Journal of Alzheimer’s Disease 41 (2014) 1193–1205DOI 10.3233/JAD-140066IOS Press

1193

Tubastatin A/ACY-1215 Improves Cognitionin Alzheimer’s Disease Transgenic Mice

Ling Zhang, Cui Liu, Jie Wu, Jing-jing Tao, Xiao-long Sui, Zhi-gang Yao, Yan-feng Xu, Lan Huang,Hua Zhu, Shu-li Sheng and Chuan Qin∗Comparative Medical Center, Peking Union Medical College (PUMC) & Institute of Laboratory Animal Science,Chinese Academy of Medical Science (CAMS), Beijing, China

Accepted 13 March 2014

Abstract. Histone deacetylase 6 (HDAC6) is currently being discussed as a promising therapeutic target for the treatment ofAlzheimer’s disease (AD). Mounting evidence indicates that increased HDAC6 expression may contribute to AD-associatedneurodegeneration, although beneficial effects have also been identified. In the present study, we tested the potential of twoselective HDAC6 inhibitors, tubastatin A and ACY-1215, to rescue cognitive deficits in a mouse model of AD. We found that bothtubastatin A and ACY-1215 alleviated behavioral deficits, altered amyloid-� (A�) load, and reduced tau hyperphosphorylationin AD mice without obvious adverse effects. Our data suggested that tubastatin A and ACY-1215 not only promoted tubulinacetylation, but also reduced production and facilitated autophagic clearance of A� and hyperphosphorylated tau. Further, thedecreased hyperphosphorylated tau and increased tubulin acetylation may account for the improved microtubule stability in ADmice after tubastatin A/ACY-1215 treatment. These preclinical results support the detrimental role of HDAC6 in AD, and offerprospective approaches for using tubastatin A/ACY-1215 as potential therapeutic strategy for AD.

Keywords: Alzheimer’s disease, amyloid, autophagy, histone deacetylase (HDAC), tau

INTRODUCTION

Alzheimer’s disease (AD), a devastating neu-rodegenerative disorder associated with progressivecognitive decline, is characterized by extracellulardeposit of amyloid-� (A�) peptides in senile plaquesand accumulation of hyperphosphorylated tau (P-tau) proteins in neurofibrillary tangles. Despite muchprogress, the underlying pathogenesis of AD remainsto be elucidated and effective treatments are not cur-rently available. Over the past decade, lots of studiesin vitro and vivo have identified many compounds oftherapeutic interest [1, 2]. Among these compounds,

∗Correspondence to: Dr. Chuan Qin, Comparative Medical Cen-ter, Peking Union Medical College (PUMC) & Institute ofLaboratory Animal Science, Chinese Academy of Medical Science(CAMS), Panjiayuan Nanli NO. 5, Beijing 100021, China. Tel./Fax:+86 10 8777 8141; E-mail: qinchuan@pumc.edu.cn.

histone deacetylase (HDAC) inhibitors such as tricho-statin A and suberoylanilide hydroxamic acid (SAHA)have shown neuroprotective effects [3–5]. However,these inhibitors are not specific for a certain HDAC.They target not only class I HDACs, but also classII, such as HDAC6. Unlike other HDACs, HDAC6 isa unique cytoplasmic HDAC that deacetylates manynon-histone proteins (e.g., �-tubulin, HSP90, cor-tactin) with two catalytical domains and a ubiquitininteracting domain [6, 7].

HDAC6 levels in brain regions related to learningand memory, such as hippocampus and cortex increasesignificantly in AD patients and models. Mountingevidence supported that HDAC6 overexpression maycontribute to the neurodegeneration in AD, althoughbeneficial effects have also been identified [8–10]. Arecent study demonstrated that reducing endogenousHDAC6 restores learning and memory abilities in a

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mouse model for AD [11]. Based on previous data, wereviewed the possible roles of HDAC6 in AD [7] andhere we investigated the potential effects of tubastatinA and ACY-1215, two selective HDAC6 inhibitors, onbehavioral deficits in AD transgenic mice [12, 13]. Wefound that both tubastatin A and ACY-1215 improvedcognitive function in A�PPswe/PS1�E9 (PAP) mice.The possible mechanisms included increased micro-tubule stability, enhanced autophagy and reducedamyloid and tau pathology. Our results suggest thattubastatin A and ACY-1215 may be potential drugs totreat AD.

MATERIALS AND METHODS

Animals

The A�PPswe/PS1�E9 (PAP) double-transgenicmice (C57BL/6J) were generated as previouslydescribed [14]. The use of animals was in compliancewith the Health Guide for care and use of Labora-tory Animals. The study protocol was approved by theAnimal Care and Use Committee of the Institute ofLaboratory Animal Science of Peking Union MedicalCollege (ILAS-PL-2012-003).

Groups and treatments

Six-month-old male and female mice were dividedinto six groups: the wild-type control (WT, n = 16),model control (PAP), vehicle (DMSO/saline = 1:10),SAHA, tubastatin A, and ACY-1215 groups. ForSAHA (Selleck, S1047), tubastatin A (Selleck,S8049), and ACY-1215 (MedKoo, 205808) treatments,we dissolved the compounds in DMSO (Sigma-Aldrich) and further diluted them in saline to the finaldoses. We treated transgenic mice for 20 consecutivedays by daily intraperitoneal injection of 50 mg/kgSAHA, 25 mg/kg tubastatin A, 25 mg/kg ACY-1215,or an equivalent amount of vehicle. The doses of thecompounds were selected based on other experimentalstudies [4, 13, 15].

Behavioral analysis

Behavioral tests were performed as described pre-viously with certain modifications [11, 14, 16]. Theanxiety of the mice was analyzed by the open fieldtest. This test utilized Noldus Ethovision XT monitor-ing and analysis software (Noldus Ltd, Holland). Eachmouse had 5 min of free movement in the open field

(50 × 50 cm), which was divided into the peripheralzone and the central zone. The time that each mousestayed in each zone, as well as the frequency withwhich each mouse was immobile, mobile, or highlymobile, were recorded. Highly mobile and immobilestates were defined as mobility rates >60% and <20%,respectively. Mice with an intermediate mobility ratewere between the two levels (>20%–<60%).

The reference memory of the mice was assessedusing the novel object recognition (NOR) task and theMorris water maze (MWM) test.

The NOR task consists of a habituation phase, atraining phase, and a test phase. Mice were firstly habit-uated to the test box measuring 45 cm × 30 cm × 20 cmfor 5 min daily on 2 consecutive days without anyobject. After habituation phase, two identical objectswere introduced. And mice were allowed to explorefreely for 5 min daily on 3 consecutive days. In the testphase, each mouse was placed in the arena with a famil-iar object they explored during the training phase anda novel object of different shape and color. The explo-ration time for the familiar (TF) or the novel object(TN) during the test phase was videotaped and analyzedusing the Noldus Ethovision XT software (Noldus Ltd,Holland). The discrimination index (DI = (TN-TF) /(TN+TF) × 100%) was used to measure memory pref-erence.

The MWM test was performed in a circular tank(100 cm in diameter) filled with water (22 ± 1◦C) thatwas made turbid with powdered milk. An escape plat-form (10 cm in diameter) was submerged 0.5 cm belowthe water surface in the center of one of the four quad-rants of the maze. Each mouse was subjected to twotrials each 1 min long for five consecutive days. Thetime taken to reach the platform (escape latency) wasrecorded and the average of two trials was determined.In the probe trial, mice swam freely in the tank with-out platform for 1 min, and the time and frequencyspent in the quadrant of the original platform and otherquadrants was recorded. Each mouse was kept dry ina plastic holding cage on an electric heater after theswim. The room temperature was constant and the lightlevels were even. Monitoring was performed with theNoldus Ethovision XT system.

Histochemical staining

Brain tissue from the mouse (n = 5) was fixed in 10%neutral buffered formalin and embedded in paraffin.After dewaxing, the cortical sections (8 �m thick-ness) were washed in PBS. For thioflavin-S staining,sections were incubated in 0.25% potassium perman-

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ganate and 1% oxalic acid until they appeared white.Then the sections immersed in water and stained for5 min with 1% thioflavin-S solution in 50% ethanol.Finally, the sections were washed and dehydrated fol-lowed by mounting with coverslips and analyzed byusing ImageJ software (1.43u, NIH, USA).

The immunohistochemistry procedures were car-ried out as the conventional methods. Sections wereprobed with primary antibody: ace-�-tubulin (1:1000,Sigma)/6E10 (1:1000, Convance) overnight at 4◦C.Then, the immunoreaction was visualized after incu-bating with the secondary antibody (HRP-labeledanti-rabbit/mouse IgG) and DAB (ZSGB-BIO, Bei-jing, China). Positive sections were scanned withAperio Scanscope XT (Aperio Technologies, Vista,CA) and analyzed using Image Pro-Plus 6.0 software(Media Cybernetics Inc., USA).

ELISA test for Aβ1-40 and Aβ1-42

Soluble and insoluble A�1-40 and A�1-42 wasextracted according to previous studies [14, 17]. Inbrief, the frozen mouse brain was weighed and homog-enized with ice-cold TBS (20 mM Tris-HCl, 150 mMNaCl, pH 7.4) to the frozen tissue at 4:1 (TBSvolume/brain wet weight). The homogenate was cen-trifuged at 4◦C for 30 min at 20,000 g. The supernatantcontaining soluble A� peptide was aliquoted and thenstored at −80◦C. The precipitate containing insolubleA� was re-homogenized in an equal volume of TBSplus 5 M guanidine HCl (pH8.0) and incubated for 4 hat room temperature. The supernatant was regarded asthe insoluble A� fraction. A�1-40 and A�1-42 levelswere quantified by ELISA according to the manufac-turer’s protocol (Invitrogen, KHB3482/3442).

Immunoblotting

Brain tissue was homogenized in RIPA buffercontaining 0.1% PMSF and 0.1% protease inhibitorcocktail (Pierce, 78428). The lysates were centrifugedat 14,000 rpm for 30 min at 4◦C. The protein concen-tration in supernatant was measured by BCA assay kit(Pierce, Rockford, IL, USA, 23225). The procedureswere performed as the conventional methods [11].Quantitative analysis was performed by densitometryusing NIH ImageJ software. Immunoblotting wascarried out with antibodies specific for ace-�-tubulin(K40), �-tubulin (1:1000, Sigma); HDAC6, SIRT1(1:500, Millipore); A�PP, sA�PP� (6E10), A�PPC-terminal (1:1000, Convance); sA�PP�, BACE1,ADAM10, PS1, LAMP2, Beclin1, HDAC1/2,

H3K9/H3, H4K8/H4 (1:500, Abcam); AT8, AT100,AT180, AT270, PHF1 (1:300, Pierce), Tau5 (1:500,Calbiochem); LC3I/3II, P-mTOR (Ser2448), mTOR,CDK5, p-GSK3� (Ser9), GSK3�, P-Akt (Ser473), Akt(1:1000, Cell Signaling). Variations in sample loadingwere corrected by normalizing to GAPDH levels.

Electron microscopy

Ultrastructural analysis was performed as previ-ous studies [16]. Briefly, brains were removed aftercardiac perfusions with 0.01 M PBS (pH 7.4) con-taining 2% paraformaldehyde. The frontal cortex andhippocampal CA1 were carefully dissected and fixedin 2.5% glutaraldehyde solution at 4◦C for 2 h. Thesamples were treated with 1% osmium tetroxide in0.1 M cacodylate buffer for an additional 1 h, fol-lowed by 1% uranyl acetate and dehydrated in ethanol.Samples were embedded in epoxy resin, sectioned(90 nm) and placed on carbon-coated copper grids.After uranyl acetate and lead citrate staining, thespecimens were observed with transmission electronmicroscopy (JEOL JEM-1400).

Assessment of toxicity of HDAC6 inhibitors

Blood was obtained from four mice in each group viacardiopuncture at the time of sacrifice. Then serum wascollected and immediately subjected to biochemicalexamination (Supplementary Table 2). Tissues of liver,kidney, and brain were collected after sacrifice. 5 �mthick sections were cut and stained with hematoxylin-eosin (H&E) for histopathological evaluation.

Statistical analysis

All data were shown as means ± SEM. The escapelatencies in the MWM test were analyzed usingtwo-way ANOVA with repeated measures. Other dif-ferences between groups were examined by one wayANOVA followed by Dunnett’s post hoc multiple com-parison tests using SPSS 19.0. In all tests, p < 0.05 wasconsidered as statistically significant.

RESULTS

Tubastatin A and ACY-1215 ameliorate cognitivedeficits in AβPPswe/PS1∆ E9 mice withoutsignificant effects on anxiety levels

The open field test was used to examine anxietylevels in the mice. The A�PPswe/PS1�E9 (PAP) and

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Fig. 1. Tubastatin A and ACY-1215 rescue memory function in A�PPswe/PS1�E9 (PAP) mice without significant effects on anxiety levels. (a)The frequency of mice exhibiting immobile, mobile, and highly mobile states; (b) The time spent in the center and periphery of the open field(n = 16) *p < 0.05, **p < 0.01, versus the vehicle-treated group; (c) Escape latency in 5 days during the training phase of the Morris water maze.(d) Representative images showing behavioral traces during the training phase (5th day) and the probe test (6th day). (e) Time spent in the targetquadrant during the probe test (QT, target quadrant). Data represent means ± SEM (n = 16). *p < 0.05, **p < 0.01, versus the vehicle-treatedgroup; #p < 0.05, ##p < 0.01, versus other quadrants. (f) Effects of tubastatin A and ACY-1215 on performance of the novel object recognitiontask. Data represent means ± SEM (n = 16). *p < 0.05, **p < 0.01, versus the vehicle-treated group.

vehicle-treated groups showed an increased frequencyof immobile, mobile and highly mobile states com-pared with WT littermates (p < 0.01 respectively)(Fig. 1a). These two groups spent more time in theperipheral zone and less time in the central zone(Fig. 1b), which indicated that the PAP mice were morehyperactive and anxious. Anxiety levels of the PAPmice were not significantly altered by the inhibitors(Fig. 1a, b).

To examine whether HDAC6 inhibitors affect cog-nitive function in PAP mice, we tested spatial learningand memory by MWM. During the whole test, thevehicle-treated group showed similar performance to

the PAP group. In the hidden platform test (Fig. 1c,d), the vehicle-treated mice showed significantlyincreased escape latency from day 2 compared to theWT littermates (p < 0.05 for day 2; p < 0.01 for day3–5). SAHA and ACY-1215 groups showed dramati-cally decreased escape latencies from day 3 (p < 0.01for day 3–5, respectively), and tubastatin A group atday 5 (p < 0.01) compared with the vehicle group.Interestingly, mice of ACY-1215 group showed shorterescape latency than those of SAHA (p < 0.01 for day 4,p < 0.05 for day 5), and tubastatin A groups (p < 0.01for day 4-5 respectively). These results revealed thatthe impaired acquisition of the spatial learning task

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Fig. 2. Tubastatin A and ACY-1215 increase the levels of tubulin acetylation and show no effects on the expression of other HDACs. (a)Representative images showing increased immonoreactivity of ace-�-tubulin (K40) in the hippocampus (left) and prefrontal cortex (right) afterTubastatin A or ACY-1215 treatment along with densitometric quantification (upper right). Scale bar: 50 �m. (b) Immunoblot analysis showingthat HDAC6 inhibitors elevated the level of �-tubulin acetylation without altered HDAC6 expression. (c) Immunoblot showing protein levels ofother HDACs by densitometry. Data represent means ± SEM (n = 5). *p < 0.05, **p < 0.01, versus the vehicle-treated group.

in PAP mice was rescued by tubastatin A and ACY-1215. ACY-1215-treated mice exhibited a better spatiallearning performance than SAHA and tubastatin A.

In the subsequent probe test, preference for the tar-get quadrant was significantly impaired in the PAPand vehicle controls compared with the WT littermates(Fig. 1e). However, this impairment can be correctedby tubastatin A or ACY-1215 treatment (Fig. 1e). Thisimplied that tubastatin A and ACY-1215 reversed thespatial memory deficit in AD mice.

In addition, we tested recognition memory by theNOR task. A one-way ANOVA analysis revealed thatthe discrimination index (DI), reduced in the modelcontrols (PAP) and vehicle-treated mice, was signif-icantly improved in HDACi-treated groups (Fig. 1f).As shown in Fig. 1f, DI was increased by nearly

88%, 75%, and 70% in SAHA, tubastatin A, andACY-1215 groups, respectively, compared with thevehicle-treated group. Collectively, these data suggestthat tubastatin A/ACY-1215 as well as SAHA canrestore cognition in AD mice.

Tubastatin A and ACY-1215 increase α-tubulinacetylation with relative specificity for HDAC6

After sacrificing the mice, we first tested the speci-ficity of tubastatin A and ACY-1215. In line withprevious studies, we observed a dramatic increase inthe level of HDAC6 in the PAP and vehicle-treatedmice compared with the WT littermates (Fig. 2b).But neither tubastatin A nor ACY-1215 has a signifi-cant influence on the expression of HDAC6 compared

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Fig. 3. Tubastatin A and ACY-1215 lighten plaque pathology in A�PPswe/PS1�E9 (PAP) mice. (a) Representative images showing A� plaques(arrow) in the hippocampus and cortex of mice by Thioflavin-S staining (upper) and 6E10 staining (lower). Scale bar: 500 �m. (b) Graphsshowing the average number of plaque, the percentage of area occupied by plaques and the mean optical density of Thioflavin-S (ThS) and6E10 staining in the cortex and hippocampus. Data represent means ± SEM (n = 5). *p < 0.05, **p < 0.01, versus the vehicle-treated group.(c) Representative electron microscopy of senile plaques surrounded by dystrophic neuritis, autophagic vacuoles (AVs) and microglial cells(asterisk) in prefrontal cortex of the three groups (vehicle, SAHA, and ACY-1215). Flame-like plaque core, present in the vehicle-treated group(1, 4), was absent in HDAC6i-treated groups (2, 3, 5, 6). The number of AVs was reduced (7–10) and axon degeneration (2, 3) was improved inHDACi-treated groups (n = 3). Scale bar: 5 �m (1, 2, 3); 1 �m (4–9); 500 nm (10).

with the PAP and vehicle-treated mice (Fig. 2b). Since�-tubulin is one of the most important substrates ofHDAC6, we examined the level of acetylated �-tubulin(K40), which is a marker of HDAC6 activity [15,18]. As expected, we found an apparent reduction inthe level of acetylated �-tubulin in the hippocampusand prefrontal cortex of the PAP and vehicle-treatedmice compared with the WT littermates, which wasreversed by tubastatin A and ACY-1215 (Fig. 2a,b). Thus, both tubastatin A and ACY-1215 inhib-ited the activity of HDAC6 rather than its expressionlevel.

Besides, SAHA treatment resulted in a marked ele-vation of histone acetylation through inhibiting class IHDACs (Fig. 2c), while neither tubastatin A nor ACY-1215 brought such result (Fig. 2c) [4].

Tubastatin A and ACY-1215 reduce Aβ depositionin AβPPswe/PS1∆E9 mice

To determine the mechanisms underlying the effectsof tubastatin A and ACY-1215 on cognitive function,we analyzed A� plaque load using thioflavin-S and6E10 staining (Fig. 3a). As shown in Fig. 3a, the WT lit-termates presented no visible plaque. However, plentyof plaques accumulated in the hippocampus and cor-tex of the PAP and vehicle-treated mice, whereas micetreated with the HDAC6 inhibitors displayed reducedplaque deposition compared with the PAP and vehicle-treated controls (Fig. 3a, b). Furthermore, we measuredthe levels of soluble and insoluble A�1-40 and A�1-42in mice brains by ELISA. Our results revealed a dra-matic decrease in levels of soluble A�1-40 and A�1-42

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Fig. 4. Tubastatin A and ACY-1215 inhibit A� production and upregulate autophagy-related proteins in the brain tissues of A�PPswe/PS1�E9

(PAP) mice. (a) The levels of soluble and insoluble A�1-40 and A�1-42 (n = 3). (b) Representative immunoblot (upper) and quantitative analysisshowing decreased �- and �-secretase cleavage of A�PP after Tubastatin A or ACY-1215 treatment (n = 5). (c) Representative immunoblot(upper) and corresponding analysis (lower) showing up-regulated levels of autophagy-related proteins (e.g., LC3 II, beclin1, and LAMP2). Datarepresent means ± SEM (n = 5). *p < 0.05, **p < 0.01, versus the vehicle-treated group.

in HDAC6i-treated groups compared with the vehiclecontrols (Fig. 4a).

Tubastatin A and ACY-1215 reduce Aβ depositionby inhibiting β/γ-secretase cleavage of AβPP andpromoting autophagy

We next examined the effects of tubastatin Aand ACY-1215 on A� production and elimination,including degradation process of A�PP (Fig. 4b)and levels of several autophagy-related proteins(Fig. 4c) by western blot analysis. Come to light,A�1-40/1-42 was derived from sequential cleavages ofA�PP by �- and �-secretase and produced neurotoxiceffect, while its cleavage by �-secretase (ADAM10)precludes neurotoxic A� peptide production andgenerates neurotrophic sA�PP� and �-CTF (C83)

[19]. Immunoblot analysis manifested that the levelsof sA�PP�, BACE1 (�-secretase), �-CTF (C99),and PS1 (the catalytic core of �-secretase) signifi-cantly decreased, whereas there were no changes in�-cleavage-related proteins in HDAC6i-treated micecompared with the vehicle controls (Fig. 4b).

Moreover, we tested the levels of several autophagy-related proteins, including LC3II/I, Beclin1, andLAMP2 (lysosomal-associated membrane protein 2)[20–23]. We found that treatment with tubastatin A orACY-1215 facilitated autophagy (referred as macroau-tophagy) by upregulating autophagy-related proteinsand inhibiting the phosphorylation of mTOR, which isa negative regulator of autophagy (Fig. 4c) [24–26].

Interestingly, using transmission electronmicroscopy, we observed atypical amyloid plaquestructures in all HDAC6i-treated groups (including

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Fig. 5. Tubastatin A and ACY-1215 attenuate tau hyperphosphorylation and restore microtubule stability. (a) Representative immunoblot (left)and quantitative analysis (right) showing reduced levels of tau hyperphosphorylation. (b) Representative immunoblot (left) and quantification(right) showing decreased level of phosphokinase CDK5 and suppressive activity of Akt/GSK3�. Data represent means ± SEM (n = 5). *p<0.05,**p<0.01, versus the vehicle-treated group. (c) Representative electron microscopy of hippocampus showing that microtubule depolymerization(arrow) and myelin damage (arrowhead) were alleviated in inhibitors-treated groups. Scale bar: 1 �m.

SAHA, tubastatin A, and ACY-1215 groups). Asshown in Fig. 3c, the plaque core seemed to bedissolved, and the dystrophic neuritis surroundedthe plaque showed a sign of revival in brains ofSAHA/ACY-1215-treated mice. Meanwhile, thenumbers of autophagic vesicles (AVs) declined sig-nificantly in all HDAC6i-treated mice compared withthe vehicle-treated controls (Fig. 3c, SupplementaryTable 1). All these data suggested that tubastatin Aand ACY-1215 may reduce A� deposition not only byinhibiting �/�-secretase cleavage of A�PP but also bypromoting autophagy.

Tubastatin A and ACY-1215 attenuate tauhyperphosphorylation via inhibiting Akt/GSK3β

signaling pathway and consequently restoremicrotubule stability

Since HDAC6 was demonstrated to be a tau-interacting protein and a modulator of tau phospho-

rylation and accumulation, we tested the level of tauhyperphosphorylation by western blot analysis [7–9,27–29]. Our results represented that tubastatin A orACY-1215 treatment reduced tau hyperphosphoryla-tion (Fig. 5a). Moreover, we examined the levels ofprotein kinases that phosphorylate tau, such as CDK5and GSK3� [30]. We found that the level of CDK5decreased in both tubastatin A and ACY-1215 groupscompared with the vehicle group. Meanwhile, all theHDAC6 inhibitors significantly increased the phospho-rylation of GSK3� at the Ser9 site, an inhibitory site ofGSK3� activity. That is, GSK3� activity was inhibitedby the HDAC6 inhibitors.

As an HDAC6-interating protein, GSK3� is also adownstream target of Akt involved in several signaltransduction pathways [31]. And then, we evaluatedthe effect of tubastatin A and ACY-1215 on the phos-phorylation of Akt (Ser473). The result showed thatphosphorylation of Akt increased in tubastatin A andACY-1215 groups as expected (Fig. 5b). Moreover,

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the ultrastructual analysis displayed that the damageof microtubule in AD transgenic mice was rescuedby tubastatin A or ACY-1215 treatment (Fig. 5c).According to the results above, the improvement ofmicrotubule stability may achieve through increasingtubulin acetylation and decreasing tau hyperphos-phorylation via inhibiting Akt/GSK3� pathway byHDAC6 inhibition.

HDAC6 inhibitors show no significant adverseeffects on mice

After administration of the HDAC6 inhibitors, a bat-tery of experiments designed to detect possible sideeffects on animals. However, there was no detectablechange in gross brain morphology and brain/bodymass (Supplementary Figure 1a). Further, liver enzymemeasurements and kidney function tests were per-formed (Supplementary Table 2). The results revealedno significant effects on liver and kidney function inmice treated with tubastatin A or ACY-1215, whilethere were dramatic increases in levels of lactic dehy-drogenase and urea nitrogen in SAHA-treated mice(Supplementary Table 2). Microscopic examinationsof different tissues, including brain, liver, and kid-ney, showed no obvious severe abnormity in allgroups except SAHA-treated group (SupplementaryFigure 1b). As Fig. 6b shows, the hepatocytes exhib-ited hyaline degeneration and inflammatory reaction inSAHA-treated mice.

DISCUSSION

Since HDAC6 overexpression was demonstrated toplay an important role in the pathogenesis of AD, it ismeaningful to study the potential of selective HDAC6inhibitors to treat AD [7–11].

Currently, many HDAC6 inhibitors have been iden-tified. Most of the known inhibitors are pan-HDACinhibitors. For instance, SAHA, approved for the treat-ment of cutaneous T-cell lymphoma, was previouslydemonstrated to reverse contextual memory deficientin A�PPswe/PS1�E9 mice [3, 4]. Thus, we used it as thepositive control. To date, a limited number of selectiveHDAC6 inhibitors have been identified [3, 32]. Amongthese selective inhibitors, tubacin was first identifiedand widely used in vitro. However, as a consequenceof its non-drug-like properties, high lipophilicity, andtedious synthesis, it is more useful as a research toolthan as a drug [32–34]. In the present study, we choosetwo selective inhibitors to treat AD mice. One is tubas-tatin A, demonstrated to show neuroprotection in vitro

and vivo with high selectivity, activity, and drug-likestructure [12, 15, 35]. The other is ACY-1215, a novelcompound under phase II clinical trial for multiplemyeloma. ACY-1215 is considered to be a promisingpotential drug due to many properties, including highselectivity, suitable oral bioavailability, cellular per-meability, metabolic stability, and minimal drug-druginteraction [13, 32].

In this study, we examined the effects of the twoselective inhibitors on the non-spatial working memoryby NOR task, hippocampus-dependent spatial learningand memory by MWM test, and some related neu-ropathies by molecular and morphological tests [11,14]. Our data indicated that the cognitive impairmentand neuropathies of AD could be halted and evenreversed by tubastatin A and ACY-1215, at least inanimal models. Such reversibility was also found inSAHA-treated group. But interestingly, tubastatin Aand ACY-1215 show less hepatotoxicity compared toSAHA, which may be attributed to their high selec-tivity and limited effects on histone acetylation. Inaddition, there is no essential difference between tubas-tatin A and ACY-1215.

Our molecular and morphological results denotethat preservation of learning and memory function byinhibiting HDAC6 is likely to involve multiple cel-lular processes. SAHA was previously confirmed tocompensate for transport deficit by increasing tubulinacetylation [6, 18], so we detected the acetylation of�-tubulin. Expectedly, the level of acetylated �-tubulinnotably increases in both tubastatin A and ACY-1215groups. This can lead to enhanced microtubule stabilityand axonal transport of BDNF/mitochondria throughrecruitment of kinesin-1 and dynein to the more acety-lated microtubule [18, 36–40]. On the other hand,acetylated microtubule is also required for fusion ofautophagosomes with lysosomes. Increased acetylated�-tubulin in tubastatin A and ACY-1215 groups mayenhance the fusion of autophagosomes with lysosomesand then promote the autophagy process (Figs. 4c, 5c,and 6e) [41].

Moreover, HDAC6, as a microtubule-associatedprotein (MAP) also interacts with another MAP—tau,which exists in abnormal hyperphosphorylated formin AD brain. And hyperphosphorylated tau alsocollapses the transport system of neurons via micro-tubule disintegration [8, 9, 29]. After treatmentwith HDAC6 inhibitors, we observed that hyper-phosphorylation of tau at Ser202/Thr205, Thr231,and Ser396/Ser404 were apparently attenuated com-pared with the vehicle-treated mice. Furthermore,genetic or pharmacological inhibition of HDAC6

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was recently demonstrated to rescue tau-inducedmicrotubule defects [29]. Collectively, tubastatin Aand ACY-1215 may improve microtubule-dependentaxonal transport through increasing acetylation of�-tubulin and reducing tau hyperphosphorylation(Fig. 6b).

To further determine why HDAC6 inhibitors reducetau hyperphosphorylation, we measured the activityof CDK5 and GSK3�, two kinases that can phos-phorylate tau at several sites referred above [30, 31]and several autophagy-associated parameters [23]. Theimmunoblot analysis represented that the activity ofthe kinases, including CDK5 and GSK3�, was inhib-ited by tubastatin A and ACY-1215. With regardto GSK3�, it has to be mentioned that its inter-action with HDAC6 may improve the activity ofHDAC6 and impair the microtubule-based transportof mitochondria in hippocampus neurons (Fig. 6b)[39]. Thus, the decreased activity of GSK3� mayfurther enhance the inhibitory effect of the HDAC6inhibitors. Besides, GSK3�, as a direct downstreamtarget of Akt, is implicated in several signaling path-ways and then regulates expression of many genes.Gavilan et al. recently reported that Akt/GSK3� sig-naling pathway determined autophagy activation intumor cell [42]. Indeed, GSK3� negatively regulatedautophagy, and elevated GSK3� phosphorylation atSer9 simulated autophagy activation and promotedthe expression of autophagy-related proteins, suchas LC3II and beclin1 [42–45]. Parr et al. reportedthat GSK3� inhibition promoted lysosomal biogen-esis and autophagic degradation of A�PP [45]. Inthe present study, we found that treatment withtubastatin A or ACY-1215 inhibited GSK3� acti-vation by enhancing phosphprylation at Ser9 withan increase phosphorylation of Akt. Simultaneously,proteins involved in autophagy process, includingLC3II, beclin1, and LAMP2 significantly increasedin tubastatin A and ACY-1215 groups comparedwith the vehicle-treated group. Meanwhile, the levelof phospho-form mTOR, an inhibitory factor ofautophagy decreased in tubastatin A and ACY-1215groups. All these data strongly imply that impairedautophagy in AD can be rescued by tubastatin A andACY-1215 (Fig. 6d).

Actually, increased p-mTOR contributes to AD-linked cognitive deficits involved in many cellularprocesses, including both the buildup and clearance oftoxic protein aggregates such as A� and P-tau [46–48].A recent study has reported that increasing mTORactivity elevated endogenous tau hyperphosphoryla-tion, while reducing mTOR signaling ameliorated tau

pathology and the associated behavioral deficits ina mouse model overexpressing mutant human tau.Furthermore, they provided compelling evidence thatthe association between mTOR and tau was linkedto GSK3� and autophagy function [47]. In addi-tion, loss of HDAC6 was demonstrated to alleviateabnormal tau accumulation via promoting tau clear-ance [28]. Given the overwhelming evidence, wespeculate that tubastatin A and ACY-1215 reducetau pathology through decreasing tau phosphoryla-tion and increasing autophagy-associated degradationvia Akt/GSK3�/mTOR signaling pathway by selectiveHDAC6 inhibition (Fig. 6d).

Here, it has to be mentioned that mTOR is also adownstream target of Akt [48]. However, the level ofphospho-form Akt increased obviously after Tubas-tatin A or ACY-1215 treatment compared with thevehicle controls. This suggested mTOR might havea negative feedback effect on Akt. The increasedphospho-form Akt elevated the level of GSK3� phos-phorylation and then induced subsequent effects. Allthese results imply that tubastatin A and ACY-1215possibly contribute to the elimination of misfoldedproteins or aggregates (A�/P-tau) in AD, throughupregulated Akt/GSK3�/mTOR-mediated autophagy(Fig. 6d).

It is worth noting that the massive accumula-tion of autophagic vesicles in AD was rescued byHDAC6 inhibitors, which seems inconsistent withthe enhanced autophagy in tubastatin A and ACY-1215 groups described above. But in fact, reducedautophagic vesicles may be a proof supporting the ele-vated autophagy in inhibitors-treated groups. Duringlast decade, mounting evidence hinted that autophagicpathology observed in AD most likely arose fromimpaired clearance of autophagic vesicles rather thanstrong autophagy induction [23, 49, 50]. Meanwhile,HDAC6 was revealed to play a crucial role in theeventual clearance of aggresomes, especially in theautophagosome maturation [51]. Moreover, HDAC6-associated acetylated microtubules are required forfusion of autophagosomes with lysosomes referredbefore (Fig. 6e) [41]. In addition, the protein levelsof LC3II/LAMP2, as evidence of autophagosome-lysosome fusion increased dramatically in tubastatinA and ACY-1215 mice [22, 23]. Based on currentevidence, we speculate that appropriate inhibition ofexcessive HDAC6 by tubastatin A or ACY-1215 mayrescue autophagic pathology through the followingways: 1) facilitating delivery of autophagic substratesto lysosomes where the contents are degraded viaimproved microtubule network; 2) elevating levels

L. Zhang et al. / HDAC6 Inhibitors Restore Cognition in AD 1203

of autophagy-related proteins via Akt/GSK3�/mTORpathway.

The enhanced autophagy induced by tubastatinA/ACY-1215 further promoted the clearance oftoxic proteins, including P-tau described above andA�/oligomers. Taking into account that A� depositionis a crucial event of AD pathogenesis, A� depo-sition was analyzed in all groups. Surprisingly, weobserved that HDAC6 inhibitors reduced A� plaqueand soluble A� (including A�1-40 and A�1-42). Ourresults indicated that tubastatin A/ACY-1215 couldreduce A� load via inhibiting A� production by alter-ing A�PP processing at the �- and �-secretase siteas well as increasing autophagic clearance (Fig. 4).The reduced A� production after tubastatin A/ACY-1215 administration also seemed to be related toGSK3� inhibition. Qing et al. demonstrated that VPA,a pan-HDAC inhibitor, decreased A� production byinhibiting GSK3� activity through altering its phos-phorylation at Ser9. And GSK3� was proved tomediate �-secretase cleavage of A�PP [52]. Interest-ingly, another study reported that specific inhibitionof GSK3� markedly reduced BACE1-mediated �-cleavage of A�PP and A� deposition, and alleviatedmemory deficits in AD mice [53]. Altogether, we con-clude that tubastatin A/ACY-1215 may reduce A�production via inhibiting both �- and �-cleavage ofA�PP by GSK3� inhibition (Fig. 6c).

Notably, it is very likely that inhibited GSK3�activity induced by HDAC6 inhibition may act anessential role in cognitive improvement of AD micethrough following ways: 1) reducing A� produc-tion by inhibiting �-/�-cleavage of A�PP (Fig. 6c);2) attenuating tau hyperphosphorylation (Fig. 6b);3) simulating autophagy activation and the expres-sion of autophagy-related proteins (Fig. 6d) [42–45];4) promoting lysosomal biogenesis and autophagicdegradation (Fig. 6e) [45]; 5) further inhibiting theactivity of HDAC6 by weakening its interaction withHDAC6 (Fig. 6b) [39].

In conclusion, our study indicates that selectiveinhibition of HDAC6 by tubastatin A/ACY-1215 ame-liorates cognitive impairment in an AD mice model.The therapeutic efficacy of selective HDAC6 inhibitorsis not limited to axonal transportation due to theimprovement of microtubule function. The underlyingmechanisms may also include the alleviation of A�and tau pathology, two major lesions involved in ADpathogenesis (Fig. 6). Though tubastatin A and ACY-1215 are not more effective, they are less toxic thanSAHA indeed. These preclinical results raise hope forthe treatment of AD.

ACKNOWLEDGMENTS

We thank Prof. Xing-dong Zhang for the construc-tive advice. This work was supported by DoctorialInnovation Fund of Peking Union Medical College(2012-1001-003).

Authors’ disclosures available online (http://www.j-alz.com/disclosures/view.php?id=2206).

SUPPLEMENTARY MATERIAL

The supplementary tables and figure are availablein the electronic version of this article: http://dx.doi.org/10.3233/JAD-140066.

REFERENCES

[1] Arrowsmith CH, Bountra C, Fish PV, Lee K, Schapira M(2012) Epigenetic protein families: A new frontier for drugdiscovery. Nat Rev Drug Discov 11, 384-400.

[2] Huang Y, Mucke L (2012) Alzheimer mechanisms and ther-apeutic strategies. Cell 148, 1204-1222.

[3] Graff J, Tsai LH (2013) The potential of HDAC inhibitors ascognitive enhancers. Annu Rev Pharmacol Toxicol 53, 311-330.

[4] Kilgore M, Miller CA, Fass DM, Hennig KM, Haggarty SJ,Sweatt JD, Rumbaugh G (2010) Inhibitors of class 1 histonedeacetylases reverse contextual memory deficits in a mousemodel of Alzheimer’s disease. Neuropsychopharmacology35, 870-880.

[5] Kazantsev AG, Thompson LM (2008) Therapeutic appli-cation of histone deacetylase inhibitors for central nervoussystem disorders. Nat Rev Drug Discov 7, 854-868.

[6] Hubbert C, Guardiola A, Shao R, Kawaguchi Y, Ito A,Nixon A, Yoshida M, Wang XF, Yao TP (2002) HDAC6 isa microtubule-associated deacetylase. Nature 417, 455-458.

[7] Zhang L, Sheng S, Qin C (2013) The role of HDAC6 inAlzheimer’s disease. J Alzheimers Dis 33, 283-295.

[8] Ding H, Dolan PJ, Johnson GV (2008) Histone deacetylase 6interacts with the microtubule-associated protein tau. J Neu-rochem 106, 2119-2130.

[9] Perez M, Santa-Maria I, Gomez de Barreda E, Zhu X, CuadrosR, Cabrero JR, Sanchez-Madrid F, Dawson HN, Vitek MP,Perry G, Smith MA, Avila J (2009) Tau–an inhibitor ofdeacetylase HDAC6 function. J Neurochem 109, 1756-1766.

[10] Odagiri S, Tanji K, Mori F, Miki Y, Kakita A, Takahashi H,Wakabayashi K (2013) Brain expression level and activityof HDAC6 protein in neurodegenerative dementia. BiochemBiophys Res Commun 430, 394-399.

[11] Govindarajan N, Rao P, Burkhardt S, Sananbenesi F, SchluterOM, Bradke F, Lu J, Fischer A (2013) Reducing HDAC6 ame-liorates cognitive deficits in a mouse model for Alzheimer’sdisease. EMBO Mol Med 5, 52-63.

[12] Butler KV, Kalin J, Brochier C, Vistoli G, Langley B,Kozikowski AP (2010) Rational design and simple chemistryyield a superior, neuroprotective HDAC6 inhibitor, tubastatinA. J Am Chem Soc 132, 10842-10846.

[13] Santo L, Hideshima T, Kung AL, Tseng JC, Tamang D,Yang M, Jarpe M, van Duzer JH, Mazitschek R, Ogier WC,Cirstea D, Rodig S, Eda H, Scullen T, Canavese M, BradnerJ, Anderson KC, Jones SS, Raje N (2012) Preclinical activ-

1204 L. Zhang et al. / HDAC6 Inhibitors Restore Cognition in AD

ity, pharmacodynamic, and pharmacokinetic properties of aselective HDAC6 inhibitor, ACY-1215, in combination withbortezomib in multiple myeloma. Blood 119, 2579-2589.

[14] Yao ZG, Zhang L, Liang L, Liu Y, Yang YJ, Huang L, Zhu H,Ma CM, Qin C (2013) The effect of PN-1, a traditional Chi-nese prescription, on the learning and memory in a transgenicmouse model of Alzheimer’s disease. Evid Based Comple-ment Alternat Med 2013, 518421.

[15] d’Ydewalle C, Krishnan J, Chiheb DM, Van Damme P, Irobi J,Kozikowski AP, Vanden Berghe P, Timmerman V, RobberechtW, Van Den Bosch L (2011) HDAC6 inhibitors reverse axonalloss in a mouse model of mutant HSPB1-induced Charcot-Marie-Tooth disease. Nat Med 17, 968-974.

[16] Zhang L, Ma Q, Yang W, Qi X, Yao Z, Liu Y, Liang L, WangX, Ma C, Huang L, Xu Y, Zhu H, Deng W, Gao Y, RuanL, Xiao Z, Qin C (2013) Recombinant DNA vaccine againstneurite outgrowth inhibitors attenuates behavioral deficits anddecreases Abeta in an Alzheimer’s disease mouse model. Neu-ropharmacology 70, 200-210.

[17] Nemirovsky A, Shapiro J, Baron R, Kompaniets A, Mon-sonego A (2012) Active Abeta vaccination fails to enhanceamyloid clearance in a mouse model of Alzheimer’s diseasewith Abeta42-driven pathology. J Neuroimmunol 247, 95-99.

[18] Dompierre JP, Godin JD, Charrin BC, Cordelieres FP, KingSJ, Humbert S, Saudou F (2007) Histone deacetylase 6 inhi-bition compensates for the transport deficit in Huntington’sdisease by increasing tubulin acetylation. J Neurosci 27, 3571-3583.

[19] O’Brien RJ, Wong PC (2011) Amyloid precursor proteinprocessing and Alzheimer’s disease. Annu Rev Neurosci 34,185-204.

[20] Kabeya Y, Mizushima N, Ueno T, Yamamoto A, KirisakoT, Noda T, Kominami E, Ohsumi Y, Yoshimori T (2000)LC3, a mammalian homologue of yeast Apg8p, is localizedin autophagosome membranes after processing. EMBO J 19,5720-5728.

[21] Jaeger PA, Wyss-Coray T (2010) Beclin 1 complex inautophagy and Alzheimer disease. Arch Neurol 67, 1181-1184.

[22] Lee JH, Yu WH, Kumar A, Lee S, Mohan PS, PeterhoffCM, Wolfe DM, Martinez-Vicente M, Massey AC, Sovak G,Uchiyama Y, Westaway D, Cuervo AM, Nixon RA (2010)Lysosomal proteolysis and autophagy require presenilin 1 andare disrupted by Alzheimer-related PS1 mutations. Cell 141,1146-1158.

[23] Nixon RA (2013) The role of autophagy in neurodegenerativedisease. Nat Med 19, 983-997.

[24] Nazio F, Strappazzon F, Antonioli M, Bielli P, Cianfanelli V,Bordi M, Gretzmeier C, Dengjel J, Piacentini M, Fimia GM,Cecconi F (2013) mTOR inhibits autophagy by controllingULK1 ubiquitylation, self-association and function throughAMBRA1 and TRAF6. Nat Cell Biol 15, 406-416.

[25] Kim J, Kundu M, Viollet B, Guan KL (2011) AMPK andmTOR regulate autophagy through direct phosphorylation ofUlk1. Nat Cell Biol 13, 132-141.

[26] Ravikumar B, Vacher C, Berger Z, Davies JE, Luo S, Oroz LG,Scaravilli F, Easton DF, Duden R, O’Kane CJ, Rubinsztein DC(2004) Inhibition of mTOR induces autophagy and reducestoxicity of polyglutamine expansions in fly and mouse modelsof Huntington disease. Nat Genet 36, 585-595.

[27] Guthrie CR, Kraemer BC (2011) Proteasome inhibition drivesHDAC6-dependent recruitment of tau to aggresomes. J MolNeurosci 45, 32-41.

[28] Cook C, Gendron TF, Scheffel K, Carlomagno Y, DunmoreJ, DeTure M, Petrucelli L (2012) Loss of HDAC6, a novel

CHIP substrate, alleviates abnormal tau accumulation. HumMol Genet 21, 2936-2945.

[29] Xiong Y, Zhao K, Wu J, Xu Z, Jin S, Zhang YQ (2013) HDAC6mutations rescue human tau-induced microtubule defects inDrosophila. Proc Natl Acad Sci U S A 110, 4604-4609.

[30] Liu F, Liang Z, Shi J, Yin D, El-Akkad E, Grundke-Iqbal I,Iqbal K, Gong CX (2006) PKA modulates GSK-3beta- andcdk5-catalyzed phosphorylation of tau in site- and kinase-specific manners. FEBS Lett 580, 6269-6274.

[31] Hur EM, Zhou FQ (2010) GSK3 signalling in neural devel-opment. Nat Rev Neurosci 11, 539-551.

[32] Dallavalle S, Pisano C, Zunino F (2012) Development andtherapeutic impact of HDAC6-selective inhibitors. BiochemPharmacol 84, 756-765.

[33] Haggarty SJ, Koeller KM, Wong JC, Grozinger CM, SchreiberSL (2003) Domain-selective small-molecule inhibitor of his-tone deacetylase 6 (HDAC6)-mediated tubulin deacetylation.Proc Natl Acad Sci U S A 100, 4389-4394.

[34] Estiu G, Greenberg E, Harrison CB, Kwiatkowski NP, Maz-itschek R, Bradner JE, Wiest O (2008) Structural origin ofselectivity in class II-selective histone deacetylase inhibitors.J Med Chem 51, 2898-2906.

[35] Kim C, Choi H, Jung ES, Lee W, Oh S, Jeon NL, Mook-Jung I (2012) HDAC6 inhibitor blocks amyloid beta-inducedimpairment of mitochondrial transport in hippocampal neu-rons. PLoS One 7, e42983.

[36] Matsuyama A, Shimazu T, Sumida Y, Saito A, YoshimatsuY, Seigneurin-Berny D, Osada H, Komatsu Y, Nishino N,Khochbin S, Horinouchi S, Yoshida M (2002) In vivo desta-bilization of dynamic microtubules by HDAC6-mediateddeacetylation. EMBO J 21, 6820-6831.

[37] Bulinski JC (2007) Microtubule modification: Acetylationspeeds anterograde traffic flow. Curr Biol 17, R18-R20.

[38] Reed NA, Cai D, Blasius TL, Jih GT, Meyhofer E, Gaertig J,Verhey KJ (2006) Microtubule acetylation promotes kinesin-1binding and transport. Curr Biol 16, 2166-2172.

[39] Chen S, Owens GC, Makarenkova H, Edelman DB (2010)HDAC6 regulates mitochondrial transport in hippocampalneurons. PLoS One 5, e10848.

[40] Asthana J, Kapoor S, Mohan R, Panda D (2013) Inhibitionof HDAC6 deacetylase activity increases its binding withmicrotubules and suppresses microtubule dynamic instabilityin MCF-7 cells. J Biol Chem 288, 22516-22526.

[41] Xie R, Nguyen S, McKeehan WL, Liu L (2010) Acetylatedmicrotubules are required for fusion of autophagosomes withlysosomes. BMC Cell Bio 11, 89.

[42] Gavilan E, Sanchez-Aguayo I, Daza P, Ruano D (2013) GSK-3beta signaling determines autophagy activation in the breasttumor cell line MCF7 and inclusion formation in the non-tumor cell line MCF10A in response to proteasome inhibition.Cell Death Dis 4, e572.

[43] Qi ZF, Luo YM, Liu XR, Wang RL, Zhao HP, Yan F, Song ZJ,Luo M, Ji XM (2012) AKT/GSK3beta-dependent autophagycontributes to the neuroprotection of limb remote ischemicpostconditioning in the transient cerebral ischemic rat model.CNS Neurosci Ther 18, 965-973.

[44] Yang Y, Wang H, Wang S, Xu M, Liu M, Liao M, Frank JA,Adhikari S, Bower KA, Shi X, Ma C, Luo J (2012) GSK3betasignaling is involved in ultraviolet B-induced activation ofautophagy in epidermal cells. Int J Oncol 41, 1782-1788.

[45] Parr C, Carzaniga R, Gentleman SM, Van Leuven F, Walter J,Sastre M (2012) Glycogen synthase kinase 3 inhibition pro-motes lysosomal biogenesis and autophagic degradation ofthe amyloid-beta precursor protein. Mol Cell Biol 32, 4410-4418.

L. Zhang et al. / HDAC6 Inhibitors Restore Cognition in AD 1205

[46] Cai Z, Yan LJ (2013) Rapamycin, autophagy, and Alzheimer’sdisease. J Biochem Pharmacol Res 1, 84-90.

[47] Caccamo A, Magri A, Medina DX, Wisely EV, Lopez-ArandaMF, Silva AJ, Oddo S (2013) mTOR regulates tau phosphory-lation and degradation: Implications for Alzheimer’s diseaseand other tauopathies. Aging Cell 12, 370-380.

[48] Johnson SC, Rabinovitch PS, Kaeberlein M (2013) mTORis a key modulator of ageing and age-related disease. Nature493, 338-345.

[49] Nixon RA, Yang DS (2011) Autophagy failure in Alzheimer’sdisease–locating the primary defect. Neurobiol Dis 43, 38-45.

[50] Wolfe DM, Lee JH, Kumar A, Lee S, Orenstein SJ, NixonRA (2013) Autophagy failure in Alzheimer’s disease and therole of defective lysosomal acidification. Eur J Neurosci 37,1949-1961.

[51] Lee JY, Koga H, Kawaguchi Y, Tang W, Wong E, Gao YS,Pandey UB, Kaushik S, Tresse E, Lu J, Taylor JP, Cuervo AM,Yao TP (2010) HDAC6 controls autophagosome maturationessential for ubiquitin-selective quality-control autophagy.EMBO J 29, 969-980.

[52] Qing H, He G, Ly PT, Fox CJ, Staufenbiel M, Cai F, Zhang Z,Wei S, Sun X, Chen CH, Zhou W, Wang K, Song W (2008)Valproic acid inhibits Abeta production, neuritic plaque for-mation, and behavioral deficits in Alzheimer’s disease mousemodels. J Exp Med 205, 2781-2789.

[53] Ly PT, Wu Y, Zou H, Wang R, Zhou W, Kinoshita A, ZhangM, Yang Y, Cai F, Woodgett J, Song W (2013) Inhibition ofGSK3beta-mediated BACE1 expression reduces Alzheimer-associated phenotypes. J Clin Invest 123, 224-235.