B R A I N R E S E A R C H 1 3 5 2 ( 2 0 1 0 ) 1 7 6 – 1 8 6

ava i l ab l e a t www.sc i enced i r ec t . com

www.e l sev i e r . com/ loca te /b ra i n res

Research Report

Effect of a centrally active angiotensin-converting enzymeinhibitor, perindopril, on cognitive performance in a mousemodel of Alzheimer's disease

Koji Yamadaa,⁎, Shinichi Uchidaa, Syusuke Takahashib, Makoto Takayamaa,Yoshinori Nagatab, Nobuyuki Suzukic, Shiro Shirakuraa, Tomoyuki Kandaa

aPharmacological Research Laboratories, Research Division, Kyowa Hakko Kirin Co., Ltd., 1188 Shimotogari, Nagaizumi-cho,Sunto-gun, Shizuoka 411-8731, JapanbPharmacokinetic Research Laboratories, Research Division, Kyowa Hakko Kirin Co., Ltd., 1188 Shimotogari, Nagaizumi-cho,Sunto-gun, Shizuoka 411-8731, JapancDrug Discovery Laboratories, Research Division, Kyowa Hakko Kirin Co., Ltd., 1188 Shimotogari, Nagaizumi-cho, Sunto-gun,Shizuoka 411-8731, Japan

A R T I C L E I N F O

⁎ Corresponding author. Fax: +81 55 986 7430.E-mail address: kohji.yamada@kyowa-kirAbbreviations: Aβ, amyloid β; APP, amyloid

i.c.v., intracerebroventricular; ORT, object rec

0006-8993/$ – see front matter © 2010 Elsevidoi:10.1016/j.brainres.2010.07.006

A B S T R A C T

Article history:Accepted 6 July 2010Available online 31 July 2010

Angiotensin-converting enzyme (ACE) inhibitors have clinically been widely used as anti-hypertensive agents. In the present study, we compared the effects of a centrally active ACEinhibitor, perindopril, with those of non-centrally active ACE inhibitors, imidapril andenalapril, on cognitive performance in amyloid β(Aβ) 25–35-injected mice, a rodent model ofAlzheimer's disease. We also determined the brain ACE activity in order to elucidate therelationship between the cognitive function and ACE inhibition in the brain. Aβ25–35-injectedmice showed a cognitive impairment in spontaneous alteration and object recognition tests,the indices of immediate working memory and relatively long-term recognition memory,respectively. As indicated by these tests, the oral administration of perindopril (0.1, 0.3 or1 mg/kg/day) significantly reversed the cognitive impairment in these mice, whereasneither imidapril (0.3, 1 or 3 mg/kg/day) nor enalapril (1, 3 or 10 mg/kg/day) had any effect oncognitive performance. Perindopril (1 mg/kg/day), imidapril (3 mg/kg/day), or enalapril(10 mg/kg/day) all inhibited the plasma ACE activities by more than 90%. Using the samedosing regimen, only perindopril inhibited the brain ACE activities by more than 50%,whereas imidapril and enalapril showed much less potent effects. These results suggestthat perindopril ameliorated the cognitive impairment in the Alzheimer's disease modelmice through the inhibition of brain ACE activity, but not peripheral ACE activity. Based onour observations, we concluded that a centrally active ACE inhibitor, perindopril, maytherefore have a beneficial effect on Alzheimer's disease as well as hypertension.

© 2010 Elsevier B.V. All rights reserved.

Keywords:Cognitive impairmentSpontaneous alteration testObject recognition testImidaprilEnalaprilAmyloid β25–35-injected mouse

in.co.jp (K. Yamada).precursor protein; ACE, angiotensin-converting enzyme; ARB, angiotensin receptor blocker;ognition test; s.i.d, single daily

er B.V. All rights reserved.

177B R A I N R E S E A R C H 1 3 5 2 ( 2 0 1 0 ) 1 7 6 – 1 8 6

1. Introduction

Angiotensin-converting enzyme (ACE) inhibitors decreaseblood pressure and systemic vascular resistance by blockingthe formation of angiotensin II, the endogenous pressorsubstance of the renin–angiotensin cascade. While theseagents have been used primarily in the treatment of hyper-tension and congestive heart failure, there are some clinicalstudies in which ACE inhibitors were shown to reduce theincidence of dementia or slow down the rate of cognitivedecline in patients with hypertension (Croog et al., 1986;Rozzini et al., 2006; Yasar et al., 2008; Sink et al., 2009).

Perindopril, a long-acting and centrally active ACE inhib-itor, has been used as an anti-hypertensive agent all over theworld and it has also been reported to reduce risks of dementiaand cognitive decline in the patients with recurrent strokes(Tzourio et al., 2003). Furthermore, Ohrui et al. (2004) showedthat treatment with centrally active ACE inhibitors, but notnon-centrally active ACE inhibitors, could slow down the rateof cognitive decline in mild-to-moderate Alzheimer's diseasepatients with hypertension. Although this suggests thatcentrally active ACE inhibitors, usually prescribed as anti-hypertensive drugs, could be expected to have secondarybenefits on the symptoms of Alzheimer's disease, themechanisms responsible for the pro-cognitive effect of thisclass of drugs have not yet been elucidated.

In the present study, we determined the effect of sub-chronic treatment with perindopril, imidapril and enalapril,on immediate working memory and relatively long-termrecognition memory in a mouse model of Alzheimer'sdisease. We also measured the brain ACE activity under thesame dosing regimen to elucidate whether ACE inhibition inthe brain may have an important effect on the cognitivefunction.

2. Results

2.1. Perindopril, but neither imidapril nor enalapril,eliminated working memory deficits in Alzheimer's diseasemodel mice as determined using the spontaneous alteration test

We first examined immediate working memory deficitsafter intracerebroventricular (i.c.v.) administration of amy-loid β (Aβ)25–35 using the spontaneous alteration test. Fourdays after Aβ25–35-injection, the percent of the spontaneous

Fig. 1 – Experimental protocol. s.i

alteration behavior in the Aβ25–35-injected mice treated withvehicle was significantly lower than that in the control micethat did not receive the Aβ25–35 injection (Figs. 2A, C, and E),while there was no significant difference observed in thenumber of total arm entries (Figs. 2B, D, and F). Thecognitive impairment in these mice lasted at least 1 weekafter Aβ25–35-injection. In addition, donepezil (1 mg kg/day),at the same dosage regimen as ACE inhibitors describedbelow, restored the decreased spontaneous alteration be-havior in these mice without affecting the number of totalarm entries (data not shown).

Daily treatment with perindopril (0.1, 0.3 or 1 mg/kg/day),which started one day before the spontaneous alteration test(Fig. 1), dose-dependently attenuated the decrease in sponta-neous alternation behavior in the Aβ25–35-injected mice, withthe 1 mg/kg/day dose significantly restoring the spontaneousalteration behavior (Fig. 2A). However, neither imidapril (0.3, 1,or 3 mg/kg/day) nor enalapril (1, 3, or 10 mg/kg/day) had asignificant effect on the behavior in the Aβ25–35-injected mice(Figs. 2C and E). None of the ACE inhibitors affected thenumber of total arm entries (Figs. 2B, D, and F).

2.2. Perindopril, but neither imidapril nor enalapril,eliminated the long-term recognition memory deficits inAlzheimer's disease model mice as assessed using the objectrecognition test

We evaluated the effect of the ACE inhibitors on theimpairment of relatively long-term memory using the objectrecognition test (ORT) 5–7 days after i.c.v. administration ofAβ25–35 (Fig. 1). During the retrieval trial, the sham-operatedmice spent a significantly longer time in exploring the novelobject than the familiar one (Figs. 3A and B: Tnovel=16.2±1.1 s,Tfamiliar=8.7±0.8 s, p<0.001; Figs. 3C and D: Tnovel=14.3±1.3 s,Tfamiliar=7.1±0.6 s, p<0.001, Figs. 3E and F: Tnovel=14.4±0.9 s,Tfamiliar=7.7±0.7 s, p<0.001), while the Aβ25–35-injected micetreated with vehicle spent almost equal amounts of timeexploring either of the two objects (Figs. 3A and B:Tnovel =11.8±0.9 s, Tfamiliar =11.4±0.9 s, p>0.05; Figs. 3C andD: Tnovel =11.5±0.8 s, Tfamiliar =10.6±0.6 s, p>0.05; Figs. 3Eand F: Tnovel =10.8±1.0 s, Tfamiliar =10.4±1.2 s, p>0.05). Thepreference for the novel object, which is represented by therecognition index, in the Aβ25–35-injected mice treated withvehicle was significantly lower than that of the sham-operated mice (Figs. 3A, C, and E). Since the total amount oftime spent in the exploration of objects did not differbetween the sham-operated mice and the Aβ25–35-injected

.d., semel in die (single daily).

Fig. 2 – Effect of perindopril, imidapril or enalapril on the impairment of working memory in spontaneous alteration tasks inAlzheimer's disease model mice. Each column represents the percent spontaneous alternation behavior (A, C, E) and the totalnumber of arm entries (B, D, F) for sham-operated mice (solid bar), or Aβ25–35-injected mice (white bars). Perindopril (A, B),imidapril (C, D) or enalapril (E, F) was administered once per day for 2 days to Aβ25–35-injected mice. The dose of each drug ispresented on the each panel. Behavioral experiments were carried out 1 h after the 2nd treatment with an ACE inhibitor orvehicle. ***p<0.001 compared with the sham-operated mice receiving vehicle (Student's t-test). †††p<0.001 compared with theAβ25–35-injected mice receiving vehicle (Dunnett's test). Error bars indicate the S.E.M. (N=18–20/group).

178 B R A I N R E S E A R C H 1 3 5 2 ( 2 0 1 0 ) 1 7 6 – 1 8 6

mice treated with vehicle (Figs. 3B, D, and F), this indicatesthat while the sham-operated mice had memorized theobjects during the acquisition trial, the Aβ25–35-injectedmice showed signs of cognitive impairment.

Daily treatment with perindopril (0.1, 0.3 or 1 mg/kg/day),which started 4 days before the retrieval trial (Fig. 1), dose-dependently attenuated the decrease in recognition index in theAβ25–35-injected mice. Furthermore, a dose of 1 mg/kg/daysignificantly restored the recognition index (Fig. 3A) withoutaffecting total time spent exploring the objects (Fig. 3B). Howev-er, neither imidapril (0.3, 1, or 3 mg/kg/day) nor enalapril (1, 3, or10 mg/kg/day) restored the recognition index in the Aβ25–35-injectedmice (Figs. 3C andE). However, Donepezil (1 mg/kg/day),

at the same dosage regimen as that described above for the ACEinhibitors, also restored the decrease in the recognition index intheAβ25–35-injectedmice in other experiments (datanot shown).

2.3. Treatment with ACE inhibitors did not affect thespontaneous locomotor activities nor the anxiety relatedbehavior in normal mice

Two-day administration of perindopril (1 mg/kg/day), imidapril(3 mg/kg/day), or enalapril (10 mg/kg/day) did not influence the1 h cumulative locomotor activity in naive mice (Table 1).

In addition, in the elevated plus maze test, the three-dayadministration of ACE inhibitors did not affect the number of

Fig. 3 – Effect of perindopril, imidapril or enalapril on the impairment of recognition reference memory in ORT in Alzheimer'sdisease model mice. Each column represents the recognition index (A, C, E) and the total exploration time (B, D, F) forsham-operated mice (solid bar) or Aβ25–35-injected mice (white bars) in the retrieval trial. Perindopril (A, B), imidapril (C, D) orenalapril (E, F)wasadministereddaily for 5 days inAβ25–35-injectedmicebefore the retrieval trial. Thedoseof eachdrug is presentedon each panel. The habituation session, acquisition trial and retrieval trial were carried out 1 h after the 3rd, the 4th and the 5thtreatment, respectively. ***p<0.001 compared with the sham-operated mice receiving the vehicle (Student's t-test). ††p<0.01compared with the Aβ25–35-injected mice receiving the vehicle (Dunnett's test). Error bars indicate the S.E.M. (N=20/group).

179B R A I N R E S E A R C H 1 3 5 2 ( 2 0 1 0 ) 1 7 6 – 1 8 6

entries into open and closed arms, or the total time spent inopen arms by naive mice (Table 1).

2.4. Effects of ACE inhibitors on the plasma and brainACE activities

The plasma ACE activities were inhibited by the 2-dayadministration of 1 mg/kg/day of perindopril, 3 mg/kg/day ofimidapril, or 10mg/kg/day of enalapril by up to 90% or more at1 h after the 2nd treatment (Fig. 4). The brainACE activitieswere

inhibited by at least 50% by the 1 mg/kg/day dose of perindopril(Fig. 4A). However, the inhibition of the ACE activity in the brainwas less than 50% in animals treated with either the imidapril(3 mg/kg/day) or enalapril (10 mg/kg/day) (Figs. 4B and C).

3. Discussion

In the present study, the effects of sub-chronic treatment witha centrally active ACE inhibitor, perindopril, on the cognitive

Table 1 – Effect of perindopril, imidapril or enalapril onspontaneous locomotor activity and anxiety relatedbehavior in elevated plus maze in normal mice.

Spontaneouslocomotor

activity (1 h)

Elevated plus maze

Entry intoclosedarm

Entry intoopenarm

Time spentin openarm (s)

Vehicle 9766±994 13.0±1.18 2.6±0.4 13.2±3.2Perindopril(1 mg/kg)

8957±697 11.7±0.86 2.7±0.4 12.8±2.9

Imidapril(3 mg/kg)

9249±813 13.3±1.02 2.6±0.5 15.9±3.8

Enalapril(10 mg/kg)

10,508±879 13.8±0.73 2.5±0.4 12.9±2.9

Cumulative value of locomotor activity was counted from 1 to 2 hafter the 2nd treatment of perindopril (1 mg/kg/day, imidapril(3 mg/kg/day, enalapril (10 mg/kg/day) or vehicle in naive mice. Inelevated plus maze test, the number of entry into open arm andclosed arm and time spent in open arm (s) weremeasured for 5 minat 1 h after the 3rd treatment of these drug or vehicle. Each valuerepresents the mean±S.EM (N=12).

180 B R A I N R E S E A R C H 1 3 5 2 ( 2 0 1 0 ) 1 7 6 – 1 8 6

performance were evaluated using two different behavioralparadigms (spontaneous alteration test and ORT) in a mousemodel of Alzheimer's disease. Moreover, the effects ofperindopril on these mice were compared with those of twonon-centrally active ACE inhibitors, imidapril or enalapril. Wealso determined the ACE activity in both the plasma and thebrain following the same dosing regimen in order to elucidatethe relationship between the inhibition of ACE activities in thebrain and the effects on cognitive function. To our knowledge,this is the first study to compare the effect of perindopril onboth the immediateworkingmemory and relatively long-termmemory in rodents with other ACE inhibitors with respect tothe effect of the drug on the brain ACE activity.

During our investigations, we first examined the effects ofperindopril on cognitive function in a well-validated Alzhei-mer's disease model, Aβ25–35-injected mice. The Aβ25–35

peptide is the core toxic fragment of Aβ1–40 and Aβ1–42,which are the major components of senile plaques, and are

Fig. 4 – Effect of perindopril, imidapril or enalapril on plasma andACE in plasma (closed circle) and in brain (opened triangle) weretreated daily with 0.1, 0.3, or 1 mg/kg/day perindopril or vehicle10 mg/kg/day enalapril or vehicle (C) for 2 days. Blood and brain sindicate the S.E.M. (N=7/group).

considered to have a causal role in the development andprogression of Alzheimer's disease (Yankner et al., 1990;Klegeris et al., 1994; Pike et al., 1995). It has been reportedthat i.c.v. administration of Aβ25–35 peptide in mice inducescognitive impairment caused by oxidative damage, inflam-mation, and/or impairment of the cholinergic system (Mauriceet al., 1996; Alkam et al., 2007; Lu et al., 2009a,b), all of whichare observed in patients with Alzheimer's disease. Thecognitive impairments in these model mice have beenevaluated using several popular behavioral experiments,including the fear conditioning test, passive avoidanceresponse, spontaneous alternation test, and ORT (Maurice etal., 1996; Meunier et al., 2006; Alkam et al., 2007; Wang et al.,2007; Lu et al., 2009a,b). Several anti-amnesic drugs, includingdonepezil, reverstigmine, and galantamine, have been dem-onstrated to ameliorate the cognitive impairments in Aβ25–35-injectedmice in a manner similar to human patients (Mauriceet al., 1996; Wang et al., 2007). Therefore, although there aresome well-established genetically engineered mouse modelsof Alzheimer's disease, Aβ25–35-injectedmice are considered tobe a convenient and feasible animal model to evaluate theeffects of compounds for Alzheimer's disease.

In the protocol validation experiments, we confirmed thata deficiency in cognitive performance was sustained duringthe testing period, and that donepezil restored the normalbehavior of these mice, both in the spontaneous alterationtest and in ORT. In the present study, we showed that sub-chronic treatment with perindopril reversed the decrease inthe percent spontaneous alteration behavior, an index ofworking memory, and the decrease in the recognition indexin the ORT, an index of long-term recognitionmemory, in theAβ25–35-injected mice.

The performances in those two behavioral tests partiallydepend on locomotor activities. However, perindopril (1 mg/kg/day) did not affect spontaneous locomotor activities in thenovel environment. The mood of the animals, especially theiranxiety level, might also affect their performances in the tests.However, we determined that perindopril had no effect on thelevel of anxiety, since there were no significant differences inany of the anxiety-related behavioral indices investigated in

brain ACE activities in normal mice. The relative activities ofdetermined by ex vivo enzyme assay. Normal ICR mice were(A), 0.3, 1 or 3 mg/kg/day imidapril or vehicle (B) or 1, 3 oramples were collected 1 h after the last treatment. Error bars

181B R A I N R E S E A R C H 1 3 5 2 ( 2 0 1 0 ) 1 7 6 – 1 8 6

the elevated plus maze test between the perindopril (1 mg/kg/day)-treated group and the vehicle-treated group. Theseresults showed that perindopril could reverse the immediateworking memory and the relatively long-term recognitionmemory deficits in the Alzheimer's disease model mice.

On the other hand, neither imidapril (0.3, 1, or 3 mg/kg/day)nor enalapril (1, 3, or 10 mg/kg/day) had any effect onspontaneous alteration behaviors or the recognition index inthe ORT in Aβ25–35-injected mice. Before the behavioralexperiments, we investigated the equivalent anti-hyperten-sive dose of these ACE inhibitors in spontaneous hypertensiverats using the telemetry system, and demonstrated that asingle treatment with perindopril at a dose of 1 mg/kgsignificantly decreased blood pressure to the same degree as3 mg/kg of imidapril or 10 mg/kg of enalapril, without anyeffect on heart rate (data not shown). Furthermore, in thepresent study, the ACE activity in the plasma was inhibited tothe same extent (>90%), in the mice treated with 1 mg/kg/dayof perindopril, 3 mg/kg of imidapril, or 10 mg/kg of enalapril.Thus, if we presume that more than 90% inhibition of ACEactivity in the plasmawas sufficient to produce adequate anti-hypertensive effects by the ACE inhibitors, it can be concludedthat the dose range of these ACE inhibitors required for theanti-Alzheimer's disease effects is the same as the requiredfor anti-hypertensive effects. We therefore concluded thatperindopril, but neither imidapril nor enalapril could improvethe cognitive impairments in the Alzheimer's disease modelmice in the same dose range as is required to induce anti-hypertensive activity.

In our study, we confirmed that perindopril was a centrallyactive ACE inhibitor, because the brain ACE activities in micewere decreased by more than 50% at an oral dosage of 1 mg/kg/day of perindopril. On the other hand, imidapril andenalapril are regarded as non-centrally active ACE inhibitors,and this was confirmed by the fact that 3 mg/kg/day ofimidapril or 10 mg/kg/day of enalapril, each of which wassufficient for anti-hypertensive activity, showed much smal-ler effects on brain ACE activities than perindopril. These factsmean that only the centrally active ACE inhibitors, such asperindopril, can enhance the cognitive abilities of mice withchemically-inducedAlzheimer's disease, and suggest that thisbeneficial effect depends on the inhibition of ACE activities inthe brain, but not in the plasma and peripheral organs.Moreover, our studies demonstrate that this anti-Alzheimer'sdisease effect occurs at the same dose level as the anti-hypertensive effect of the drug.

This is notable because these results were compatible withthose of clinical studies. For example, Ohrui et al. (2004)showed that treatment with centrally active ACE inhibitors,including perindopril and captopril, but not non-centrallyactive ACE inhibitors, including imidapril and enalapril, couldslow the rate of cognitive decline in mild-to-moderateAlzheimer's disease patients with hypertension, althoughthere were no significant differences in blood pressureamong the three groups. Sink et al. (2009) also reported thatcentrally active ACE inhibitors, which included perindopril,captopril, fosinopril, lisinopril, ramipril, and trandolapril, wereassociated with slower rates of cognitive decline in thepatients with hypertension. Although there was no directcomparison of ACE activity in the brain during their studies,

these results suggest that the beneficial effects of ACEinhibitors on cognitive function depend on the ACE inhibitionin the central nervous systems.

ACE inhibitors have already been shown to have positiveeffects on cognitive performance in normal rats (Jenkins andChai, 2007), streptozotocin-diabetic rats (Manschot et al.,2003), hypertensive aged rats (Wyss et al., 2003) and so on.Therefore, it is not clear whether the improvement in thecognitive function in the Aβ25–35-injectedmice depends on thesame mechanism(s) as the above animals or whether there isa mechanism that is specific for Alzheimer's disease. It willtherefore be necessary to further analyze the mechanism(s)underlying the pro-cognitive effect of perindopril in order toanswer this question.

Several reports have shown the activation of renin–angio-tensin system in the brain of patients with Alzheimer's disease.The ACE activity was increased in the frontal cortex, and theactivity correlated directly with the parenchymal Aβ load inpatients with Alzheimer's disease (Miners et al., 2008, 2009).Increases in the expression of ACE, angiotensin II and the AT1receptor in the parietal cortex were reported using immunohis-tochemical methods (Savaskan et al., 2001), and it has beenshown that the density of the ACE inhibitor recognition site alsoaugmented in the temporal cortex (Barnes et al., 1991). The up-regulation of ACE activity may cause an increase in the amountof angiotensin II, which was reported to inhibit the potassium-induced acetylcholine release in slices of rat entorhinal cortex(Barnes et al., 1989) and of human temporal cortex (Barnes et al.,1990). Furthermore, angiotensin II blocks the induction of long-term potentiation (LTP) in perforant path-stimulated dentategranule cells when injected on the hippocampus in rats (Dennyet al., 1991). In fact, it has been reported that angiotensin IIinfusion into the hippocampus disrupted the consolidation andretrieval of aversive memory in the inhibitory avoidance test(Kerr et al., 2005; Bonini et al., 2006). Based on these previousstudies and our current findings, one possible mechanismexplaining thepro-cognitiveeffectofperindopril onAlzheimer'sdisease is the inhibition of the up-regulation of angiotensin II inthis disease.

Several studies have reported that the amnesic effect ofangiotensin II is blocked by the AT2 antagonist, PD123319, butnot by the AT1 antagonist, losartan, which means that theeffects of angiotensin II are mediated via the AT2 receptor(Kerr et al., 2005; Bonini et al., 2006). Another study reportedthat the suppression of hippocampal LTP by angiotensin II wasreversed by losartan, but not by the AT2 antagonist, PD123319(Wayner et al., 1993). Therefore, the receptor subtypes that areresponsible for the amnesic effect of angiotensin II still remaincontroversial.

AT1 antagonists, which are generally called angiotensinreceptor blockers (ARBs), have been widely prescribed asantihypertensive agents as well as ACE inhibitors. Recently,it was reported that treatments with ARBs were associatedwith a reduction in the incidence and progression ofAlzheimer's disease in human retrospective cohort analysis(Li et al., 2010). Mogi et al. (2008) and Tsukuda et al. (2009)also showed that a non-hypotensive dose of telmisartan, anARB, inhibited the cognitive decline in mice that received aninjection of Aβ1–40 into their cerebral ventricle. However, thepreventive effect of telmisartan on cognitive impairment

182 B R A I N R E S E A R C H 1 3 5 2 ( 2 0 1 0 ) 1 7 6 – 1 8 6

depended on the drug's peroxisome proliferator-activatedreceptor (PPAR)-γagonism. Another ARB, losartan, which hasa less potent PPAR-γ agonistic effect, inhibited Aβ1–40-injection-induced cognitive decline to a lesser than telmi-sartan. Therefore, it is not clear whether ARBs improve thecognitive dysfunction in Alzheimer's disease by the samemechanism as that identified for ACE inhibitors.

Since ACE has been reported to be a promiscuousenzyme, an alternative perspective is that one of the othersubstrates of ACE, apart from angiotensin I, may beassociated with the mechanisms of cognitive improvementinduced by perindopril. It has been reported that substance Pis cleaved and deactivated by ACE in vitro (Skidgel et al.,1984), and that the levels of substance P in several region ofthe central nervous system increased following i.c.v. injec-tion of the ACE inhibitor (Hanson and Lovenberg, 1980), andsubstance P-induced salivation was potentiated by system-ically administered ACE inhibitors (Cascieri et al., 1984).Substance P, after peripheral or central application, hasmemory facilitating effects inducing place preference (Hase-nöhrl et al., 2000; Oitzl et al., 1990) and avoidance learning(Tomaz and Nogueira, 1997). Therefore, an increase insubstance P may be another candidate that could mediatethe pro-cognitive effects of perindopril.

Several reports have indicated that ACE can degrade andeliminate Aβ in vitro (Hu et al., 2001) and in cellular over-expression experiments (Hemming and Selkoe, 2005; Oba etal., 2005). These studies raise the question whether ACEinhibitors, especially those with centrally active properties,can elevate Aβ levels in the brain and exacerbate the disease.However, cerebral Aβ accumulation and plaque depositionwere unaltered in ACE-deficient mice and in normal micetreated with ACE inhibitors (Eckman et al., 2006). In transgenicmice harboring the human amyloid precursor protein (APP)gene, captopril affected neither the cerebral Aβ levels norplaque deposition in two lines of APP transgenic mice(Hemming & Selkoe, 2007). However, captopril promoted thepredominant Aβ deposition in another subset of APP mice(Zou et al., 2007). It thus remains controversial whether Aβ is asubstrate of ACE in model animals (Kehoe et al., 2009).However, it is unlikely that ACE inhibitors are responsible forall of the symptoms of Alzheimer's disease brought on byinhibiting the Aβ-degradation in humans, because severalclinical studies have demonstrated a beneficial effect of ACEinhibitors on the dementia (Ohrui et al., 2004; Rozzini et al.,2006; Yasar et al., 2008; Sink et al., 2009).

Alzheimer's disease is one of the most prevalent neuro-degenerative diseases in aging societies. However, thesatisfaction rate for medical treatment is very low level.This is partly because the efficacy of clinically-approvedanti-dementia drugs, such as cholinesterase inhibitors, isinsufficient, and tolerance to these drugs limits longitudinalpharmacological interventions (Courtney et al., 2004). As aresult, there has been great hope for alternative drugs whichhave other mechanisms of action. However, there have beenonly a few new drugs, other than the cholinesteraseinhibitors, which have shown any effect on this disease.

In this study, we confirmed that perindopril improved thecognitive dysfunction of Aβ-injected mice similar to theresult shown in clinical studies. Although a mouse model of

Alzheimer's disease may not reflect all of the properties ofpatients with that disease, our results suggest that ACEinhibitors have beneficial effects on cognitive function ofAlzheimer's patients, but only if the ACE inhibitors arecentrally active agents such as perindopril. Because peri-ndopril has already achieved satisfactory results as an anti-hypertensive agent in the clinic with regard to its safety andanti-hypertensive efficacy, it may be the best choice for thetreatment of hypertension in patients with Alzheimer'sdisease, because the agent may also prevent the progressionof their Alzheimer's disease.

4. Experimental procedures

4.1. Materials

Perindopril erbumine (Nihon Servier, Tokyo, Japan), imidaprilhydrochloride (LGM Pharmaceutical Inc., FL, USA) andenalapril maleate (Wako Pure Chemical Industries, Kyoto,Japan), were dissolved in distilled water before use. Aβ25–35

(Bachem AG., Bubendorf, Switzerland) was dissolved indistilled water at a concentration of 1 mg/mL and stored at−20 °C.

4.2. Animals

All experiments on animals were approved by the Commit-tee for Animal Experiments of Kyowa Hakko Kirin Co., Ltd.Male ICR mice were purchased from Nihon SLC Co.(Shizuoka, Japan). All mice were aged 5 to 6 weeks at thebeginning of the ACE inhibitor-treatment. The mice werehoused in a controlled environment (19–25 °C, 30–70%humidity) and allowed food and water ad libitum. Roomlights were kept on between 7:00 a.m. and 7:00 p.m. Behav-ioral experiments were carried out in a sound-attenuated,air-regulated, and dim-lighted experimental room, to whichmice were habituated for at least 12 h.

4.3. Intracerebroventricular injection of Aβ25–35

In the present study, the concentration and dose of Aβ25–35

were selected according to previous studies, which showedthat i.c.v. administration at the dose of 3 nmol (approxi-mately 3 μg) leads to brain dysfunction (Maurice et al., 1996).Aβ25–35 (1 mg/mL) was incubated at 37 μ for 4 days beforeinjection in order to promote the formation of aggregates(Gitter et al., 1995; Yu et al., 2005). Aggregated Aβ25–35 (3 μg/mouse) or distilled water (for sham-operated controls) wasinjected into the cerebral ventricle in a volume of 3 μL asdescribed previously (Maurice et al., 1996; Alkam et al., 2007;Lu et al., 2009a,b). Briefly, a micro-syringe with a 27-gaugestainless-steel needle, 2 mm in length, was used for microinjection. The mice were anesthetized with pentobarbitalintraperitoneally (50 mg/kg), and the needle was insertedunilaterally 1 mm to the right of the midline point equidis-tant from each eye, at an equal distance between the eyesand the ears and perpendicular to the plane of the skull(anteroposterior, −0.22 mm from the bregma; lateral, 1 mm

183B R A I N R E S E A R C H 1 3 5 2 ( 2 0 1 0 ) 1 7 6 – 1 8 6

from the bregma). Aβ25–35 was delivered gradually within30 s. The needle was taken out after waiting 30 s. All miceexhibited normal behavior after they recovered from theanesthetic. Neither the insertion of the needle nor injectionof 3 μL of distilled water had a significant influence on thesurvival, behavioral responses, or cognitive functions (datanot shown), as described previously (Maurice et al., 1996;Alkam et al., 2007; Lu et al., 2009a,b). To verify that theinjection was carried out properly, the same volume ofEvans blue was i.c.v. injected into another group of mice. Wethereafter confirmed the distribution of the injected Evansblue throughout both sides of the ventricles.

4.4. Test drug treatment

All mice received once-daily treatment with the ACEinhibitors or vehicle by oral gavage in a volume of 10 mL/kg. For the spontaneous alteration test (N=18–20/group) andORT (N=20/group), the treatment with ACE inhibitors orvehicle started from 3 days after acute i.c.v. injection ofdistilled water or Aβ25–35 peptide into the ICR mice (Day 1).Perindopril (0.1, 0.3, or 1 mg/kg), imidapril (0.3, 1, or 3 mg/kg),enalapril (1, 3, or 10 mg/kg), or vehicle was administered tothe Aβ25–35-injected mice, and vehicle to the sham-operatedmice, on Day 1; 1 h before performing the spontaneousalteration test on Day 2; and 1 h before habituation, theacquisition trial, and the retrieval trial of ORT on Days 3, 4,and 5, respectively (Fig. 1).

For the measurement of spontaneous locomotor activitiesand anxiety-related behavior in the elevated plus maze test(N=12/group), perindopril (1 mg/kg/day), imidapril (3 mg/kg/day), enalapril (10 mg/kg), or vehicle was administered to thenaive ICR mice on Day 1, 1 h before the measurement ofspontaneous locomotor activities on Day 2, and 1 h beforeperforming the elevated plus maze test on Day 3.

To measure the plasma and brain ACE activities (N=7/group), perindopril (0.1, 0.3, or 1 mg/kg), imidapril (0.3, 1, or3 mg/kg), enalapril (1, 3, or 10 mg/kg), or vehicle was admin-istered to naive ICR mice on Day 1, and 1 h before the bloodand brain sampling on Day 2.

4.5. Spontaneous alteration test

The spontaneous alteration test was carried out as describedpreviously (Alkam et al., 2007). Briefly, the experimentalapparatus (Y-maze) consisted of 3 black colored arms, eachof which was 250 mm long, 200 mm high, and 50 mm wide,and positioned at equal angles. The apparatus was locatedin a dimly-lit room and a video camera was mounted on theceiling above the apparatus. Each mouse was placed at theend of an arm and allowed to move freely through the mazefor a 7-min session, and an observer recorded the sequenceof arm entry through the video camera. Spontaneousalteration behavior was defined as the consecutive entry ofa mouse into all three different arms (i.e., arm A, arm B, armC) to form a triplet of non-repeated components. Thepercent spontaneous alteration behavior was calculated asthe ratio of actual to possible alterations (defined as:100%×number of spontaneous alteration behaviors/ (totalnumber of arm entries−2)).

4.6. Object recognition test

The ORT was carried out as described previously (Alkam etal., 2007; Lu et al., 2009a). Briefly, the experimental apparatusconsisted of an open-field box (385 mm wide, 385 mm long,and 400 mm high), all walls of which were covered withbrown paper for screening. The apparatus was placed in adimly-lit room and a video camera was mounted on theceiling above the apparatus. Three kinds of objects wereused in the present experiment: a blue wooden brick in theshape of triangular-column, a yellow wooden brick in theshape of square-pyramid, and a white golf ball with a rubberpedestal. The ORT procedure consisted of three sessions:namely, habituation, an acquisition trial, and a retrieval trial.Each mouse was individually habituated to the box with10 min of exploration time in the absence of objects on Day3 (habituation session). During the acquisition trial on Day 4,two objects (objects A and B) were placed in the back cornerof the box, 10 cm from the side wall. A mouse was thenplaced in the middle front of the box for 10 min and thenreturned to its home cage. During the retrieval trial on Day 5(24 h after the acquisition trial), the mouse was placed backinto the same box, in which one (e.g. object A) of the familiarobjects used during the acquisition trial was kept in thesame position and another (e.g. object B) of the familiarobjects was replaced by a novel object (object C). The animalwas then allowed to explore freely for 5 min. Time spentsniffing the novel object and familiar objects during theretrieval trial were defined as the exploration time for eachobject (Tnovel and Tfamiliar, respectively). Although we did notdetect any significant preference between every pair of twoobjects in the acquisition trial in normal mice, the objectswere used in a counterbalanced manner throughout theexperiments to ensure their neutrality. The total explorationtime (Ttotal) was calculated as the sum of the explorationtime for each object (defined as Tnovel +Tfamiliar). Therecognition index was calculated as the ratio of theexploration time of the novel object to the total explorationtime (defined as Tnovel /Ttotal).

4.7. Spontaneous locomotor activities

The spontaneous locomotor activity was measured in atransparent bucket cage, with a bottom diameter of 240 mmand a height of 250 mm, equipped with a Supermex infraredsensor (Muromachi Kikai Co., Tokyo, Japan), to detectthermal radiation from the animals. The Supermex sensorcan monitor movements in all three planes of motion(sagittal, coronal and horizontal) as one movement, andthis instrument was connected to a behavioral analysissystem, CompACT AMS (Muromachi Kikai). One hour afterthe 2nd treatment with an ACE inhibitor or vehicle, eachmouse was located individually on the experimental bucketcage, and the spontaneous activity was assessed for 1 h,without prior acclimatization to the cage.

4.8. Anxiety-related behavior in the elevated plus maze

The elevated plus maze test was carried out 1 h after the 3rdtreatment with an ACE inhibitors or vehicle. Briefly, the

184 B R A I N R E S E A R C H 1 3 5 2 ( 2 0 1 0 ) 1 7 6 – 1 8 6

elevated plus maze apparatus consisted of two open arms(300 mm long and 40 mm wide) and two enclosed arms(300 mm long, 40 mm wide, and 300 mm high), all of whichextended from a common central platform (40 mm long and40 mm wide). The configuration formed the shape of a plussign, with similar arms arranged opposite to each other, andthe apparatus was elevated 300 mm above the floor. A videocamerawasmounted on the ceiling above the apparatus. Aftermice were placed onto the central platform facing an openarm, a 5-min trial was performed. The behaviors of mice wererecorded through the video camera and the following con-ventional parameters were used: number of open and closedarm entries (arm entry defined as all four paws entering anarm) and time spent on open arms (excluding the centralplatform).

4.9. Ex vivo ACE activity in plasma and brain

One hour after the 2nd treatment with an ACE inhibitor orvehicle, mice were anesthetized with isoflurane using asmall animal anethetizer, MK-A110 (Muromachi Kikai). Bloodwas collected from the abdominal vein using a heparin-coated syringe, and the mouse was then killed by decapita-tion. The plasma was separated from the blood by centrifu-gation at 800×g for 15 min at 4 ºC. The forebrain wasremoved and washed gently in ice-cold saline in order toremove blood and then was frozen in liquid nitrogen. Thefrozen forebrain was homogenized in 9 volumes (w/v) of ice-cold 10 mmol/L Tris–HCl buffer, pH 7.5, containing200 mmol/L sucrose, using a Polytoron homogenizer (Kine-matica, Littau, Switzerland). The homogenate was centri-fuged at 800×g for 30 min at 4 ºC and the supernatant wasused for further experiments. The protein concentrations inplasma and brain homogenate were measured by BCAProtein Assay Kit (PIERCE, Rockford, USA).

The ACE activity was measured using a syntheticsubstrate, hippuryl-His-Leu (HHL, Peptide Institute Inc.,Osaka, Japan). Plasma containing 1 mg protein or brainhomogenate containing 0.3 mg protein was incubated for2 h or 4 h respectively at 37 ºC with 5 mmol/L HHL in 200 μLof 10 mmol/L Tris–HCl buffer, pH 8.3, containing 300 mmol/LNaCl. The reaction was terminated by addition of 200 μL of1 mol/L HCl and then 600 μL of ethyl acetate was added tothe mixture. After vigorous vortexing, the mixture wascentrifuged at 100×g for 5 min at room temperature. Theupper layer was collected and 200 μL was transferred to anew tube and evaporated under N2 gas. Distilled water(200 μL) was added to the tube and then hippuric acid wasdetected by ultraviolet absorbance at 228 nm. The percentinhibition of the ACE activity was determined by comparingthe activity in each sample to that of the vehicle controlgroup.

4.10. Statistical analysis

Statistical analyses were performed using the SAS Release9.1.3 software program (SAS Institute Inc., NC, USA). In thespontaneous alteration test and the ORT, data for theinduction of cognitive impairment between sham-operatedmice and Aβ25–35-injected mice were analyzed using Stu-

dent's t-test. The statistical analysis of the ACE inhibitors orvehicle-treated groups of Aβ25–35-injected mice was per-formed using a 1-way analysis of variances (ANOVA) testfollowed by Dunnett's test. The difference between the timespent in exploring the novel object and that of the familiarobject in the ORT were analyzed by paired t-test. For themeasurement of spontaneous locomotor activities andanxiety-related behavior in the elevated plus maze, afterBartlett's test, a 1-way ANOVA test and Tukey test orKruskal–Wallis and Steel–Dwass test were performed. Differ-ences were considered to be statistically significant atp<0.05.

R E F E R E N C E S

Alkam, T., Nitta, A., Mizoguchi, H., Itoh, A., Nabeshima, T., 2007. Anatural scavenger of peroxynitrites, rosmarinic acid, protectsagainst impairment of memory induced by Aβ25–35. Behav.Brain Res. 180, 139–145.

Barnes, J.M., Barnes, N.M., Costall, B., Horovitz, Z.P., Naylor, R.J.,1989. Angiotensin II inhibits the release of [3H]acetylcholinefrom rat entorhinal cortex in vitro. Brain Res. 491,136–143.

Barnes, J.M., Barnes, N.M., Costall, B., Horovitz, Z.P., Ironside, J.W.,Naylor, R.J., Williams, T.J., 1990. Angiotensin II inhibitsacetylcholine release from human temporal cortex:implications for cognition. Brain Res. 507, 341–343.

Barnes, N.M., Cheng, C.H., Costall, B., Naylor, R.J., Williams, T.J.,Wischik, C.M., 1991. Angiotensin converting enzyme density isincreased in temporal cortex from patients with Alzheimer'sdisease. Eur. J. Pharmacol. 200, 289–292.

Bonini, J.S., Bevilaqua, L.R., Zinn, C.G., Kerr, D.S., Medina, J.H.,Izquierdo, I., Cammarota, M., 2006. Angiotensin II disruptsinhibitory avoidance memory retrieval. Horm. Behav. 50,308–313.

Cascieri, M.A., Bull, H.G., Mumford, R.A., Patchett, A.A.,Thornbeny, N.A., Liang, T., 1984. Carboxyl-terminaltripeptidyl carboxyterminal hydrolysis of substanceP by purified rat lung angiotensin-converting enzymeand the potentiation of substance P activity invivo by captopril and MK-422. Mol. Pharmacol. 25,287–293.

Courtney, C., Farrell, D., Gray, R., Hills, R., Lynch, L., Sellwood, E.,Edwards, S., Hardyman, W., Raftery, J., Crome, P., Lendon, C.,Shaw, H., Bentham, P., 2004. Long-term donepezil treatment in565 patients with Alzheimer's disease (AD2000): randomiseddouble-blind trial. Lancet 363, 2105–2115.

Croog, S.H., Levine, S., Testa, M.A., Brown, B., Bulpitt, C.J., Jenkins,C.D., Klerman, G.L., Williams, G.H., 1986. The effects ofantihypertensive therapy on the quality of life. N Engl J. Med.314, 1657–1664.

Denny, J.B., Polan-Curtain, J., Wayner, M.J., Armstrong, D.L., 1991.Angiotensin II blocks hippocampal long-term potentiation.Brain Res. 567, 321–324.

Eckman, E.A., Adams, S.K., Troendle, F.J., Stodola, B.A., Kahn, M.A.,Fauq, A.H., Xiao, H.D., Bernstein, K.E., Eckman, C.B., 2006.Regulation of steady-state β-amyloid levels in the brain byneprilysin and endothelin-converting enzyme but notangiotensin-converting enzyme. J. Biol. Chem. 281,30471–30478.

Gitter, B.D., Cox, L.M., Rydel, R.E., May, P.C., 1995. Amyloid β peptidepotentiates cytokine secretion by interleukin-1β-activatedhuman astrocytoma cells. Proc. Natl Acad. Sci. USA 92,10738–10741.

185B R A I N R E S E A R C H 1 3 5 2 ( 2 0 1 0 ) 1 7 6 – 1 8 6

Hanson, G.R., Lovenberg, W., 1980. Elevation of substance P-likeimmunoreactivity in rat central nervous system by proteaseinhibitors. J. Neurochem. 35, 1370–1374.

Hasenöhrl, R.U., Souza-Silva, M.A., Nikolaus, S., Tomaz, C.,Brandao, M.L., Schwarting, R.K., Huston, J.P., 2000. Substance Pand its role in neural mechanisms governing learning, anxietyand functional recovery. Neuropeptides 34, 272–280.

Hemming, M.L., Selkoe, D.J., 2005. Amyloid β-protein is degradedby cellular angiotensin-converting enzyme (ACE) and elevatedby an ACE inhibitor. J. Biol. Chem. 280, 37644–37650.

Hu, J., Igarashi, A., Kamata, M., Nakagawa, H., 2001.Angiotensin-converting enzyme degrades Alzheimer amyloidβ-peptide (Aβ ); retards A beta aggregation, deposition, fibrilformation; and inhibits cytotoxicity. J. Biol. Chem. 276,47863–47868.

Jenkins, T.A., Chai, S.Y., 2007. Effect of chronic angiotensinconverting enzyme inhibition on spatial memory andanxiety-like behaviours in rats. Neurobiol. Learn. Mem. 87,218–224.

Kehoe, P.G., Miners, S., Love, S., 2009. Angiotensins in Alzheimer'sdisease — friend or foe? Trends Neurosci. 32, 619–628.

Kerr, D.S., Bevilaqua, L.R., Bonini, J.S., Rossato, J.I., Köhler, C.A.,Medina, J.H., Izquierdo, I., Cammarota, M., 2005. Angiotensin IIblocks memory consolidation through an AT2receptor-dependent mechanism. Psychopharmacology (Berl)179, 529–535.

Klegeris, A., Walker, D.G., McGeer, P.L., 1994. Activation ofmacrophages by Alzheimer β amyloid peptide. Biochem.Biophys. Res. Commun. 199, 984–991.

Li, N.C., Lee, A., Whitmer, R.A., Kivipelto, M., Lawler, E., Kazis, L.E.,Wolozin, B., 2010. Use of angiotensin receptor blockers and riskof dementia in a predominantly male population: prospectivecohort analysis. BMJ. 340, b5465.

Lu, P., Mamiya, T., Lu, L.L., Mouri, A., Zou, L., Nagai, T., Hiramatsu,M., Ikejima, T., Nabeshima, T., 2009a. Silibinin preventsamyloid β peptide-inducedmemory impairment and oxidativestress in mice. Br. J. Pharmacol. 157, 1270–1277.

Lu, P., Mamiya, T., Lu, L.L., Mouri, A., Niwa,M., Hiramatsu,M., Zou, L.B., Nagai, T., Ikejima, T., Nabeshima, T., 2009b. Silibininattenuates amyloid β25–35 peptide-induced memoryimpairments: implication of inducible nitric-oxide synthase andtumornecrosis factor-alpha inmice. J. Pharmacol. Exp. Ther. 331,319–326.

Manschot, S.M., Biessels, G.J., Cameron, N.E., Cotter, M.A., Kamal, A.,Kappelle, L.J., Gispen,W.H., 2003. Angiotensin converting enzymeinhibition partially prevents deficits in water maze performance,hippocampal synaptic plasticity and cerebral blood flow instreptozotocin-diabetic rats. Brain Res. 966, 274–482.

Maurice, T., Lockhart, B.P., Privat, A., 1996. Amnesia induced inmice by centrally administered β-amyloid peptides involvescholinergic dysfunction. Brain Res. 706, 181–193.

Meunier, J., Ieni, J., Maurice, T., 2006. The anti-amnesic andneuroprotective effects of donepezil against amyloid β25–35

peptide-induced toxicity in mice involve an interaction withthe sigma1 receptor. Br. J. Pharmacol. 149, 998–1012.

Miners, J.S., Ashby, E., VanHelmond, Z., Chalmers, K.A., Palmer, L.E.,Love, S., Kehoe, P.G., 2008. Angiotensin-converting enzyme (ACE)levels and activity in Alzheimer's disease, and relationship ofperivascular ACE-1 to cerebral amyloid angiopathyNeuropathol. Appl. Neurobiol. 34, 181–193.

Miners, S., Ashby, E., Baig, S., Harrison, R., Tayler, H., Speedy, E.,Prince, J.A., Love, S., Kehoe, P.G., 2009. Angiotensin-convertingenzyme levels and activity in Alzheimer's disease: differencesin brain and CSF ACE and associationwith ACE1 genotypes. AmJ Transl Res. 1, 163–177.

Mogi, M., Li, J.M., Tsukuda, K., Iwanami, J., Min, L.J., Sakata, A.,Fujita, T., Iwai, M., 2008. Telmisartan prevented cognitivedecline partly due to PPAR-γ activation. Biochem. Biophys. Res.Commun. 375, 446–449.

Oba, R., Igarashi, A., Kamata, M., Nagata, K., Takano, S.,Nakagawa, H., 2005. The N-terminal active centre of humanangiotensin-converting enzyme degrades Alzheimer amyloidβ-peptide. Eur. J. Neurosci. 21, 733–740.

Ohrui, T., Tomita, N., Sato-Nakagawa, T., Matsui, T., Maruyama,M., Niwa, K., Arai, H., Sasaki, H., 2004. Effects of centrally activeACE inhibitors on Alzheimer disease progression. Neurology63, 1324–1325.

Oitzl, M.S., Hasenöhrl, R.U., Huston, J.P., 1990. Reinforcing effectsof peripherally administered substance P and its C-terminalsequence pGlu6-SP6-11 in the rat. Psychopharmacology (Berl)100, 308–315.

Pike, C.J., Walencewicz-Wasserman, A.J., Kosmoski, J., Cribbs, D.H.,Glabe, C.G., Cotman, C.W., 1995. Structure-activity analyses ofβ-amyloid peptides: contributions of the β25–35 region toaggregation and neurotoxicity. J. Neurochem. 64,253–265.

Rozzini, L., Chilovi, B.V., Bertoletti, E., Conti, M., Del Rio, I.,Trabucchi, M., Padovani, A., 2006. Angiotensin convertingenzyme (ACE) inhibitors modulate the rate of progression ofamnestic mild cognitive impairment. Int. J. Geriatr. Psychiatry21, 550–555.

Savaskan, E., Hock, C., Olivieri, G., Bruttel, S., Rosenberg, C., Hulette,C., Müller-Spahn, F., 2001. Cortical alterations of angiotensinconverting enzyme, angiotensin II and AT1 receptor inAlzheimer's dementia. Neurobiol. Aging 22, 541–546.

Sink, K.M., Leng, X., Williamson, J., Kritchevsky, S.B., Yaffe, K.,Kuller, L., Yasar, S., Atkinson, H., Robbins, M., Psaty, B., Goff Jr.,D.C., 2009. Angiotensin-converting enzyme inhibitors andcognitive decline in older adults with hypertension: resultsfrom the cardiovascular health study. Arch. Intern. Med. 169,1195–1202.

Skidgel, R.A., Engelbrecht, S., Johnson, A., Erdos, E.G., 1984.Hydrolysis of substance P and neurotensin by convertingenzyme and neutral endopeptidase. Peptides 5, 769–776.

Tomaz, C., Nogueira, P.J., 1997. Facilitation ofmemory by peripheraladministration of substance P. Behav. Brain Res. 83, 143–145.

Tsukuda, K., Mogi, M., Iwanami, J., Min, L.J., Sakata, A., Jing, F., Iwai,M., Horiuchi, M., 2009. Cognitive deficit in amyloid b-injectedmice was improved by pretreatment with a low dose oftelmisartan partly because of peroxisome proliferator-activatedreceptor-γ activation. Hypertension 54, 782–787.

Tzourio, C., Anderson, C., Chapman, N., Woodward, M., Neal, B.,MacMahon, S., Chalmers, J., 2003. PROGRESS CollaborativeGroup. Effects of blood pressure lowering with perindopril andindapamide therapy on dementia and cognitive decline inpatients with cerebrovascular disease. Arch. Intern. Med. 163,1069–1075.

Wang, D., Noda, Y., Zhou, Y., Mouri, A., Mizoguchi, H., Nitta, A.,Chen, W., Nabeshima, T., 2007. The allosteric potentiation ofnicotinic acetylcholine receptors by galantamine amelioratesthe cognitive dysfunction in β amyloid25-35 i.c.v.-injected mice:involvement of dopaminergic systemsNeuropsychopharmacology 32, 1261–1271.

Wayner, M.J., Armstrong, D.L., Polan-Curtain, J.L., Denny, J.B., 1993.Role of angiotensin II and AT1 receptors in hippocampal LTP.Pharmacol. Biochem. Behav. 45, 455–464.

Wyss, J.M., Kadish, I., van Groen, T., 2003. Age-related decline inspatial learning and memory: attenuation by captopril. Clin.Exp. Hypertens. 25, 455–474.

Yankner, B.A., Duffy, L.K., Kirschner, D.A., 1990.Neurotrophic and neurotoxic effects of amyloid β protein:reversal by tachykinin neuropeptides. Science 250,279–282.

Yasar, S., Zhou, J., Varadhan, R., Carlson, M.C., 2008. The use ofangiotensin-converting enzyme inhibitors and diuretics isassociated with a reduced incidence of impairment oncognition in elderly women. Clin. Pharmacol. Ther. 84,119–126.

186 B R A I N R E S E A R C H 1 3 5 2 ( 2 0 1 0 ) 1 7 6 – 1 8 6

Yu, M.S., Leung, S.K., Lai, S.W., Che, C.M., Zee, S.Y., So, K.F.,Yuen, W.H., Chang, R.C., 2005. Neuroprotective effectsof anti-aging oriental medicine Lycium barbarum againstβ-amyloid peptide neurotoxicity. Exp. Gerontol. 40,716–727.

Zou, K., Yamaguchi, H., Akatsu, H., Sakamoto, T., Ko, M., Mizoguchi,K., Gong, J.S., Yu, W., Yamamoto, T., Kosaka, K., Yanagisawa, K.,Michikawa, M., 2007. Angiotensin-converting enzyme convertsamyloid β-protein 1–42 (Aβ1–42) to Aβ1–40, and its inhibitionenhances brain Aβ deposition. J. Neurosci. 27, 8628–8635.