lable at ScienceDirect

Neurobiology of Aging xxx (2013) 1e10

Contents lists avai

Neurobiology of Aging

journal homepage: www.elsevier .com/locate/neuaging

Selective benefits of simvastatin in bitransgenic APPSwe,Ind/TGF-b1 mice

Panayiota Papadopoulos, Xin-Kang Tong, Edith Hamel*

Laboratory of Cerebrovascular Research, Montreal Neurological Institute, McGill University, Montréal, Québec, Canada

a r t i c l e i n f o

Article history:Received 20 December 2012Received in revised form 13 June 2013Accepted 15 July 2013

Keywords:SimvastatinMemoryTransforming growth factor-b1PI3/Akt pathwayAmyloidosisAlzheimer’s disease

* Corresponding author at: Montreal NeurologicaStreet, Montréal, Québec, Canada H3A 2B4. Tel.: þ1398 8106.

E-mail address: edith.hamel@mcgill.ca (E. Hamel).

0197-4580/$ e see front matter � 2013 Elsevier Inc. Ahttp://dx.doi.org/10.1016/j.neurobiolaging.2013.07.010

a b s t r a c t

Cognitive and cerebrovascular deficits are 2 landmarks of Alzheimer’s disease (AD) to target for effectivetherapy. Here, we evaluated the efficacy of simvastatin in bitransgenic A/T mice overexpressing amutated form of the human amyloid precursor protein (APPSwe,Ind) and a constitutively active form oftransforming growth factor-b1. These mice feature the AD amyloid beta (Ab) and cerebrovascular pa-thology. Simvastatin significantly decreased insoluble Ab peptide levels and Ab plaque load despite noeffect on b-site amyloid precursor protein-cleaving enzyme and Ab-degrading enzyme neprilysin proteinlevels. However, simvastatin failed to improve spatial learning and memory deficits and the decreasedbaseline levels of the memory-related protein early growth response-1 (Egr-1) in the hippocampus CA1area. The impaired hyperemic response to whisker stimulation in A/T mice was not improved withtreatment, but simvastatin fully restored constitutive nitric oxide synthesis in vessel walls and exacer-bated agonist-mediated dilatory deficits. These findings point to the efficacy of simvastatin on selectiveAD features in a complex model of the disease, likely reflecting the challenges faced by recent clinicaltrials in assessing statin efficacy.

� 2013 Elsevier Inc. All rights reserved.

1. Introduction

Alzheimer’s disease (AD), the most common form of progres-sive dementia with increasing age, is characterized at the neuro-pathological exam by amyloid beta (Ab) plaques, neurofibrillarytangles, synaptic loss, glial cell activation, and a vascular pathology(Zlokovic, 2011). The latter is defined by the presence of cerebralamyloid angiopathy and thickening of the vascular basementmembrane because of accumulation of extracellular matrixproteins, a fibrotic process imputed to increased levels of trans-forming growth factor (TGF)-beta 1 (TGF-b1) (Wyss-Coray et al.,2000). Clinically, AD is associated with a chronic cerebral hypo-perfusion and decreased brain metabolism (Iadecola, 2004).

Statins have shown promise in decreasing the incidence of ADamong statin-prescribed patients (Cramer et al., 2008; Haag et al.,2009; Jick et al., 2000; Wolozin et al., 2000; Yaffe et al., 2002), ben-efits imputed to their pleitropic effects rather than their ability tolower cholesterol (Simons et al., 2002; Sparks et al., 2005). Similarly,statins normalize several AD hallmarks such as impaired brainglucose metabolism, glial activation, cerebrovascular dysfunction,and most importantly, memory deficits depending on age and

l Institute, 3801 University514 398 8928; fax: þ1 514

ll rights reserved.

treatment duration in ADmousemodels (Kurata et al., 2011; Li et al.,2006; Tong et al., 2009, 2012). However, studies in AD patients havegenerated mixed results (Arvanitakis et al., 2008; Butterfield et al.,2011; Sano et al., 2011), and it was argued that statins might exertbenefits only if administered early in the disease process (Sano et al.,2011; Shepardson et al., 2011a, 2011b; Sparks, 2011). Such contra-dictory results point to the need for a better understanding of statinefficacy in ADmousemodels that better recapitulate the complexityof the human disease.

Thus, we investigated the therapeutic value of simvastatin inbitransgenic mice that display concurrent overexpression of amutated form of the human amyloid precursor protein (hAPPS-we,Ind, line J20) and a constitutively active form of TGF-b1 (lineT64) (A/T mice). These mice exhibit the Ab-associated neuronaldeficits and the comorbid TGFeb1-induced AD cerebrovascularpathology (Grammas and Ovase, 2002; Ongali et al., 2010; Wyss-Coray et al., 2000). We assessed the capacity of simvastatin (40mg/kg/d) in rescuing spatial learning and memory in adult A/Tmice (3-month-old, tested after 3 and 6 months of treatment), andthe evoked cerebral blood flow (CBF) responses to increasedneuronal activity as an index of functional hyperemia. Cerebralarterial reactivity, astroglial and microglial activation, amyloidosis,and proteins regulating vascular structure or function were eval-uated at the end point (9-month-old A/T mice). The data indicatethat simvastatin, in contrast to its high efficacy in APP mice (Liet al., 2006; Tong et al., 2012), has more limited benefits in A/T

P. Papadopoulos et al. / Neurobiology of Aging xxx (2013) 1e102

mice even after extended treatment (6 months). Together, thesefindings highlight the importance of testing potentially promisingtherapies in experimental models that recapitulate multiple facetsof AD because they might better predict efficacy in a disease ascomplex as AD.

2. Methods

2.1. Animals

Bitransgenic A/T mice (Ongali et al., 2010) concomitantlyoverexpress a mutated form of the human amyloid precursorprotein (APPSwe,Ind) (APP mice) driven by the platelet-derivedgrowth factor b promoter (line J20; Mucke et al., 2000), and theconstitutively active form of TGF-b1 (TGF mice) driven by glialfibrillary acidic protein (GFAP) promoter on a C57BL/6J back-ground (line T64; Wyss-Coray et al., 1995). Unlike the APP mice,TGF micedwhich recapitulate the cerebrovascular pathology ofAD (Tong et al., 2005; Wyss-Coray et al., 2000)ddo not displaymemory impairments or parenchymal and vascular Ab plaques(Nicolakakis et al., 2011; Papadopoulos et al., 2010). Therefore,genetically engineered A/T mice recapitulate the full spectrum ofthe multifaceted human AD, including chronic cerebral hypo-perfusion, vascular fibrosis, progressive loss of endothelial func-tion and degenerating capillaries, and age-dependent failure ofcognitive function (Ongali et al., 2010). Three-month-old A/Tmice and wild type (WT) littermates (body weight, approxi-mately 30 g) were treated or not for a total of 6 months (9months old at end point) with simvastatin (sv; 40 mg/kg/d indrinking water), a dose proven efficacious in APP mice (Tonget al., 2012). Male and female mice were randomly and equallyassigned in each group. Water and chow were available ad libi-tum. There was no significant difference in body weight gain overthe course of the experiment between the groups (in g: WT, 13.8� 2.0; WTsv, 10.0 � 1.7; A/T, 9.6 � 1.3; A/Tsv, 7.1 � 2.4). Similarly,total blood cholesterol levels did not differ between groups at theend point (in mmol/L: WT, 4.25 � 0.07; WTsv, 4.19 � 0.04; A/T,4.20 � 0.05; A/Tsv, 4.19 � 0.05), measured in blood collected fromthe tail tip with a commercial cholesterol meter, Accutrend GCmeter (Roche Diagnostic, Laval, Canada). Animal use was inaccordance with the Animal Ethics Committee of the MontrealNeurological Institute, and abided by the guidelines of the Ca-nadian Council on Animal Care.

2.2. Morris water maze

After 3 and 6 months of treatment, mice were tested in theMorris water maze. The first test was performed at the age of 6months: mice were trained to locate a visible platform during a3-day session (Days 1e3; familiarization session), followed by a5-day hidden platform training session (Days 4e8; platform sub-merged approximately 1 cm below the surface of the water;learning phase of the test) in a 1.4-m circular pool filled withopaque water (18 � 1 �C). The location of the platform and of thedistal visual cues was changed between the 2 training sessions, aspreviously described (DeIpolyi et al., 2008). Mice that passed thetime limit were directed to the platform, and all mice were given10 seconds on the platform on the first day of each training ses-sion. Spatial memory was evaluated 24 hours after the last hiddenplatform session in the probe trial (Day 9; platform removed, 60seconds). Performance was compared according to escape la-tencies (Days 4e8; learning curve), and percent time spent,percent distance traveled, and the number of crossings over theprevious location of the platform (Day 9; probe trial). All param-eters together with swim speed were recorded using the 2020 Plus

tracking system and Water 2020 software (Ganz FC62D videocamera; HVS Image, Buckingham, UK). Mice were kept warmthroughout the sessions with a heating lamp and subsequently leftto rest for 2 days before additional experiments resumed. Threemonths later, the same mice underwent a second Morris watermaze test. Because the mice were already familiar with the task,only the hidden platform session was performed (Day 1e5),whereby the location of the platform and that of the distal visualcues differed from those used in the first Morris water maze test(Aboulkassim et al., 2011). A probe trial was performed 24 hourslater (Day 6).

2.3. CBF

CBF increases during whisker stimulation were measured inketamine/xylazine-anesthetized mice using laser Doppler flow-metry (Transonic Systems Inc, Ithaca, NY, USA), as previouslydescribed in detail (Tong et al., 2012). Whiskers on the right snoutwere stimulated with an electric toothbrush (8e10 Hz, 4e5stimulation blocks of 20 seconds each interspaced by approxi-mately 30 seconds). Increases in CBF were recorded with a laserprobe positioned on the thinned bone over the barrel cortex. Re-sults are expressed as percent increase over baseline and representaverages of all evoked CBF responses acquired during the stimu-lation blocks. The identity of the mouse was unknown to theexperimenter.

2.4. Tissue collection and preparation

On completion of the in vivo experiments, a subset of mice wassacrificed using cervical dislocation for reactivity studies of theposterior cerebral artery (see section 2.5). The remaining arteriesof the circle of Willis together with their small branches and theattached pial membrane were removed, together with the cortexand hippocampus of 1 hemibrain. These tissues were frozen on dryice and stored at �80 �C for subsequent enzyme-linked immu-nosorbent assay (ELISA) and Western blot experiments. Theother hemibrain was immersion-fixed overnight in 4% para-formaldehyde (in 0.1 M phosphate buffer, pH 7.4, 4 �C), cry-oprotected overnight (30% sucrose, 0.1 M phosphate buffer, 4 �C),and then frozen in isopentane and stored at �80 �C until cuttinginto free-floating thick sections (25 mm) on a freezing microtome.The other subset of mice was anesthetized with pentobarbital (80mg/kg, intraperitoneally) and perfused transcardially (4% para-formaldehyde solution at 4 �C, approximately 200 mL per mouse).One brain hemisphere was used for thick sections as describedabove (in section 2.4), and the other was embedded in paraffin forsectioning of thin (5-mm thick) brain sections.

2.5. Vascular reactivity

Posterior cerebral artery segments (40e70 mm average intra-luminal diameter) were cannulated, pressurized (60 mm Hg), andsuperfused with Krebs solution (37 �C) for measurement of vaso-motor reactivity using online videomicroscopy (Tong et al., 2005).Vessels were preconstricted submaximally with serotonin (2�10�7

M) for themeasurement of dilatory responses to acetylcholine (ACh;10�10 � 10�5 M), calcitonin gene-related peptide (CGRP; 10�10 �10�6M), and the nitric oxide (NO) donor sodiumnitroprusside (SNP,10�10 � 10�4 M). Contractile responses to endothelin-1 (ET-1;10�10 � 10�6 M), and the tonic production of NO evaluated inconditions of inhibition of NO synthase (NOS) with Nu-nitro-L-arginine (L-NNA; 10�5 M, for 35minutes) weremeasured on vesselsat basal tone. Vessel diameter changeswere expressed inpercentagerelative to either the basal or preconstricted tone, and plotted as a

P. Papadopoulos et al. / Neurobiology of Aging xxx (2013) 1e10 3

function of agonist concentration or time of L-NNA superfusion. Themaximal vessel response (EAmax) and half maximal effective con-centration (pD2 ¼ �EC50 value) were used to compare the agonist’sefficacy and potency, respectively.

2.6. ELISA

An ELISA (BioSource International) was used to measure thelevels of insoluble Ab1e40 and Ab1e42 species in cortex and hip-pocampus from treated and untreated A/T mice. Tissue was ho-mogenized using sonication (approximately 20 seconds in 20 mMTris buffer containing 1 mM ethylenediaminetetraacetic acid(EDTA), 1 mM ethylene glycol tetraacetic acid (EGTA), 250 mMsucrose, and protease inhibitors) and centrifuged (100,000g, 60minutes, 4 �C). The pellet containing insoluble Ab was then soni-cated (approximately 20 seconds) in 70% formic acid and centri-fuged (100,000g, 60 minutes, 4 �C). The Ab levels were measuredaccording to the manufacturer (BioSource International), and wereexpressed as micrograms per gram of protein in formic acid-insoluble fraction.

2.7. Western blot analysis

The levels of total soluble Ab species were measured in proteinextracts (20 mg) from cerebral cortex and hippocampus loaded ontoa 15% Tris/tricine sodium dodecyl sulfate polyacrylamide gel elec-trophoresis (SDS PAGE), transferred to nitrocellulose membranesand detected with a mouse anti-Ab1e16 antibody (6E10; 1:1000,Covance, Emeryville, CA, USA). The same protein extracts were usedto measure the levels of the anti-b-site APP cleaving enzyme 1 andthe Ab-degrading enzyme neprilysin (1:2000; Santa Cruz Biotech-nology [Santa Cruz, CA, USA] and Chemicon, respectively). Cere-brovascular proteins (12e15 mg) were loaded onto 10% SDS PAGEand probed with goat anti-connective tissue growth factor (1:400;Abcam), rabbit anti-vascular endothelial growth factor (1:500;Santa Cruz Biotechnology), mouse anti-actin (A5441; 1:8000; SantaCruz Biotechnology), or anti-endothelial NOS (eNOS; 1:500; BDTransduction) (for details, see Tong et al., 2009). Blots were incu-bated (1 hour) with horseradish peroxidase-conjugated secondaryantibodies (1:2000; Jackson ImmunoResearch, West Grove, PA,USA) and visualized using enhanced chemiluminescence (ECL Pluskit; Amersham, Baie d’Urfé, Quebec, Canada) using phosphorImager(Scanner STORM 860; GE Healthcare, Baie d’Urfé Quebec, Canada).Bands were quantified using ImageQuant 5.0 (Molecular Dynamics,Sunnyvale, CA, USA) using actin for loading normalization.

2.8. Immunohistochemical and histochemical stainings

Thick sections were immunostained with a rabbit anti-GFAPantibody, a marker of astrocyte activation (1:1000; DAKO), visual-ized with a donkey anti-rabbit cyanin 2-conjugated secondaryantibody (1:400; Jackson ImmunoResearch). Thioflavin S (1%, 8minutes) staining of mature, dense core Ab plaques was also per-formed on thick sections. Dewaxed paraffin sections were used forimmunostaining of diffuse and dense-core Ab plaques (6E10,1:2000), the memory-linked immediate early gene Egr-1/Zif268(1:200; Santa Cruz Biotechnology) and the marker of microglialactivation ionized calcium binding adaptor molecule 1 (Iba-1;1:300;Wako Chemicals). These were detected with species-specificbiotinylated IgGs (1 5 hours; Vector Labs) and the AB Complex (1.25hours, Vector Labs), and the reaction visualized with 3,3’-dia-minobenzidine (brown precipitate, 6E10, and Iba-1, Vector Labs) orthe SG kit (blue-gray precipitate, Egr-1; Vector Labs).

2.9. Data analysis

Brain sections (2e3 sections per mouse, 4 mice per group) wereobserved using a Leitz Aristoplan light microscope either usingbright field or epifluorescence and an FITC filter (Leica, Montréal,Quebec, Canada). Digital pictures were taken using a Nikon camera(Coolpix 4500, Montréal, Québec, Canada) and used for quantifi-cation using MetaMorph 6.1r3 (Universal Imaging, Downingtown,PA, USA). The areas of interest (cingulate and somatosensory cortex,hippocampus) were manually outlined to quantify the percent areaoccupied by GFAP-, thioflavin S-, and 6E10-immunostaining. Thenumber of immunopositive Egr-1 nuclei in the CA1 area of thehippocampus was counted in a 0.062 mm2 surface area. The in-tensity of Egr-1-positive nuclei in the CA1 region of the hippo-campus was measured as total gray level using MetaMorph 6.1r3.The surface area of individual Ibae1-positive microglial cell bodiesand proximal extensions (1 section per mouse, approximately 20microglial cells per section, 4 mice per group) was quantified usingImageJ (National Institutes of Health, Bethesda, MD, USA) andexpressed as a percent of WT.

2.10. Statistical analysis

Data, expressed as mean � standard error of the mean, wereanalyzed using 2-way analysis of variance taking genotype andtreatment as the 2 variables, followed by Newman-Keuls post hocmultiple comparison test (Statistica Academic, Tulsa, OK, USA).Newman-Keulss p values were indicated when the interaction or atleast 1 factor was significant. Student t test was usedwhen 2 groupswere compared (GraphPad Prism 4, GraphPad Software, San Diego,CA, USA). A p � 0.05 was considered significant.

3. Results

3.1. Simvastatin and spatial memory

A/Tmicewere tested twice in theMorris water maze (test 1: at 6months of age after 3months of treatment; test 2: at 9months of ageafter 6months of treatment). In test 1, all animals located the visibleplatform with ease ruling out visual or motor disabilities, and anypossible lack of motivation. During the 5 days of hidden platformtraining (days 4e8), A/T mice exhibited learning impairments,depicted by their increased escape latencies relative to age-matchedWT control mice (Fig. 1A). This deficit, however, did not reach sta-tistical significance despite a clear trend on days 7 and 8. Similarly,during the probe trial (day 9), despite decreased percentage of timespent and distance traveled in the target quadrant, and a smallernumberof platformcrossings inA/Tmice comparedwithWTcontrolmice, these parameters of memory deficits did not reach statisticalsignificance and A/T mice had no swimming disability (Fig. 1B). Intest 2, 9-month-old A/T mice were significantly impaired in thelearning and memory components of the test (Fig. 1C and D). Sim-vastatin failed to improve learning andmemory performance in A/Tmice treated for 3 (Fig. 1A and B) or 6 (Fig. 1C and D) months, butenhanced memory performance in WT mice treated for 3 months(Fig. 1B), as previously reported (Li et al., 2006).

3.2. Simvastatin and baseline protein levels of Egr-1

The baseline (Dickey et al., 2003; Tong et al., 2012) or induced(Blanchard et al., 2008) messenger RNA or protein levels of thememory-related immediate early gene Egr-1 are decreased in APPmice and, particularly in the CA1 area of the hippocampus involvedwith memory formation. Consistent with these findings, baselinelevels of Egr-1 in the CA1 area of the hippocampus were lower in A/

Fig. 1. Simvastatin did not improve spatial learning and memory in A/T mice. (A) A/T mice (6-month-old, closed triangles) had no deficit in the visible platform session, butdisplayed impaired although not significant learning capacity during the hidden-platform test compared with WT littermates (closed circles). Simvastatin treatment for 3 monthshad no effect on the learning capacity of A/T (open triangles) or WT (open circles) mice. (B) A/T mice treated or not with simvastatin for 3 months displayed a slight, not significantdeficit in spatial memory compared with untreated WT control mice, indicated according to the reduced percentage of time spent and distance travelled in the target quadrant, andfewer crossings over the previously located platform during the probe trial. However, these deficits were significant compared with simvastatin-treated WT control mice thatshowed enhanced memory performance compared with untreated WT control mice. (C) A/T mice (9-month-old) with (open triangles) and without (closed triangles) extendedsimvastatin treatment (6 months) had clear learning deficits compared with WT control mice, and this extended treatment did not affect the performance of WT mice (open circles).(D) Spatial memory deficits in A/T mice (probe trial) were not recovered even after extended simvastatin treatment. Error bars represent SEM (n ¼ 9e13 mice per group). One star,p < 0.05; 2 stars, p < 0.01; 3 stars, p < 0.001, using repeated measures ANOVA (learning curve) and 2-way ANOVA (probe trial parameters) followed by Newman-Keuls post hoc test.Abbreviations: ANOVA, analysis of variance; SEM, standard error of the mean; sv, simvastatin; WT, wild type.

P. Papadopoulos et al. / Neurobiology of Aging xxx (2013) 1e104

T mice compared with WT control mice, as shown by the signifi-cantly decreased number and staining intensity of Egr-1 immuno-stained nuclei (Fig. 2). Recently, upregulation of baseline levels ofEgr-1 in the CA1 areawas found to correlate with memory rescue inadult APP mice treated with simvastatin whereas aged APP micethat did not recover memory did not display normalization of Egr-1protein levels (Tong et al., 2012). Accordingly, in our study, simva-statin failed to improve Egr-1-immunoreactivity in CA1 neurons ofA/T mice with persistent memory deficits (Fig. 2).

3.3. Simvastatin decreased insoluble Ab species and Ab plaque load

In line with previous studies (Ongali et al., 2010), A/T micefeatured increased levels of Ab species and Ab plaque load (Fig. 3).Simvastatin significantly reduced the levels of insoluble Ab1e40and Ab1e42 in cortex and hippocampus of A/T mice, measuredusing ELISA (Fig. 3A and B). Additionally, total Ab plaque loaddetermined according to 6E10 immunolabeling on thin sectionswas significantly decreased in cortex and hippocampus (�59%and �53%; p < 0.05, respectively) (Fig. 3C), whereas the small de-creases in dense-core Ab plaques detected using thioflavin Sstaining did not reach significance in either brain region (Fig. 3D).The levels of soluble Ab species measured using Western blotanalysis remained unchanged after simvastatin treatment (Fig. 3E),including the 4-kDa band of monomeric Ab. The protein levels ofAb synthesizing and degrading enzymes, b-site APP cleavingenzyme 1 and neprilysin, respectively, were similarly not altered by

simvastatin (Fig. 3F). Together, these results indicate that simva-statin promoted a decrease in parenchymal amyloidosis in A/Tmice,in line with some (Shinohara et al., 2010; Tamboli et al., 2010), butnot all studies (Li et al., 2006; Tong et al., 2009, 2012) in APP mice.

3.4. Simvastatin effects on the neurovascular coupling response towhisker stimulation

Impaired functional hyperemia on sensory activation, indicativeof an imbalance between neuronal demand and blood supply to theactivated brain area, has been reported in A/T mice of different ages(Ongali et al., 2010). We confirmed that the CBF response towhiskerstimulation was significantly impaired in A/T mice at 9 months ofage compared withWTcontrol mice, andwe found that simvastatintreatment did not improve this deficit (Fig. 4). Surprisingly, therewas a slight although significant deterioration in the evoked CBFresponse in simvastatin-treatedWTmice compared with untreatedcontrol mice (WT: 22.0 � 1.9% vs. WTsv: 16.6 � 1.2%, p < 0.05), aneffect that was not observed in previous cohorts of adult or agedWTmice treated with simvastatin (Tong et al., 2009, 2012).

3.5. Simvastatin and neuroinflammation

Astrocytesandmicroglia are critical inorchestrating the immunityof the brain in response to pathogenic molecules such as Ab (Rivest,2011). A/T mice displayed increased GFAP immunoreactivity in thecortex and hippocampus comparedwith that inWTmice (p< 0.001),

Fig. 2. Simvastatin did not upregulate baseline Egr-1 protein levels in A/T mice. Egr-1immunopositive nuclei were significantly decreased in the CA1 area of the hippo-campus of A/T mice relative to WT control mice (delineated by arrows in left panelsand magnified in right panels). Simvastatin failed to normalize the number and in-tensity of Egre1-positive nuclei even after 6 months of treatment (n ¼ 4 mice pergroup). Three stars, p < 0.001 using 2-way ANOVA followed by Newman-Keuls posthoc test. Scale bar, 50 mm. Abbreviations: ANOVA, analysis of variance; Egr-1, earlygrowth response-1; sv, simvastatin; WT, wild type.

P. Papadopoulos et al. / Neurobiology of Aging xxx (2013) 1e10 5

and simvastatin did not reduce such astroglial activation in eitherregion (Fig.5).Microgliawerealsohighlyactivated inA/Tmousebrainasevidencedby the swellingofmicroglial cell bodiesand theextentoftheir ramifications (Fig. 6). Simvastatin failed to exert any protectiveeffects in minimizing microglial activation in the cortex and thehippocampus of A/T mice (Fig. 6).

3.6. Simvastatin, cerebrovascular reactivity, and fibrosis

As reported before (Ongali et al., 2010), cerebral arteries of A/Tmice featured intact contractile responses, as shown herein for ET-1,

but were significantly impaired in their dilatory capacity comparedwith their control counterparts, determined for ACh- and CGRP-induced dilatations (Fig. 7, Table 1). A small decrease in baselineNO synthesis was also evidenced by the smaller diameter decreaseinduced by NOS inhibition with L-NNA superfusion. However, dila-tory responses to the NO donor SNP were intact in A/T mice, indic-ative of a preserved capacity of the smooth muscle cells to dilate(Fig. 7). Simvastatin fully normalized constitutiveNO-mediated toneby the end of L-NNA incubation time (Fig. 7), and did not affect thecontractile responses induced by ET-1. However, simvastatinsignificantlyworsened the dilatory deficits toACh andCGRP. Indeed,the dilatory responses were completely abolished at low agonistconcentrations and reversed to small contractions at higher con-centrations (Fig. 7). Likewise, the response to the NO donor SNPwasaltered in simvastatin-treated A/T mice and resulted in constrictionat low concentrations that tended toward dilatation at higher con-centrations (Fig. 7). Simvastatin didnot alter the arterial responses inWT mice (Fig. 7).

In search for an explanation for the aggravated cerebrovascularfunction in simvastatin-treated A/T mice, we assessed simvastatineffects on markers of vascular fibrosis and on the NO-synthesizingenzyme, eNOS, in protein extracts of pial vessels. The levels of theseproteins did not differ between 9-month-old A/T mice and WTcontrol mice, and simvastatin, despite its worsening vasomotoreffects, did not alter any of these proteins (Fig. 8).

4. Discussion

We report that simvastatin failed to salvage mnemonic, hemo-dynamic, and neuroinflammatory function in adult A/T mice.Interestingly, the learning and memory deficits persisted despitesignificant decreases in insoluble Ab species and in 6E10 immu-nodetected Ab plaque load, but with persistent elevated soluble Abspecies. The results also show that simvastatin had limited benefitson cerebrovascular function in A/T mice, but rather exacerbated thevasodilatory deficits. These results highlight the potential limita-tions for simvastatin therapy in AD patients with a pathology notlimited to Ab but also encompassing TGF-b1-mediated inflamma-tion and cerebrovascular fibrosis.

4.1. TGF-b1 as an exacerbating factor in memory

Unlike our recent findings in adult APP mice (Tong et al., 2012),simvastatin did not protect spatial learning and memory in adult A/T mice despite extension of treatment duration to 6 months. Datasuggest that the chronic upregulation of TGF-b1 added a level ofcomplexity to the Ab-mediated neuronal pathology and renderedA/T mice resistant to a statin therapy proven effective in APP mice(Li et al., 2006; Tong et al., 2012). Interestingly, although neuro-protective effects of TGF-b1 in AD mice or patients have been re-ported (Caraci et al., 2008; Tesseur and Wyss-Coray, 2006; Tesseuret al., 2006), it has also been identified as an important contributorto AD pathogenesis (Flanders et al., 1995; van der Wal et al., 1993;Wyss-Coray et al., 1997, 2000). Particularly, chronic increases ofTGF-b1 have been associated with age-related reductions of hip-pocampal neurogenesis with up to a 60% decrease in the number ofimmature neurons (Buckwalter et al., 2006). A causal relationshipbetween Ab-induced neurogenesis and memory deficits has beenshown in various AD mouse models (Lazarov et al., 2010; Valeroet al., 2011; Verret et al., 2007), and further substantiated by re-ports of improved memory after pharmacological stimulation ofneurogenesis in these mice (Chen et al., 2000; Fiorentini et al.,2010). TGF-b1 upregulation did not induce spatial memory im-pairments in TGF mice (Nicolakakis et al., 2011; Papadopoulos et al.,2010), nor did it result in more severe learning andmemory deficits

Fig. 3. Simvastatin reduced insoluble Ab levels and Ab plaque load in A/T mice. The levels of insoluble Ab1-40 (A) and Ab1-42 (B) were significantly reduced in simvastatin-treated A/Tmice, assayed in cortex and hippocampus using ELISA. (C) Diffuse and dense-core Ab plaques immunostained with the 6E10 antibody in 5-mm thick paraffin sections weresignificantly reduced in simvastatin-treated A/T mice (n ¼ 4 mice per group). (D) However, thioflavin S-stained dense-core Ab plaque load was unaltered by simvastatin in cortex andhippocampus of A/T mice. (E) Western blot analysis revealed no effect of simvastatin on soluble Ab species in cortex and hippocampus of A/T mice. (F) Similarly, BACE and neprilysinprotein levels were not altered by treatment, measured using Western blot analysis in cortex and hippocampus of A/T mice compared with their untreated counterparts. Actin wasused as a reference for loading (n ¼ 4 mice per group). Error bars represent SEM (n ¼ 4 mice per group). One star, p < 0.05 for comparison with A/T mice using 2-way ANOVAfollowed by Newman-Keuls post hoc test. Abbreviations: Ab, amyloid beta; ANOVA, analysis of variance; BACE, b-site amyloid precursor protein cleaving enzyme; Co, cortex; ELISA,enzyme-linked immunosorbent assay; Hi, hippocampus; SEM, standard error of the mean; sv, simvastatin.

P. Papadopoulos et al. / Neurobiology of Aging xxx (2013) 1e106

in A/T mice compared with age-matched APP mice (Ongali et al.,2010). Hence, TGF-mediated inhibition of adult neurogenesismight not be sufficient to harmmemory processes, but might act asan exacerbating factor to the already impaired neurogenesis causedby Ab in APP mice (Lazarov et al., 2010; Valero et al., 2011),rendering neurogenesis in A/T mice to levels unresponsive topharmacotherapy.

Interestingly, simvastatin is known to promote neurogenesis andimprove cognitive function through activation of the PI3k/Aktpathway (Wu et al., 2008) in experimental models of stroke (Chenet al., 2003; Karki et al., 2009) and traumatic brain injury (Lu et al.,2007; Wu et al., 2008). Activation of PI3k/Akt signaling was alsosuggested as a possible mechanism for the memory-rescuing effectof simvastatin in APPmice (Li et al., 2006; Tong et al., 2012). BecauseTGF-b1 is an activator of PI3k/Akt signaling in the hippocampus (Zhuet al., 2004), this pathway might have been overstimulated in A/Tmice because of chronic TGF-b1 overproduction, thereby preventingany further activation by simvastatin. Such a possibility would becompatible with the failure of simvastatin to upregulate Egr-1protein levels in A/T mice, Egr-1 being downstream of PI3k/Akt

signaling (Bozon et al., 2003; Davis et al., 2003; Jones et al., 2001),and linked to memory recovery in adult simvastatin-treated APPmice (Tong et al., 2012), or memory retrieval in newborn dentategyrus neurons of adult mice (Trouche et al., 2009).

4.2. Insoluble Ab reduction and persistent memory deficits

Simvastatin failed to normalize cognitive performance despitesignificantly reduced brain levels of insoluble Ab and Ab plaque load,in agreement with previous studies (Fassbender et al., 2001;Petanceska et al., 2002). However, this finding is not undisputedbecause other studies found cognitive improvement after simva-statin treatment in Tg2576 (Li et al., 2006) and APPSwe/Ind (Tong et al.,2012) mice, or in asymptomatic middle-aged adults at risk for AD(Carlsson et al., 2008) independently from reductions in Ab plaqueload or cerebrospinal fluid Ab42 levels. Cognitively intact elderly in-dividuals having Ab plaque densities equivalent to those in AD pa-tients suggested that Ab plaquesmight be necessary for initiation butnot maintenance of progressive neurodegeneration (Davis et al.,1999). It could be argued that the sustained high levels of soluble

Fig. 6. Simvastatin did not reduce microglial activation. Simvastatin had no reducingeffects on the cell body and process expansion of Ibae1-positive microglial cells in Coand Hi of A/T mice compared with WT mice (n ¼ 4 mice per group). Error bars

Fig. 4. Simvastatin did not restore the stimulus-evoked CBF increase in A/T mice. (A)Traces of averaged evoked CBF responses acquired before, during, and after whiskerstimulation (WT: blue; WTsv: purple; A/T: green; and A/Tsv: pink). (B) Histogramrepresenting the maximum values of the evoked CBF responses for each group (averagefrom 4e5 stimulations), measured using LDF. The impaired hyperemic response towhisker stimulation in A/T mice was not rescued by simvastatin (n ¼ 4 mice pergroup). Values represent the percent increase of CBF response relative to baseline. Errorbars represent SEM. One star, p < 0.05; 2 stars, p < 0.01; 3 stars, p < 0.001, using 2-wayANOVA followed by Newman-Keuls post hoc test. Abbreviations: ANOVA, analysis ofvariance; CBF, cerebral blood flow; LDF, laser Doppler flowmetry; SEM, standard errorof the mean; sv, simvastatin; WT, wild type.

P. Papadopoulos et al. / Neurobiology of Aging xxx (2013) 1e10 7

Ab species in simvastatin-treated A/T mice might explain theirpersistent memory deficit because Ab oligomers are presumably themost harmful to neuronal and synaptic function (Lesne et al., 2008;Mucke et al., 2000). Yet, simvastatin-treated APP mice recoveredmemory without any reduction in soluble Ab (Li et al., 2006; Tonget al., 2012), and aged Tg2576 mice lacking the catalytic subunit ofnicotinamide adenine dinucleotide phosphate (NADPH) oxidase(Park et al., 2008) or treated with cyclooxygenase-2 (COX-2) in-hibitors (Kotilinek et al., 2008) had preserved memory despite high

represent SEM. One star, p < 0.05; 2 stars, p < 0.01, using 2-way ANOVA followed byNewman-Keuls post hoc test. Right scale bar, 50 mm; left scale bar, 10 mm. Abbrevia-tions: ANOVA, analysis of variance; Co, cortex; Hi, hippocampus; Iba-1, ionized calciumbinding adaptor molecule 1; SEM, standard error of the mean; sv, simvastatin; WT,wild type.

Fig. 5. Simvastatin did not reduce astrocytosis. The percent of area occupied by GFAP-immunopositive astrocytes in cortex and hippocampus of A/T mice compared with WTlittermates remained elevated after simvastatin treatment (n ¼ 4 mice per group). Onestar, p < 0.05; 2 stars, p < 0.01, using 2-way ANOVA followed by Newman-Keuls posthoc test. Abbreviations: ANOVA, analysis of variance; Co, cortex; GFAP, glial fibrillaryacidic protein; Hi, hippocampus; sv, simvastatin; WT, wild type.

levels of soluble and insoluble Ab, further pointing to the dichotomybetween memory impairment and amyloidosis.

4.3. Simvastatin and neuroinflammation

The abnormal production of proinflammatory cytokines andchemokines by glial cells can disrupt nerve terminal activitycausing dysfunction and loss of synapses, and eventually memorydecline (Ferretti and Cuello, 2011; Frank-Cannon et al., 2009; Raoet al., 2012). Furthermore, astrocytes can be activated by TGF-b1 togenerate more Ab, further aggravating the pathology (Burton et al.,2002). Thus, treatments directed at controlling glial cell activationmight be valuable to counter AD neurodegeneration. In A/T mice,simvastatin did not display anti-inflammatory properties because itwas ineffective in silencing Ab plaque-associated microglia andastrocytes. This contrasted with our previous study in APP mice(Tong et al., 2009), and might thus have contributed to the persis-tent memory deficit in treated A/T mice. Enduring glial activationmight also account for the drug’s failure to normalize the neuro-vascular coupling response to whisker stimulation that criticallydepends on astrocytes for the synthesis and release of vasoactivemessengers (Koehler et al., 2009).

4.4. Simvastatin and cerebrovascular reactivity

We anticipated normalization of cerebrovascular function withsimvastatin based on its significant benefits in adult and aged APP

Fig. 7. Simvastatin worsened dilatory function in A/T mice. Concentration-dependent contractile responses to ET-1 were unaltered in treated (open triangles) and untreated (closedtriangles) A/T mice compared with treated (closed circles) and untreated (open circles) WT control mice. In contrast, the impaired cerebrovascular dilatations to ACh and CGRP in A/Tmice (closed triangles) were aggravated and even reversed to weak constrictions at high agonist concentrations in simvastatin-treated A/T mice (open triangles). Baseline NOsynthesis measured during NOS inhibition (L-NNA, 10�5 M) was fully recovered in simvastatin-treated A/T mice, whereas the dilatations induced by the NO donor SNP wereimpaired by treatment. Error bars represent SEM. n ¼ 4 for each group. One star, p < 0.05; 2 stars, p < 0.01; 3 stars, p < 0.001, of A/T mice relative to their WT littermates, anduntreated versus treated A/T mice. * p < 0.05, ** p < 0.01, *** p < 0.001, using repeated measures ANOVA followed by Newman-Keuls post hoc test. Abbreviations: ACh, acetylcholine;ANOVA, analysis of variance; CGRP, calcitonin gene-related peptide; ET-1, endothelin-1; L-NNA, Nu-nitro-L-arginine; NO, nitric oxide; NOS, nitric oxide synthase; SEM, standarderror of the mean; SNP, sodium nitroprusside; sv, simvastatin; WT, wild type.

P. Papadopoulos et al. / Neurobiology of Aging xxx (2013) 1e108

(Tong et al., 2009, 2012) or TGF (Tong and Hamel, 2013) mice. Themost surprising finding was that cerebral arteries from simvastatin-treated A/T mice did not respond to the vasodilators, ACh and CGRP,or displayed small constrictions at high agonist concentrations. Thisunexpected response indicated deleterious effects of simvastatin onreceptor-mediated vasodilatory responses through endothelial andsmooth muscle cells. Such a finding could be explained by theability of simvastatin to elicit NO-mediated dilations via direct

Table 1Effect of simvastatin on cerebrovascular responses in A/T mice

Agent WT WTsv A/T A/Tsv

AChEAmax 49.1 � 3.6 41.3 � 3.7 26.1 � 3.3++ 16.6 � 13.6++,**pD2 8.1 � 0.2 8.3 � 0.3 7.9 � 0.4 ND

CGRPEAmax 46.0 � 3.7 35.5 � 4.1 21.3 � 1.0+++ 14.4 � 3.9+++,***pD2 8.2 � 0.2 8.3 � 0.3 8.6 � 0.1 ND

SNPEAmax 64.1 � 6.1 57.8 � 3.0 58.4 � 3.2 3.4 � 10.1+++,***

ET-1EAmax 57.3 � 7.0 54.2 � 4.2 41.6 � 6.8 54.1 � 4.7pD2 8.3 � 0.4 8.0 � 4.2 8.2 � 0.5 8.0 � 0.3

L-NNAEAmax 50.0 � 4.3 36.1 � 6.0 27.2 � 6.9+ 41.8 � 6.8+,*

Data are mean � SEM (n ¼ 3e4 mice per group) and are expressed as the agonistmaximal response (EAmax) or potency (pD2, �[logEC50]). EAmax is the percentmaximal dilatation to ACh, CGRP, and SNP, or the percent maximal diameterdecrease to ET-1, 5-HT, or after 35 minutes of inhibition with 10�5 M L-NNA.Key: 5-HT, serotonin; ACh, acetylcholine; ANOVA, analysis of variance; CGRP,calcitonin gene-related peptide; EC50, half maximal effective concentration; ET-1,endothelin-1; L-NNA, Nu-nitro-L-arginine; ND, not determined; SEM, standarderror of the mean; SNP, sodium nitroprusside; sv, simvastatin; WT, wild type.+, * p < 0.05; ++, ** p < 0.01, +++, *** p < 0.001 when compared with untreatedWT control mice (+) or A/T mice (*) using 2-way ANOVA followed by Newman-Keuls post hoc multiple comparison test.

activation of soluble guanylyl cyclase (sGC), as shown in retinalarteries (Nagaoka et al., 2007). Simvastatinmight thus have acted asa competitive agonist for the ACh-mediated NO signaling cascade,thereby chronically exhausting the soluble guanylyl cyclase/cyclic

Fig. 8. Simvastatin had no effect on profibrotic and eNOS protein levels. Protein levelsof VEGF, CTGF, and eNOS were not altered in pial vessels from A/T mice compared withWT control mice, and simvastatin did not affect any of these proteins, measured usingWestern blot analysis. Actin was used as a reference for loading (n ¼ 4 mice per group).Error bars represent SEM. One star, p < 0.05; 2 stars, p < 0.01; 3 stars, p < 0.001, 2-wayANOVA followed by Newman-Keuls post hoc test. Abbreviations: ANOVA, analysis ofvariance; CTGF, connective tissue growth factor; eNOS, endothelial nitric oxide syn-thase; SEM, standard error of the mean; sv, simvastatin; VEGF, vascular endothelialgrowth factor; WT, wild type.

P. Papadopoulos et al. / Neurobiology of Aging xxx (2013) 1e10 9

guanosine monophosphate (sGC/cGMP) pathway. The vasocon-striction observed during incubationwith the NO donor SNP furthersupports a disruption of sGC signalling because it required higherconcentrations of SNP to activate and induce dilatation. Whethersimilar effects of simvastatin on Kþ channels that mediate theCGRP-induced dilation (Vedernikov et al., 2002) are involved in A/Tmouse diseased vessels will require further investigation. Upregu-lation of the protein levels of the NO-synthesizing enzyme, eNOS,has been reported in brain of APP mice treated with simvastatin (Liet al., 2006). However, we did not find such an effect in A/T mice,further pointing to the complexity of their cerebrovascularpathology.

4.5. Conclusion

Our results suggest that the concurrent upregulation of Ab andTGF-b1 in A/T mice might represent an advanced stage of the dis-ease or the complex interaction between cerebrovascular pathologyand Ab-induced neuronal deficits, which is beyond the therapeuticcapacity of simvastatin (Butterfield et al., 2011; Li et al., 2010). Ourdata further highlight the importance of studying drug efficacy inanimal models that reproduce multiple aspects of the human ADpathology for a reliable screening of potential new treatmentstrategies.

Disclosure statement

The authors have no conflicts of interest.Animal use was in accordance with the Animal Ethics Commit-

tee of the Montreal Neurological Institute, and abided by theguidelines of the Canadian Council on Animal Care.

Acknowledgements

This work was supported by the Canadian Institutes of HealthResearch (CIHR grants MOP-84275 and MOP-126001 to EH) and aJeanne Timmins Costello Fellowship (PP). We are grateful toDr Lennart Mucke (Gladstone Institute of Neurological Disease andDepartment of Neurology, UCSF, CA) and the J. David GladstoneInstitutes for the hAPPSwe,Ind and TGF-b1 transgenic mousebreeders.

References

Aboulkassim, T., Tong, X.K., Tse, Y.C., Wong, T.P., Woo, S.B., Neet, K.E., Brahimi, F.,Hamel, E., Saragovi, H.U., 2011. Ligand-dependent TrkA activity in brain differ-entially affects spatial learning and long-term memory. Mol. Pharmacol. 80,498e508.

Arvanitakis, Z., Schneider, J.A., Wilson, R.S., Bienias, J.L., Kelly, J.F., Evans, D.A.,Bennett, D.A., 2008. Statins, incident Alzheimer disease, change in cognitivefunction, and neuropathology. Neurology 70, 1795e1802.

Blanchard, J., Martel, G., Guillou, J.L., Nogues, X., Micheau, J., 2008. Impairment ofspatial memory consolidation in APP(751SL) mice results in cue-guidedresponse. Neurobiol. Aging 29, 1011e1021.

Bozon, B., Davis, S., Laroche, S., 2003. A requirement for the immediate early genezif268 in reconsolidation of recognition memory after retrieval. Neuron 40,695e701.

Buckwalter, M.S., Yamane, M., Coleman, B.S., Ormerod, B.K., Chin, J.T., Palmer, T.,Wyss-Coray, T., 2006. Chronically increased transforming growth factor-beta1strongly inhibits hippocampal neurogenesis in aged mice. Am. J. Pathol. 169,154e164.

Burton, T., Liang, B., Dibrov, A., Amara, F., 2002. Transcriptional activation and in-crease in expression of Alzheimer’s beta-amyloid precursor protein gene ismediated by TGF-beta in normal human astrocytes. Biochem. Biophys. Res.Commun. 295, 702e712.

Butterfield, D.A., Barone, E., Mancuso, C., 2011. Cholesterol-independent neuro-protective and neurotoxic activities of statins: perspectives for statin use inAlzheimer disease and other age-related neurodegenerative disorders. Phar-macol. Res. 64, 180e186.

Caraci, F., Battaglia, G., Busceti, C., Biagioni, F., Mastroiacovo, F., Bosco, P., Drago, F.,Nicoletti, F., Sortino, M.A., Copani, A., 2008. TGF-beta 1 protects against Abeta-

neurotoxicity via the phosphatidylinositol-3-kinase pathway. Neurobiol. Dis. 30,234e242.

Carlsson, C.M., Gleason, C.E., Hess, T.M., Moreland, K.A., Blazel, H.M., Koscik, R.L.,Schreiber, N.T., Johnson, S.C., Atwood, C.S., Puglielli, L., Hermann, B.P.,McBride, P.E., Stein, J.H., Sager, M.A., Asthana, S., 2008. Effects of simvastatin oncerebrospinal fluid biomarkers and cognition in middle-aged adults at risk forAlzheimer’s disease. J. Alzheimers Dis. 13, 187e197.

Chen, G., Rajkowska, G., Du, F., Seraji-Bozorgzad, N., Manji, H.K., 2000. Enhancementof hippocampal neurogenesis by lithium. J. Neurochem. 75, 1729e1734.

Chen, J., Zhang, Z.G., Li, Y., Wang, Y., Wang, L., Jiang, H., Zhang, C., Lu, M.,Katakowski, M., Feldkamp, C.S., Chopp, M., 2003. Statins induce angiogenesis,neurogenesis, and synaptogenesis after stroke. Ann. Neurol. 53, 743e751.

Cramer, C., Haan, M.N., Galea, S., Langa, K.M., Kalbfleisch, J.D., 2008. Use of statinsand incidence of dementia and cognitive impairment without dementia in acohort study. Neurology 71, 344e350.

Davis, D.G., Schmitt, F.A., Wekstein, D.R., Markesbery, W.R., 1999. Alzheimerneuropathologic alterations in aged cognitively normal subjects. J. Neuropathol.Exp. Neurol. 58, 376e388.

Davis, S., Bozon, B., Laroche, S., 2003. How necessary is the activation of the im-mediate early gene zif268 in synaptic plasticity and learning? Behav. Brain Res.142, 17e30.

DeIpolyi, A.R., Fang, S., Palop, J.J., Yu, G.Q., Wang, X., Mucke, L., 2008. Alterednavigational strategy use and visuospatial deficits in hAPP transgenic mice.Neurobiol. Aging 29, 253e266.

Dickey, C.A., Loring, J.F., Montgomery, J., Gordon, M.N., Eastman, P.S., Morgan, D.,2003. Selectively reduced expression of synaptic plasticity-related genes inamyloid precursor protein þ presenilin-1 transgenic mice. J. Neurosci. 23,5219e5226.

Fassbender, K., Simons, M., Bergmann, C., Stroick, M., Lutjohann, D., Keller, P.,Runz, H., Kuhl, S., Bertsch, T., von Bergmann, K., Hennerici, M., Beyreuther, K.,Hartmann, T., 2001. Simvastatin strongly reduces levels of Alzheimer’s diseasebeta -amyloid peptides Abeta 42 and Abeta 40 in vitro and in vivo. Proc. Natl.Acad. Sci. U.S.A 98, 5856e5861.

Ferretti, M.T., Cuello, A.C., 2011. Does a pro-inflammatory process precede Alz-heimer’s disease and mild cognitive impairment? Curr. Alzheimer Res. 8,164e174.

Fiorentini, A., Rosi, M.C., Grossi, C., Luccarini, I., Casamenti, F., 2010. Lithium im-proves hippocampal neurogenesis, neuropathology and cognitive functions inAPP mutant mice. PloS One 5, e14382.

Flanders, K.C., Lippa, C.F., Smith, T.W., Pollen, D.A., Sporn, M.B., 1995. Alteredexpression of transforming growth factor-beta in Alzheimer’s disease.Neurology 45, 1561e1569.

Frank-Cannon, T.C., Alto, L.T., McAlpine, F.E., Tansey, M.G., 2009. Does neuro-inflammation fan the flame in neurodegenerative diseases? Mol. Neurodegener.4, 47.

Grammas, P., Ovase, R., 2002. Cerebrovascular transforming growth factor-betacontributes to inflammation in the Alzheimer’s disease brain. Am. J. Pathol.160, 1583e1587.

Haag, M.D., Hofman, A., Koudstaal, P.J., Stricker, B.H., Breteler, M.M., 2009. Statins areassociated with a reduced risk of Alzheimer disease regardless of lipophilicity.The Rotterdam Study. J. Neurol. Neurosurg. Psychiatry 80, 13e17.

Iadecola, C., 2004. Neurovascular regulation in the normal brain and in Alzheimer’sdisease. Nat. Rev. Neurosci. 5, 347e360.

Jick, H., Zornberg, G.L., Jick, S.S., Seshadri, S., Drachman, D.A., 2000. Statins and therisk of dementia. Lancet 356, 1627e1631.

Jones, M.W., Errington, M.L., French, P.J., Fine, A., Bliss, T.V., Garel, S., Charnay, P.,Bozon, B., Laroche, S., Davis, S., 2001. A requirement for the immediate earlygene Zif268 in the expression of late LTP and long-term memories. Nat. Neu-rosci. 4, 289e296.

Karki, K., Knight, R.A., Han, Y., Yang, D., Zhang, J., Ledbetter, K.A., Chopp, M.,Seyfried, D.M., 2009. Simvastatin and atorvastatin improve neurologicaloutcome after experimental intracerebral hemorrhage. Stroke 40, 3384e3389.

Koehler, R.C., Roman, R.J., Harder, D.R., 2009. Astrocytes and the regulation of ce-rebral blood flow. Trends Neurosci. 32, 160e169.

Kotilinek, L.A., Westerman, M.A., Wang, Q., Panizzon, K., Lim, G.P., Simonyi, A.,Lesne, S., Falinska, A., Younkin, L.H., Younkin, S.G., Rowan, M., Cleary, J.,Wallis, R.A., Sun, G.Y., Cole, G., Frautschy, S., Anwyl, R., Ashe, K.H., 2008.Cyclooxygenase-2 inhibition improves amyloid-beta-mediated suppression ofmemory and synaptic plasticity. Brain 131, 651e664.

Kurata, T., Miyazaki, K., Kozuki, M., Panin, V.L., Morimoto, N., Ohta, Y., Nagai, M.,Ikeda, Y., Matsuura, T., Abe, K., 2011. Atorvastatin and pitavastatin improvecognitive function and reduce senile plaque and phosphorylated tau in agedAPP mice. Brain Res. 1371, 161e170.

Lazarov, O., Mattson, M.P., Peterson, D.A., Pimplikar, S.W., van Praag, H., 2010.When neurogenesis encounters aging and disease. Trends Neurosci. 33,569e579.

Lesne, S., Kotilinek, L., Ashe, K.H., 2008. Plaque-bearing mice with reduced levels ofoligomeric amyloid-beta assemblies have intact memory function. Neurosci-ence 151, 745e749.

Li, G., Shofer, J.B., Rhew, I.C., Kukull, W.A., Peskind, E.R., McCormick, W., Bowen, J.D.,Schellenberg, G.D., Crane, P.K., Breitner, J.C., Larson, E.B., 2010. Age-varying as-sociation between statin use and incident Alzheimer’s disease. J. Am. Geriatr.Soc. 58, 1311e1317.

Li, L., Cao, D., Kim, H., Lester, R., Fukuchi, K., 2006. Simvastatin enhances learningand memory independent of amyloid load in mice. Ann. Neurol. 60, 729e739.

P. Papadopoulos et al. / Neurobiology of Aging xxx (2013) 1e1010

Lu, D., Qu, C., Goussev, A., Jiang, H., Lu, C., Schallert, T., Mahmood, A., Chen, J., Li, Y.,Chopp, M., 2007. Statins increase neurogenesis in the dentate gyrus, reducedelayed neuronal death in the hippocampal CA3 region, and improve spatiallearning in rat after traumatic brain injury. J. Neurotrauma 24, 1132e1146.

Mucke, L., Masliah, E., Yu, G.Q., Mallory, M., Rockenstein, E.M., Tatsuno, G., Hu, K.,Kholodenko, D., Johnson-Wood, K., McConlogue, L., 2000. High-level neuronalexpression of abeta 1-42 in wild-type human amyloid protein precursortransgenic mice: synaptotoxicity without plaque formation. J. Neurosci. 20,4050e4058.

Nagaoka, T., Hein, T.W., Yoshida, A., Kuo, L., 2007. Simvastatin elicits dilation ofisolated porcine retinal arterioles: role of nitric oxide and mevalonate-rho ki-nase pathways. Invest. Ophthalmol. Vis. Sci. 48, 825e832.

Nicolakakis, N., Aboulkassim, T., Aliaga, A., Tong, X.K., Rosa-Neto, P., Hamel, E., 2011.Intact memory in TGF-beta1 transgenic mice featuring chronic cerebro-vascular deficit: recovery with pioglitazone. J. Cereb. Blood Flow Metab. 31,200e211.

Ongali, B., Nicolakakis, N., Lecrux, C., Aboulkassim, T., Rosa-Neto, P.,Papadopoulos, P., Tong, X.K., Hamel, E., 2010. Transgenic mice overexpressingAPP and transforming growth factor-beta1 feature cognitive and vascularhallmarks of Alzheimer’s disease. Am. J. Pathol. 177, 3071e3080.

Papadopoulos, P., Ongali, B., Hamel, E., 2010. Selective in vivo antagonism ofendothelin receptors in transforming growth factor-beta1 transgenic mice thatmimic the vascular pathology of Alzheimer’s disease. Can. J. Physiol. Pharmacol.88, 652e660.

Park, L., Zhou, P., Pitstick, R., Capone, C., Anrather, J., Norris, E.H., Younkin, L.,Younkin, S., Carlson, G., McEwen, B.S., Iadecola, C., 2008. Nox2-derived radicalscontribute to neurovascular and behavioral dysfunction in mice overexpressingthe amyloid precursor protein. Proc. Natl. Acad. Sci. U.S.A 105, 1347e1352.

Petanceska, S.S., DeRosa, S., Olm, V., Diaz, N., Sharma, A., Thomas-Bryant, T., Duff, K.,Pappolla, M., Refolo, L.M., 2002. Statin therapy for Alzheimer’s disease: will itwork? J. Mol. Neurosci. 19, 155e161.

Rao, J.S., Kellom, M., Kim, H.W., Rapoport, S.I., Reese, E.A., 2012. Neuroinflammationand synaptic loss. Neurochem. Res. 37, 903e910.

Rivest, S., 2011. The promise of anti-inflammatory therapies for CNS injuries anddiseases. Exp. Rev. Neurother. 11, 783e786.

Sano, M., Bell, K.L., Galasko, D., Galvin, J.E., Thomas, R.G., van Dyck, C.H., Aisen, P.S.,2011. A randomized, double-blind, placebo-controlled trial of simvastatin totreat Alzheimer disease. Neurology 77, 556e563.

Shepardson, N.E., Shankar, G.M., Selkoe, D.J., 2011a. Cholesterol level and statin usein Alzheimer disease: I. Review of epidemiological and preclinical studies. Arch.Neurol. 68, 1239e1244.

Shepardson, N.E., Shankar, G.M., Selkoe, D.J., 2011b. Cholesterol level and statin usein Alzheimer disease: II. Review of human trials and recommendations. Arch.Neurol. 68, 1385e1392.

Shinohara, M., Sato, N., Kurinami, H., Takeuchi, D., Takeda, S., Shimamura, M.,Yamashita, T., Uchiyama, Y., Rakugi, H., Morishita, R., 2010. Reduction of brainbeta-amyloid (Abeta) by fluvastatin, a hydroxymethylglutaryl-CoA reductaseinhibitor, through increase in degradation of amyloid precursor protein C-ter-minal fragments (APP-CTFs) and Abeta clearance. J. Biol. Chem. 285,22091e22102.

Simons, M., Schwarzler, F., Lutjohann, D., von Bergmann, K., Beyreuther, K.,Dichgans, J., Wormstall, H., Hartmann, T., Schulz, J.B., 2002. Treatment withsimvastatin in normocholesterolemic patients with Alzheimer’s disease: a 26-week randomized, placebo-controlled, double-blind trial. Ann. Neurol. 52,346e350.

Sparks, D.L., 2011. Alzheimer disease: statins in the treatment of Alzheimer disease.Nat. Rev. Neurol. 7, 662e663.

Sparks, D.L., Sabbagh, M.N., Connor, D.J., Lopez, J., Launer, L.J., Browne, P., Wasser, D.,Johnson-Traver, S., Lochhead, J., Ziolwolski, C., 2005. Atorvastatin for thetreatment of mild to moderate Alzheimer disease: preliminary results. Arch.Neurol. 62, 753e757.

Tamboli, I.Y., Barth, E., Christian, L., Siepmann, M., Kumar, S., Singh, S., Tolksdorf, K.,Heneka, M.T., Lutjohann, D., Wunderlich, P., Walter, J., 2010. Statins promote thedegradation of extracellular amyloid {beta}-peptide by microglia via stimula-tion of exosome-associated insulin-degrading enzyme (IDE) secretion. J. Biol.Chem. 285, 37405e37414.

Tesseur, I., Wyss-Coray, T., 2006. A role for TGF-beta signaling in neurodegeneration:evidence from genetically engineered models. Curr. Alzheimer Res. 3, 505e513.

Tesseur, I., Zou, K., Esposito, L., Bard, F., Berber, E., Can, J.V., Lin, A.H., Crews, L.,Tremblay, P., Mathews, P., Mucke, L., Masliah, E., Wyss-Coray, T., 2006. Defi-ciency in neuronal TGF-beta signaling promotes neurodegeneration and Alz-heimer’s pathology. J. Clin. Invest. 116, 3060e3069.

Tong, X.K., Hamel, E., 2013. Simvastatin restored endothelial and smooth muscle cellfunction and reduced string vessel pathology in transgenic transforming growthfactor-b1 mice Abstract # 620 Presented at: Brain 2013. May 20e23, 2013;Shanghai.

Tong, X.K., Lecrux, C., Hamel, E., 2012. Age-dependent rescue by simvastatin ofAlzheimer’s disease cerebrovascular and memory deficits. J. Neurosci. 32,4705e4715.

Tong, X.K., Nicolakakis, N., Fernandes, P., Ongali, B., Brouillette, J., Quirion, R.,Hamel, E., 2009. Simvastatin improves cerebrovascular function and counterssoluble amyloid-beta, inflammation and oxidative stress in aged APP mice.Neurobiol. Dis. 35, 406e414.

Tong, X.K., Nicolakakis, N., Kocharyan, A., Hamel, E., 2005. Vascular remodelingversus amyloid beta-induced oxidative stress in the cerebrovascular dysfunc-tions associated with Alzheimer’s disease. J. Neurosci. 25, 11165e11174.

Trouche, S., Bontempi, B., Roullet, P., Rampon, C., 2009. Recruitment of adult-generated neurons into functional hippocampal networks contributes toupdating and strengthening of spatial memory. Proc. Natl. Acad. Sci. U.S.A 106,5919e5924.

Valero, J., Espana, J., Parra-Damas, A., Martin, E., Rodriguez-Alvarez, J., Saura, C.A.,2011. Short-term environmental enrichment rescues adult neurogenesis andmemory deficits in APP(Sw, Ind) transgenic mice. PloS One 6, e16832.

van der Wal, E.A., Gomez-Pinilla, F., Cotman, C.W., 1993. Transforming growthfactor-beta 1 is in plaques in Alzheimer and Down pathologies. Neuroreport 4,69e72.

Vedernikov, Y.P., Fulep, E.E., Saade, G.R., Garfield, R.E., 2002. Calcitonin gene-relatedpeptide dilates the pregnant rat uterine vascular bed via guanylate cyclase, ATP-and Ca-sensitive potassium channels and gap junctions. Curr. Med. Res. Opin.18, 465e470.

Verret, L., Jankowsky, J.L., Xu, G.M., Borchelt, D.R., Rampon, C., 2007. Alzheimer’s-type amyloidosis in transgenic mice impairs survival of newborn neuronsderived from adult hippocampal neurogenesis. J. Neurosci. 27, 6771e6780.

Wolozin, B., Kellman, W., Ruosseau, P., Celesia, G.G., Siegel, G., 2000. Decreasedprevalence of Alzheimer disease associated with 3-hydroxy-3-methyglutarylcoenzyme A reductase inhibitors. Arch. Neurol. 57, 1439e1443.

Wu, H., Lu, D., Jiang, H., Xiong, Y., Qu, C., Li, B., Mahmood, A., Zhou, D., Chopp, M.,2008. Simvastatin-mediated upregulation of VEGF and BDNF, activation of thePI3K/Akt pathway, and increase of neurogenesis are associated with therapeuticimprovement after traumatic brain injury. J. Neurotrauma 25, 130e139.

Wyss-Coray, T., Feng, L., Masliah, E., Ruppe, M.D., Lee, H.S., Toggas, S.M.,Rockenstein, E.M., Mucke, L., 1995. Increased central nervous system productionof extracellular matrix components and development of hydrocephalus intransgenic mice overexpressing transforming growth factor-beta 1. Am. J.Pathol. 147, 53e67.

Wyss-Coray, T., Lin, C., Sanan, D.A., Mucke, L., Masliah, E., 2000. Chronic over-production of transforming growth factor-beta1 by astrocytes promotes Alz-heimer’s disease-like microvascular degeneration in transgenic mice. Am. J.Pathol. 156, 139e150.

Wyss-Coray, T., Masliah, E., Mallory, M., McConlogue, L., Johnson-Wood, K., Lin, C.,Mucke, L., 1997. Amyloidogenic role of cytokine TGF-beta1 in transgenic miceand in Alzheimer’s disease. Nature 389, 603e606.

Yaffe, K., Barrett-Connor, E., Lin, F., Grady, D., 2002. Serum lipoprotein levels,statin use, and cognitive function in older women. Arch. Neurol. 59,378e384.

Zhu, Y., Culmsee, C., Klumpp, S., Krieglstein, J., 2004. Neuroprotection by trans-forming growth factor-beta1 involves activation of nuclear factor-kappaBthrough phosphatidylinositol-3-OH kinase/Akt and mitogen-activated proteinkinase-extracellular-signal regulated kinase1,2 signaling pathways. Neurosci-ence 123, 897e906.

Zlokovic, B.V., 2011. Neurovascular pathways to neurodegeneration in Alzheimer’sdisease and other disorders. Nat. Rev. Neurosci. 12, 723e738.