spent and distance travelled in the target quadrant were
recorded along with swim speed. Probe trials were conducted 2 h following the last hidden-platform session on day 8 (probe 1) and subsequently 48 h later (probe 2). Mice were tracked with the 2020 Plus
tracking system and data analyzed with the Water 2020 software
(Ganz FC62D video camera; HVS Image, Buckingham, UK). Animals
were dried under a heating lamp after each trial, and experiments
started at the same time every day.

Animals and treatments


Experiments were in compliance with the Canadian Council on Animal Care and were approved by the Animal Ethics Committee of the
Montreal Neurological Institute, McGill University. We used approximately equal numbers of male and female wild-type (WT) and littermate heterozygous transgenic APP mice carrying the Swedish (K670N,
M671L) and Indiana (V717F) familial AD mutations driven by the
platelet-derived growth factor beta (PDGF) promoter on a C57BL/6
background (line J20, Mucke et al., 2000). Mice were screened for transgene expression by touchdown PCR using tail-extracted DNA (Mucke
et al., 2000; Tong et al., 2005). They were housed under a 12 h lightdark cycle under controlled temperature (23 C) and humidity (50%),
with food and tap water ad libitum. The centrally acting AT1 receptor
antagonist losartan (10 mg/kg per day, Merck Frost Canada Lte,
Kirkland, QC, Canada or Cedarlane, Burlington, ON, Canada) (Li et al.,
1993; Michel et al., 2013) was administered in drinking water for
three months starting at ~15 months of age (~18 months at endpoint,
aged mice). Prophylactic treatment with losartan was administered to
~ 2 month-old pups lacking A plaques (Aucoin et al., 2005; Mucke
et al., 2000). Losartan was started at 1 mg/kg per day until the age of
eight months, and then increased to 10 mg/kg per day until end of treatment (~12 months at endpoint, adult mice). This progressive dose increase was used to prevent the death of young mice when put directly
on high-dose losartan, as seen in preliminary tests. Age-matched control WT and APP mice received unmedicated drinking water. Body
weights were monitored weekly or monthly. Weight gain was similar
across age-matched groups, except for 10-month losartan-treated WT
mice in which it was slightly but signicantly larger than in the other
groups of the prophylactic cohort (Table 1).
Morris water maze
Mice were tested in a modied Morris water maze (deIpolyi et al.,
2008) previously used by us (Papadopoulos et al., 2010; Tong et al.,

Laser Doppler owmetry


One week following the Morris water maze, a subset of aged
losartan- and vehicle-treated mice was evaluated for the CBF increase
evoked during whisker stimulation, using laser Doppler owmetry
(Transonic Systems Inc., Ithaca, NY), as described previously
(Nicolakakis et al., 2008; Ongali et al., 2010). Mice were anesthetised
with a mixture of ketamine (85 mg/kg, intraperitoneal , i.p., Bioniche,
Belleville, ON, Canada) and xylazine (3 mg/kg, i.p., Bayer Inc, Toronto,
ON, Canada) and xed in a stereotaxic frame for thinning of the bone
over the left somatosensory cortex. Changes in CBF before, during and
after stimulation (electric toothbrush, 20 s at 8-10Hz) of the right whiskers were recorded with four to six recordings acquired every 30-40 s
and averaged for each mouse. Cortical CBF change was expressed as percentage increase relative to pre-stimulation baseline. Body temperature
was kept stable at 37 C with a heating pad. Experiments were performed blind to the identity of mice, and lasted less than 20 min during
which mouse physiological parameters remained stable (Tong et al.,
2012).
[18F]uoro-2-deoxy-D-glucose ([18F]FDG)-PET
Aged losartan- and vehicle-treated mice were fasted 24 h prior to
PET scanning for cerebral uptake of [18F]uoro-2-deoxy-D-glucose
([18F]FDG) during whisker stimulation (8-10Hz, electric toothbrush,
45 min) under isourane sedation (1-2% in medical air) in a CTI
Concorde R4 microPET scanner (Siemens Preclinical Solutions, Knoxville, TN). Procedures have been described in detail elsewhere
(Nicolakakis et al., 2008; Ongali et al., 2010). Animals were kept warm
with a heating lamp, and physiological parameters maintained stable
through online monitoring of heart rate, respiration, and temperature
(Biopac Inc., Goleta, CA). Fasting glycemia prior to scanning was measured with a commercial glucometer (OneTouch Ultra, LifeScan,

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B. Ongali et al. / Neurobiology of Disease 68 (2014) 126136

Burnaby, BC, Canada), and was comparable for all groups (WT: 8.62
0.53 mmol/L, APP: 5.82 0.90 mmol/L, APP-losartan: 6.21
0.93 mmol/L; F(2,16) = 3.236, p = 0.066, one-way ANOVA followed
by Newman-Keuls). CGU was corrected for body weight and injected radioactivity dose, and was expressed as an activation ratio of the somatosensory cortex contralateral to whisker stimulation to the analogous
ipsilateral cortex.
Blood pressure measurement
Mean arterial blood pressure (MAP) was measured through the femoral artery in aged treated APP mice and untreated WT and APP mice.
Animals were anesthetised with isourane (5% in medical air during
2-min induction, 1.52% during ~20 min surgery) and the femoral artery was catheterized. Mice were then placed in a restraining cylinder
and anesthesia was switched off for measurement of MAP with a
pressure transducer for a period of 30 min (Powerlab, ADinstruments,
St.-Laurent QC, Canada). Body temperature, heart and respiratory rates
were monitored throughout the procedure. Blood gases (pO2 and
pCO2) and pH were measured at the end of the experiment (RapidLab
348, Bayer).
Vascular and brain tissue collection
A subset of adult and aged mice was euthanized by cervical dislocation, and the middle cerebral artery (MCA) was removed for reactivity
studies (below). Remaining pial vessels (circle of Willis and ramications) were dissected and frozen on dry ice, along with cortex and hippocampus from one hemisphere, heart, lung, aorta and kidney from
untreated WT and APP mice. Tissues were stored ( 80 C) until use
in Western blotting experiments. The other hemisphere was
immersion-xed overnight (4 C, 4% paraformaldehyde (PFA) in 0.1 M
phosphate-buffered saline, pH = 7.4), cryoprotected, frozen in
isopentane, and stored ( 80 C) until cutting of 25 m-thick freeoating coronal sections using a freezing microtome. A subset of animals was intracardially perfused (4% PFA) under deep anaesthesia
(65 mg/kg sodium pentobarbital, i.p.). Right hemibrains were processed for cutting of 25 m-thick sections as above. Left hemibrains
were parafn-embedded and cut into 5 m-thin microtome sections.
Vascular reactivity
Isolated, pressurized MCA segments were preconstricted with
serotonin (2 107 M; Sigma-Aldrich, St.-Louis, MO), and tested for dilatations to acetylcholine (ACh; 1010-105 M; Sigma-Aldrich) and calcitonin gene-related peptide (CGRP; 1010-106 M; American Peptide,
Vista, CA). Constriction to endothelin-1 (ET-1; 10 10-10 6 M;
American Peptide) and passive diameter decrease during nitric oxide
synthase (NOS) inhibition with N-nitro-L-arginine (L-NNA; 105 M;
35 min; Sigma-Aldrich) were tested on vessels at basal tone, using online videomicroscopy as detailed before (Tong et al., 2005). Percent
changes in vessel diameter were plotted as a function of agonist concentration or time course of NOS inhibition. The maximal response
(EAmax) and concentration eliciting half of the EAmax (EC50 or pD2
value = -[log EC50]) were generated from the dose-response curves
using the GraphPad Prism software (version 4, San Diego, CA), and
used to compare agonist efcacy and potency, respectively.

for separation of A monomers and oligomers according to Wiltfang


et al. (1997). Membranes were incubated overnight with either rabbit
anti-SOD1, -SOD2 (1:1000; Stressgen, Ann Arbor, MI), p67phox
(1:200; Abcam, Cambridge, MA), -AT4 or -insulin-regulated aminopeptidase (Albiston et al., 2001) (IRAP, 1:500; Chemicon, Temecula, CA,
USA), mouse monoclonal anti-AT1, -AT2 (1:200; Frei et al., 2001),
mouse anti-6E10 (detects A and full-length APP; 1:1000; Covance,
Emeryville, CA), -ACE (1:100; Abcam) or --actin (1:10000; SigmaAldrich). Membranes were further incubated (2 h) with horseradish
peroxidase-conjugated secondary antibodies (1:2000; Jackson
ImmunoResearch, Westgrove, PA) and proteins visualized with Enhanced ChemiLuminescence (ECL Plus kit; Amersham, Mississauga,
ON, Canada) using phosphorImager (Scanner STORM 860; GE
Healthcare, Piscataway, NJ) and densitometric quantication with
ImageQuant 5.0 (Molecular Dynamics, Sunnyvale, CA) or Scion image
software (NIH, Bethesda, MD).
Histochemistry and immunohistochemistry
Free-oating thick (25 m) and dewaxed thin (5 m) sections were
stained with 1% thioavine S (8 min) to reveal mature, dense core A
plaques or were incubated with an antibody against A (overnight
with mouse anti-6E10, 1:2000 Covance) followed by species-specic
biotinylated IgG, and the avidin-biotin complex to reveal total A
plaque burden. The reaction was visualized with 0.05% 3,3diaminobenzedene-Nickel (Vector Laboratories, Burlingame, CA). Thin
sections were incubated with goat anti-choline acetyltransferase
(ChAT; 1:250; Millipore, Temecula, CA) followed by immunodetection
as above, or incubated with rabbit anti-glial brillary acidic protein
(GFAP; 1:1000; DAKO, Mississauga, ON, Canada) detected with a donkey anti-rabbit cyanin 2 (Cy2)-conjugated secondary antibody (1:400;
Jackson ImmunoResearch). Sections were observed under light or
epiuorescence microscopy (Leitz Aristoplan microscope, Leica,
Montral, QC, Canada) and pictures acquired with a digital camera
(Coolpix 4500; Nikon, Tokyo, Japan) or under confocal microscopy
(Zeiss LSM 510, Zeiss, Jena, Germany). Captured images (two to three
sections/mouse, three to ve mice/group) were analyzed with
MetaMorph 6.1r3 software (Universal Imaging, Downingtown, PA).
The areas of interest (somatosensory/cingulate cortex, hippocampus)
containing thioavin S-, A- and GFAP-positive elements were manually outlined in low-power images, while high-power microscope images
of layers II to IV of the somatosensory cortex were used for quantication of ChAT-immunoreactive bres. The number and/or surface area
occupied by thioavin S- or A-positive plaques, GFAP-positive astrocytes and ChAT-positive cholinergic bres was quantied in the delineated areas of interest.
Statistical analysis
Data are expressed as mean SEM. Student t-test was used for twogroup comparisons (GraphPad Prism) and, for multiple comparisons,
one-way or two-way (transgene and treatment as factors) analysis of
variance (ANOVA) followed by Newman-Keuls post-hoc tests (Statistica
10, Statsoft, Tulsa, OK). Reactivity dose-response curves, Morris
water maze latency curves and probe trials were analyzed by
repeated-measures one-way or two-way ANOVA. A p b 0.05 was considered signicant.

Western blot

Results

Tissues were sonicated in Laemmli buffer and protein concentration


measured as detailed previously (Tong et al., 2005). Proteins (~ 35 g
cortex, 6-15 g all other tissues) were separated by SDS-gel electrophoresis and transferred to nitrocellulose membranes incubated (1 h) in
TBST blocking buffer (50 mM Tris-HCl, pH = 7.5; 150 mM NaCl; 0.1%
Tween 20) containing 5% skim milk. Tricine or gradient gels were used

Benecial effects of losartan on memory, neurovascular and


neurometabolic coupling responses
Aged and adult J20 APP mice exhibited signicant learning and
memory decits in the Morris water maze (Figs. 1A,B) in keeping with
documented impairments in 18 (Nicolakakis et al., 2008) and 12

B. Ongali et al. / Neurobiology of Disease 68 (2014) 126136

129

Fig. 1. Losartan (L) exerted benecial effects on cognitive performance in both aged and adult APP mice. A. Following three-month losartan therapy, aged APP mice () displayed similar
escape latencies (top panel) to their untreated APP counterparts () in the hidden platform training sessions. In the probe trials (bottom panel), although aged losartan-treated and untreated APP mice were comparable on the rst trial (day 8), treated APP mice performed as well as WT controls in the second probe trial (day 10). They devoted more time and swim
distance in the target quadrant compared to untreated APP mice. B. In adult APP-treated mice, losartan prevented the decline in learning memory (upper panel) and in the acquired memory (lower panel). Although losartan-treated adult APP mice performed better than untreated counterparts in the rst probe trial, the benet of losartan reached signicance in probe 2
(day 10, bottom panel). Losartan had no effect on the performance of treated wild-type (WT) () relative to untreated WT mice () at any age. There was no difference in swim speed
among the groups. Comparisons to WT mice are indicated with p b 0.05, p b 0.01, p b 0.001, and to APP mice with *p b 0.05, **p b 0.01, ***p b 0.001. N = 7-18 mice/group.
Analyzed by repeated-measures two-way ANOVA.

(Tong et al., 2012) month-old J20 mice. In aged APP mice (Fig. 1A),
losartan had limited therapeutic benets on the learning decit, but it
improved memory performance, which was signicantly better compared to untreated APP mice in probe trial 2 (day 10). This was depicted
by a free swim signicantly focused in the target quadrant relative to
untreated APP counterparts (Fig. 1A, bottom). Aged APP mice were
slightly hypotensive compared to WT mice (Table 2), but they did not
differ from losartan-treated littermate APP mice, indicating that the
memory-enhancing effects of losartan were unrelated to changes in

blood pressure. A similar conclusion was reached with the AT1 receptor
antagonist olmesartan in a comparative study with other classes of antihypertensive drugs, which were inconsequential on memory (Takeda
et al., 2009) or with eprosartan that had no or minimal time specic effects on blood pressure (Ferrington et al., 2011, 2012).
In adult APP mice on long-term treatment, losartan completely
prevented the onset of learning and memory decits (Fig. 1B). Treated
APP mice displayed comparable latencies to nd the hidden platform
to those of WT mice, indicating preserved spatial learning. They also

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B. Ongali et al. / Neurobiology of Disease 68 (2014) 126136

Table 2
Effects of losartan (L) treatment on physiological parameters in aged wild-type (WT) and APP mice.
Group (n)

MAP (mmHg)

Heart rate (beats/min)

Respiratory rate (breaths/min)

pCO2

pO2

pH

WT (7)
APP (5)
APP-L (4)

126.1 2.8
93.5 3.6
110.3 11.0

475.6 35.7
527.9 27.2
484.7 54.9

176.9 21.1
182.7 8.5
196.7 27.0

36.9 1.5
46.1 3.7
35.2 1.5*

100.2 7.5
95.8 3.0
86.4 4.6

7.4 0.01
7.3 0.03
7.3 0.12

Data represent mean SEM. p b 0.01 for comparison to untreated WT mice, and *: p b 0.05 for comparison to untreated APP mice, when using one-way ANOVA followed by
Newman-Keuls post-hoc test. MAP: mean arterial blood pressure. PO2 and pCO2 are expressed as mmHg.

exhibited superior probe trial performance compared to untreated APP


mice (signicant in probe 2, Fig. 1B, bottom) as depicted by the % time
spent and distance travelled in the target quadrant. Together these
data suggest that both prophylactic and curative losartan treatments
consolidated acquisition and recall memory.
Given its benecial outcome on memory in aged APP mice, we tested
the effects of losartan on evoked CBF and CGU responses, respectively
neurovascular and neurometabolic coupling responses that have
been shown previously to be impaired in aged J20 APP mice
(Nicolakakis et al., 2008) and AD patients (for review see Nicolakakis
and Hamel, 2011). Losartan completely normalized the neuronallydriven CBF increase to whisker stimulation (% increase, WT: 27.5
2.9, n = 5; WT-L: 25.2 2.5, n = 5; APP: 13.3 1.0, n = 5; APP-L:
26.0 2.1, n = 5), as measured with laser Doppler owmetry in the somatosensory cortex (Fig. 2A). Similarly, losartan recovered the reduced
CGU response to increased neuronal activity in the sensory cortex
(activation ratio, WT: 1.099 0.015, n = 4; APP: 1.010 0.007, n =
6; APP-L: 1.108 0.027, n = 6) measured by [18F]FDG-PET (Fig. 2B).
These ndings demonstrate the ability of losartan to normalize the supply of blood and energy to activated brain areas even at this advanced
stage of the pathology.

Losartan protected cerebrovascular responsiveness by inhibiting oxidative


stress
In line with previous data (Nicolakakis et al., 2008; Tong et al., 2005,
2012), the ability of cerebral arteries to dilate in response to ACh and
CGRP was signicantly decreased in adult and aged APP mice (by
~ 50%) (Fig. 3A, Table 3). Losartan completely restored dilatory responses in aged APP arteries (Fig. 3A, Table 3) and prevented the
onset of decits when given in prophylaxis (Table 3), suggesting the involvement of the RAS or AT1 receptor-mediated pathological mechanisms in the cerebrovascular dysfunction of APP mice. Decits were
not due to desensitization of cerebrovascular receptors since pD2 values
were comparable between WT and APP arteries in most cases (Table 3).
However, losartan in aged APP mice increased the ACh pD2 values, raising the possibility that part of the recovery occurred through sensitization of vasodilatory muscarinic ACh receptors (Table 3). As expected, we
found no contractile decits to ET-1-mediated contractions in either
adult or aged APP mice (Table 3). The constitutive synthesis and release
of endothelial nitric oxide to maintain basal vessel tone was unaltered in
aged APP arteries (L-NNA response, Table 3), but was reduced in adult
APP mice (Table 3). This is consistent with litter-specic variations in

Fig. 2. Losartan reinstated the increases in cerebral blood ow (CBF) and cerebral glucose uptake (CGU) evoked by whisker stimulation in aged APP mice. A. The impaired neurovascular
coupling response to sensory stimulation was completely restored in losartan-treated APP mice (APP-L). Responses of wild-type (WT) mice were unaffected by treatment (WT-L). Curves
represent CBF measured in the somatosensory cortex with Laser Doppler owmetry during 20 s stimulation of the whiskers (black bar on the x axis). Traces are WT: green, WT-L: black,
APP: blue, APP-L: red. Histogram represents percent maximal increase in CBF from pre-stimulation baseline. B. Losartan rescued the neurometabolic response induced by whisker stimulation, as measured with the cerebral glucose uptake (CGU) using [18F]FDG-PET. Functional metabolic PET images were co-registered to WT or APP MRI templates (left panel), and CGU
expressed as an activation ratio of the activated somatosensory cortex (indicated by arrows) to the analogous ipsilateral cortex (right panel). and **p b 0.01, and ***p b 0.001,
when compared to WT and treated APP mice, respectively, using two-way ANOVA. N = 4-6 mice/group.

B. Ongali et al. / Neurobiology of Disease 68 (2014) 126136

131

Whereas it had no effect on cerebrovascular proteins in WT mice,


losartan reversed p67phox up-regulation in pial vessels of aged APP
mice (Fig. 3B). In APP mouse vessels, losartan also countered and even
reversed the up-regulation of SOD2 protein levels (Fig. 3B) and further
increased (+ 47.8%, p b 0.01) those of SOD1 (Fig. 3B). This points to
the high antioxidant capacity of losartan in both mitochondrial and cytoplasmic compartments that respectively contain the SOD2 and SOD1
isoenzymes. These benecial effects are consistent with a role for
p67phox-containing NADPH oxidase and its product superoxide,
which induces SOD2 (Wong and Goeddel, 1988) in the dilatory impairments of APP mice (Park et al., 2005; Tong et al., 2005).
Losartan effects on AD neuropathology

Fig. 3. Losartan restored cerebrovascular responsiveness and decreased cerebrovascular oxidative stress. A. Dilatory impairments to acetylcholine (ACh) and calcitonin gene-related
peptide (CGRP) in aged APP mice () were completely normalized by three-month losartan
therapy (), as evaluated in segments of the middle cerebral artery (MCA). Losartan did not
enhance the dilatory capacity of arteries from treated wild-type (WT) mice () relative to
untreated WT littermates (), but it slightly, though signicantly, diminished CGRP dilatations. No impairments were observed in passive diameter decrease during nitric oxide
synthase inhibition with L-NNA and in the contractile response to ET-1 (not shown,
see Table 3). B. Losartan countered p67phox upregulation, reversed the enhanced SOD2
protein levels, and potentiated the SOD1 increase in pial vessels, as measured by Western
blot. Protein expression was normalized to -actin and displayed as percent change relative
to WT mice. Comparisons to untreated WT mice indicated with p b 0.05, p b 0.01,

p b 0.001, and to APP mice with *p b 0.05, **p b 0.01, ***p b 0.001 using repeatedmeasures two-way ANOVA. N = 3-8 mice/group.

Aged APP mice exhibited increased GFAP-positive cortical and hippocampal areas relative to WT controls (Fig. 4A), with a predominant
association with A plaques in the cerebral cortex and both diffuse
and plaque-associated labeling in the hippocampus, consistent with
glial activation characteristic of AD brains and APP mice (Wyss-Coray,
2006). This activation was signicantly, but not completely blocked by
losartan therapy as quantied in the cortex (Fig. 3A). However, losartan
failed to rescue the neocortical reduction in the density of ChAT-positive
nerve bres (-49.1%, p b 0.01) (B), a marker of the cholinergic decit reported in these mice (Aucoin et al., 2005; Nicolakakis et al., 2008).
In addition, losartan had no effect on the amyloid pathology. Indeed,
losartan did not reduce the levels of the A1-42 monomer (4-kDa band,
see Rangachari et al., 2007; Fukumoto et al., 2010) and oligomers (9and 56-kDa bands) detected by Western blot (Figs. 5A,C). Correspondingly, despite trends toward an increase, brain A plaque burden was
not signicantly changed in APP mice treated with losartan, as seen by
the cortical and hippocampal surface area occupied by dense core
thioavin S-positive plaques or by total amyloid beta 6E10-positive
plaques in both curative and prophylactic treatments (Figs. 5B,D).
Losartan effects on RAS components

nitric oxide levels (Nicolakakis et al., 2008), but it could also represent
an age-related alteration in nitric oxide synthesis at 12 months that is
corrected with aging. Given in prophylaxis, losartan protected baseline
nitric oxide synthesis in adult APP mice, as shown by the L-NNAinduced decrease in vessel diameter, comparable to that seen in treated
and untreated WT controls (Table 3).

No alterations in the peripheral RAS were detected in aged APP mice


with respect to ACE, AT1 and AT2 receptors, evaluated by Western blot
in kidney (Fig. 6A), heart and aorta (not shown). In contrast, cortical and
cerebrovascular AT1 receptor levels were signicantly increased in aged
APP mice (Fig. 6A), whereas those of AT2 receptor and ACE were

Table 3
Cerebrovascular responses to ACh, CGRP, ET-1 and L-NNA in aged and adult mice treated with losartan (L).
Aged mice
Vasoactive agent
ACh
CGRP
ET-1
L-NNA

WT (4)
EAmax
pD2
EAmax
pD2
EAmax
pD2
EAmax

60.4
7.76
65.6
8.31
63.6
8.47
75.7

2.8
0.13
3.1
0.12
4.0
0.16
3.2

WT-L (5)

APP (3)

56.0
7.84
48.7
8.59
67.3
8.68
69.9

33.6
7.50
29.2
8.14
68.6
8.55
71.1

2.1
0.10
1.8
0.10
2.8
0.11
4.2

APP-L (5)
1.7
0.14
2.5
0.20
3.8
0.15
2.0

57.3
8.42
52.0
8.54
74.6
8.60
77.9

2.5
0.30
2.1
0.17
1.8
0.09
5.7

69.0
8.27
59.5
8.49
66.8
8.78
65.0

2.6 ***
0.14,**,
3.2**
0.16
2.8
0.10
1.3

Adult mice
Vasoactive agent
ACh
CGRP
ET-1
L-NNA

WT (3)
EAmax
pD2
EAmax
pD2
EAmax
pD2
EAmax

62.8
7.88
69.6
8.43
57.8
8.69
71.7

3.3
0.14
2.4
0.09
1.7
0.08
1.9

WT-L (3)

APP (3)

57.1
8.52
67.6
8.15
69.9
8.62
68.7

23.4
8.03
29.5
8.27
52.4
8.67
27.6

2.5
0.14
2.7
0.10
3.1
0.12
7.2

APP-L (3)

2.1***
0.09
2.5 ***
0.11
3.5*
0.14
2.2***

Maximal responses (EAmax) and agonist concentrations eliciting half of the EAmax (pD2 values) are expressed as mean SEM of the number of arteries in parentheses. p b 0.05,

p b 0.01, p b 0.001 for comparisons to untreated wild-type (WT) mice; *p b 0.05, **p b 0.01, ***p b 0.001 for comparisons to untreated APP mice; p b 0.05 for comparison to
WT-L. Repeated measures two-way ANOVA followed by Newman-Keuls post-hoc test.

132

B. Ongali et al. / Neurobiology of Disease 68 (2014) 126136

Fig. 4. Treatment effects on glial brillary acidic protein (GFAP) and cortical choline acetyltransferase (ChAT)-immunoreactive cholinergic bres in aged APP mice. A. Losartan (L) decreased astrocyte activation in 18 month-old APP mice, as measured by the signicant
decrease in GFAP-positive cortical area. Comparisons to untreated APP mice are indicated
by ***p b 0.001. B. Losartan did not restore the signicantly reduced density of ChATimmunodetected nerve bres in the neocortex. Comparisons to wild-type (WT) mice
are indicated by p b 0.01 p b 0.001, using two-way ANOVA. Scale bars 20 m.
N = 3-6 mice/group.

unaltered in pial vessels (Fig. 6A) and cortex (not shown). Losartan signicantly reversed this cerebrovascular and cortical AT1 receptor upregulation in both aged and adult APP mice (Fig. 6B). Moreover, losartan
normalized the signicantly reduced levels of cortical AT4 receptors in
aged APP mice, an effect that was conrmed in the preventive cohort
of adult mice (Fig. 6C).
Discussion
Salient new ndings of the present study are: (1) the novel ability of
losartan to consolidate the acquisition and recall memory of aged in addition to adult APP mice, and this, despite unrelenting high levels of soluble A species and A plaque load; (2) the capacity of losartan to fully
rescue evoked arterial, hemodynamic and neurometabolic responses
even at an advanced pathological stage; and (3) the selective downregulation of AT1 and restoration of AT4 receptor levels as the most likely
molecular underpinnings of losartan-based improvements.
The AT1 receptor antagonist losartan conferred protection against
the onset of cognitive dysfunction in adult mice treated preventively
for 10 months. This protective potency of losartan was mitigated in
old APP mice, but even at this advanced age, losartan maintained its
ability to consolidate memory acquisition and recall after a three
months treatment (see Fig. 1). Such restorative effects of losartan on
spatial memory in both adult and aged APP mice are not unique.
Other positive effects on cognition have been observed in young A40-

injected mice treated with losartan (Mogi et al., 2008) or other ARBs
like olmesartan (Takeda et al., 2009) and telmisartan (Tsukuda et al.,
2009). Valsartan (Wang et al., 2007) was reported to attenuate the development of spatial learning decits in adult AD mice (11 months
old, treated 5 months), but there was no reference to memory. However, eprosartan failed to improve learning and memory in aged triple
transgenic AD mice (Ferrington et al., 2012), possibly due to its low penetrance across the blood brain barrier at therapeutic doses (Michel et al.,
2013). Hence, our study conrms the cognitive benets of ARBs in adult
AD mice, and extends them to elderly AD mice, further emphasizing the
possibility of a promising intervention at a late stage of the disease.
Studies with losartan or different ARBs have yielded conicting data
on the amyloid pathology, varying from no effect in young and aged AD
mice treated for one, two or six months (Ferrington et al., 2011, 2012;
Hemming et al., 2007) to signicant reductions in soluble or deposited
A in adult AD mice after several months of treatment (two to ve
months) (Danielyan et al., 2010; Wang et al., 2007). This apparent discrepancy might be explained by the distinct capacity of different ARBs
to affect the generation of A40 and A42, losartan being amongst those
that do not alter the levels of A species (Liu et al., 2014). Surprisingly, except for one study that did not nd cognitive improvement (Ferrington
et al., 2012), none of the other investigations evaluated memory.
Our ndings of no reduction in the amyloid pathology in both
losartan-treated adult and aged APP mice with rescued memory are
thus unique. However, they agree with studies in A1-40-injected mice
demonstrating the capacity of losartan to enhance cognition in
prophylactically-treated adult animals without affecting A1-40 deposition (Mogi et al., 2008). Our data not only conrm unchanged amyloidosis in losartan-treated adult mice but also highlight that cognitive
rescue, particularly the consolidation of the acquisition and recall memory, is attainable in spite of unchanged amyloid burdens. These ndings
were replicated in both adult and aged APP mice, and are in agreement
with normal learning and memory in mice engineered to have prominent A plaque load (Cheng et al., 2007). Our ndings are also supported by reports of cognitive recovery with other therapeutic approaches,
despite no measured change in the amyloid pathology, for example
high soluble A levels in aged Tg2576 AD mice following deletion of
the Nox2 NADPH oxidase subunit (Park et al., 2008) or following treatment with COX-2 inhibitors (Kotilinek et al., 2008), and unchanged A
plaque load and levels of soluble A1-40/1-42 (Tong et al., 2012) or A*56
(unpublished data) in adult J20 APP mice after simvastatin treatment.
The latter amyloid species, likewise unaffected in our study, refers to
the 56-kDa A1 -42 oligomer linked to memory decits in Tg2576 AD
mice (Lesn et al., 2006) and AD pathogenesis in patients (Lesn et al.,
2013). These ndings further stress the dissociation between cognitive
failure and amyloidosis, and support the unequivocal results of Elan
Pharmaceuticals A1-42 immunization trial: despite plaque clearance,
patients declined cognitively until death (Holmes et al., 2008; Liu
et al., 2012).
Losartan completely normalized neurovascular and neurometabolic
coupling in aged APP mice, responses that are similarly impaired in AD
patients (Machulda et al., 2003; Melrose et al., 2009; Mentis et al., 1998;
Nicolakakis and Hamel, 2011). These ndings corroborate the reported
benet of the AT1R antagonist olmesartan on neurovascular coupling to
sensory stimulation in young AD mice (Takeda et al., 2009). The localized CBF and CGU increases during neuronal activation depend upon coordinated signaling between neuronal, vascular and astrocytic cells, the
latter considered bridges between neuronal activity and perfusion
(Haydon and Carmignoto, 2006). Hence, the reduction of GFAP up-regulation by losartan indicated attenuated astrocyte activation, suggesting
improved astrocytic function that may have contributed to the normalization of hemodynamic and metabolic responses. In addition, it may
have resulted in subdued astroglial release of inammatory mediators,
as reported for telmisartan (Tsukuda et al., 2009; Washida et al., 2010).
Losartan improved responsiveness of isolated arteries to vasoactive
stimuli and suppressed an oxidative stress pathway involving

B. Ongali et al. / Neurobiology of Disease 68 (2014) 126136

133

Fig. 5. Losartan did not affect the amyloid pathology in APP mice. A, C. In both adult and aged APP mice, losartan (L) did not affect levels of the A1-42 monomer (4-kDa band) and oligomers
(9- and 56-kDa bands) detected by Western blot. -actin was used to normalize loading variation. B, D. Losartan had no signicant effect on the cortical and hippocampal surface area
occupied by diffuse (6E10) and dense core (thioavine S) A plaques in both adult and old APP mice. Analysed by Students t test. Scale bars 20 m. N = 3-6 mice/group.

p67phox-containing NADPH oxidase. Downregulation of the p47phox


subunit of NADPH oxidase has likewise been reported with losartan in
major arteries of spontaneously hypertensive rats (Zhu et al., 2007).
A-mediated ROS production by NADPH oxidase constitutes the major
deleterious pathway in A-induced cerebrovascular dysfunction, with
free radicals sequestering vasodilatory nitric oxide (Iadecola et al.,
1999; Park et al., 2005, 2008; Tong et al., 2005) and disrupting cerebrovascular K+ channel signalling pathways (Tong et al., 2012; Zhang et al.,

2013). Superoxide is a known inducer of SOD2 (Wong and Goeddel,


1988) that then acts quickly to eliminate its substrate. Hence SOD2
downregulation likely signalled a decrease in vascular oxidative stress
as shown before in brain vessels from young APP mice treated with
olmesartan (Takeda et al., 2009), and may also reect a reduction in various cytokines known to induce SOD2 (Wong and Goeddel, 1988). In
support of this argument, losartan decreased blood serum levels of
interleukin-1 in APP/PS1 mice (Danielyan et al., 2010). The SOD1

134

B. Ongali et al. / Neurobiology of Disease 68 (2014) 126136

Fig. 6. Losartan (L) countered AT1 upregulation and restored cortical AT4 receptor levels assessed by Western blot. A. AT1 receptor protein levels were signicantly increased in cortex and
pial vessels of aged APP mice, while those of AT2 receptors and ACE were unchanged as shown in pial vessels Pia. RAS components were unaltered in kidney, heart and aorta, as shown
here in the kidney (kid) for AT1 receptors. B. Losartan signicantly reversed cerebrovascular and cortical AT1 receptor upregulation in both aged and adult APP mice. The signicant decrease (p b 0.05) in APP-L compared to WT-L is not illustrated on the Figure for clarity purposes. C. Furthermore, losartan restored the signicantly reduced cortical levels of AT4 receptors
seen in both aged and adult APP mice. -actin was used to normalize loading variation. Comparisons to untreated WT mice are indicated with p b 0.05, p b 0.01, and to APP mice with
*p b 0.05, **p b 0.01, ***p b 0.001, using two-way ANOVA. N = 3-6 mice/group.

increase in aged APP vessels likely contributed to safeguard basal nitric


oxide levels shown by the preserved L-NNA responses in aged APP mice,
and SOD1 further increase by losartan may reect a boost in endogenous antioxidant reserve.
Binding of AngII to AT1 receptors triggers superoxide production
through NADPH oxidase (Kazama et al., 2004). We document for the
rst time AT1 receptor upregulation in APP cerebral vessels and cortex,
in line with receptor increases in AD patients (Kehoe et al., 2009), and
show that selective and chronic blockade of these receptors with
losartan countered their upregulation. Combined with the p67phox
and SOD2 downregulation, these data suggest that losartan prevented
the AT1-NADPH oxidase-superoxide deleterious pathway, and provide
a molecular substrate for improved neuronal and vascular function,
through inhibition of oxidative stress. Recent data indicate that reducing superoxide production is indeed not only benecial for Ainduced impairments in cerebrovascular function, but also in synaptic
plasticity (Ma et al., 2011).
It has been proposed that blocking AT1 receptors favours conversion
of AngII to downstream active metabolite angiotensin IV (AngIV) that
binds with high afnity and specicity the AT4 receptor. AT4 receptor,
identied as insulin-regulated aminopeptidase (IRAP, Albiston et al.,
2001, 2010), is heavily distributed in neurons of cognitive-processing

areas, including neocortex, hippocampus, amygdala and basal forebrain,


and has been shown to facilitate learning and memory through yet unidentied mechanisms (Gard, 2008; Wright and Harding, 2008, 2010).
Accordingly, AT4 knockout mice displayed accelerated spatial memory
decline (Albiston et al., 2010), and selective AT4 receptor activation
reversed the scopolamine-induced spatial memory decit, favoured
hippocampal synaptogenesis (Benoist et al., 2011), facilitated
hippocampal glucose uptake (Fernando et al., 2008), and enhanced
LTP in hippocampal CA1 neurons (Wayner et al., 2001). In adult and
aged APP mice, we found cortical losses of AT4 receptor that were
counteracted by prophylactic and curative losartan treatments, thus
providing a mechanistic basis for the improvement of memory
by losartan through restoration or preservation of the neuronal Ang
IV/AT4 receptor signalling cascade (Braszko et al., 2006; Wright and
Harding, 2008).
Together, our ndings demonstrate potent cerebrovascular and cognitive benets of losartan in aged and adult APP mice and point to alterations in AT1 and AT4 receptors of the brain RAS as underlying culprits
in AD pathogenesis. Most importantly, our ndings highlight the need
for further preclinical and clinical studies with losartan and, possibly,
for testing AngIV analogs as a potential new therapeutic option for the
prevention and treatment of AD.

B. Ongali et al. / Neurobiology of Disease 68 (2014) 126136

Sources of funding
Supported by grants from the Canadian Institutes of Health Research
(CIHR, MOP-84275 and MOP-126001) and la Fondation des maladies du
Coeur du Qubec (to E.H.), by fellowships from the Heart and Stroke
Foundation of Canada/Canadian Stroke Network (C.L.) and les Fonds
de la Recherche en Sant du Qubec (FRSQ, B.O.), as well as a Jeanne
Timmins Costello studentship (P.P.) and fellowship (B.O.).

Conict of Interest/Disclosure
The authors have no conict of interest to disclose.

Acknowledgments
We thank Dr. L. Mucke (Gladstone Institute of Neurological Disease
and Department of Neurology, UCSF, CA, USA) and the J. David
Gladstone Institutes for APP transgenic mouse breeders, and Merck
Frost Canada Lte, Kirkland, QC, Canada for our original supply of
losartan. We also thank Ms. P. Fernandes (Montreal Neurological
Institute) for performing the laser Doppler owmetry experiments,
and Drs J. Brouillette and R. Quirion (Douglas Hospital Research Centre,
McGill University, Montral, QC, Canada) for initial training and assistance in the Morris water maze.

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