# paper.pdf ## Metadata - PDFFormatVersion=1.7 - IsLinearized=false - IsAcroFormPresent=false - IsXFAPresent=false - IsCollectionPresent=false - IsSignaturesPresent=false - Author=蔡成云 - Custom.SourceModified=D:20260403101210+08'00' - CreationDate=D:20260403101210+08'00' - Creator=WPS 文字 - ModDate=D:20260403101210+08'00' - Trapped=False ## Contents ### Page 1 1Title: Simvastatin rescues cognitive impairment in an Aβ1-42-induced model of1Alzheimer’s disease through the HDAC2-BDNF signaling pathway23Chengyun Cai1,*, Yee Song Chong8,*, Haiying Liang2, Yimin Hu1, Qi Hu1, Jiamin4Chen1, Sreedharan Sajikumar8,9,10,#, Fang Liu1,3,4,5,6,7,#561Institute of Mental Health and Drug Discovery, Oujiang Laboratory (Zhejiang Lab7for Regenerative Medicine, Vision and Brain Health), School of Mental Health,8Wenzhou Medical University, Wenzhou, Zhejiang, 325000, China.9 2Department of Pharmacy, Longyan First Affiliated Hospital of Fujian Medical10University, Longyan, 364000, China.113Campbell Family Mental Health Research Institute, Centre for Addiction and Mental12Health, Toronto, Ontario, M5T 1R8 Canada.134Institutes of Medical Science and 5Department of Pharmacology and Toxicology,14 6Psychiatry, 7Physiology, University of Toronto, Toronto, M5T 1R8 Canada.15 8Department of Physiology, National University of Singapore, Singapore 117597,16Singapore.17 9Neurobiology Programme, Life Sciences Institute, National University of Singapore,18Singapore 117456, Singapore.19 10Healthy Longevity Translational Research Programme, Yong Loo Lin School of20Medicine, National University of Singapore, Singapore 117456, Singapore.21*These authors contributed equally: Chengyun Cai and Yee Song Chong.2223#Author for correspondence:24Sreedharan Sajikumar, Department of Physiology, Yong Loo Lin School of Medicine,25National University of Singapore, Singapore 117597, Singapore.26Email: phssks@nus.edu.sg27Fang Liu, Institute of Mental Health and Drug Discovery, Oujiang Laboratory28(Zhejiang Lab for Regenerative Medicine, Vision and Brain Health), School of29Mental Health, Wenzhou Medical University, Wenzhou, Zhejiang, 325000, China.30Email: fang.liu@camh.ca31 ### Page 2 2Materials and Methods32Animals33Adult (7-8 weeks) male C57BL/6 mice (from Oujiang Laboratory, Zhejiang,34China) and male Wistar rats (5-7 weeks old) (from National University of Singapore,35Singapore) were used in this study. The mice were maintained at a controlled36temperature (20 ± 2℃) and group-housed (12-h light/dark cycle) with access to food37and water ad libitum. All experimental and animal procedures were approved by the38Institutional Animal Care and Use Committees of Oujiang Laboratory39(OJLAB24091816) and the National University of Singapore (Protocol40R22-1084(A4)24). Every effort was made to minimize the number of animals used41and their suffering.4243Establishment of the AD model44The AD model was established according to the procedures reported in our45previous study[1]. The Aβ1-42 (Sigma, MO, USA) was prepared as previously46described[1,2]. Briefly, Aβ1-42 was dissolved in the hexafluoroisopropanol (HFIP, Sigma47Aldrich), flash-frozen in the liquid nitrogen, and then lyophilized to completely48remove the solvent. The lyophilized Aβ1-42 peptides were then dissolved in 100 mM49NaOH at a concentration of 6 mg/ml, aliquoted, flash-frozen in liquid nitrogen and50stored at -80 ℃ until use.51For intracerebroventricular (i.c.v.) injection of soluble Aβ1-42, mice were52anesthetized with isoflurane and then placed in a stereotactic apparatus (KOPE53instruments, USA). Freshly prepared Aβ1-42 (0.3 nmol/2 μl in 0.1 M54 ### Page 3 3phosphate-buffered saline (PBS)) was injected into the bilateral ventricles55(coordinates: AP +0.3 mm, ML ±1.0 mm, DV -2.5 mm) using a 10 μl microsyringe56mounted on a microinfusion pump (Harvard Apparatus) at a rate of 0.2 μl/min. The57mice injected with an equal volume of vehicle served as controls.58For the incubation of soluble Aβ1-42, slices in electrophysiological experiments,59slices were treated with 200 nM Aβ1-42 oligomers as described in previous reports[3].60The oligomers were perfused at a flow rate of 1 ml/min with artificial cerebral spinal61fluid (ACSF) and continuously oxygenated with carbogen at 16 L/hr for three hours62during the incubation period.6364Stereotaxic microinjections and cannulae implantation65Mice were anesthetized with isoflurane and fixed into a stereotaxic apparatus66(KOPE instruments, USA). Viral solution was microinjected into brain area using67microinjection pump (WPI, USA) with a glass pipette at a flow rate of 2 nl/s. The68pipette was left in place for 15 minutes after injection to prevent backflow and ensure69localized viral diffusion. A total of 0.5μl of virus solution was infused bilaterally into70the dHPC. Mice were used for experiments four weeks after microinjection.71For cannula implantation into the dHPC, 26-guage guide cannula (RWD Life72Science, Shenzhen, China) were used. Immediately after insertion into target region,73the cannula were secured using the dental cement. Cannula were implanted bilaterally.74After viral microinjection and cannula implantation surgery, mice were placed on a75warm blanket until awake. The coordinates used for both virus microinjection and76 ### Page 4 4cannula implantation were: AP -1.6 mm, ML ±1.2 mm, and DV -2.0 mm for dHPC[4].77Following surgery, each mouse was housed in an individual cage for 7 days to prevent78damage to the cannulas by other mice.7980Drug treatment81SV (Sigma-Aldrich, USA, #S6196) was converted from its inactive lactone82prodrug form to its active dihydroxy open acid form by alkaline hydrolysis[5]. SV (5083mg) was dissolved in 1 ml ethanol (100%), and then 0.813 ml 1 N NaOH was added.84This stock solution was stored in aliquots at -20 ℃ (for up to 1 month). For the in vivo85experiments, SV was neutralized with 1 N HCl to a pH of 7.4 and diluted with sterile86saline. The mice were given intragastric injection (i.g.) of SV at a dose of 20 mg/kg87for 14 days. The vehicle solution (0.01% ethanol) was used as a controls. BrdU was88purchased from Sigma-Aldrich and injected intraperitoneally (50mg/kg,12h interval ×894 times)[6] at 24 h after the last SV injection. For the in vitro experiment, the stock90solution was neutralized with l N HCl to a pH of 7.4 and diluted in ACSF. Slices were91prepared as described[7]. For each mouse, six hippocampal slices were obtained: three92slices were incubated at room temperature in 10 μM SV, and three slices were93incubated in ACSF. Mans et al.[8] reported that treatment of slices with SV for 2-4 h94significantly increased the magnitude of LTP at CA3-CA1 synapses.95Recombinant mouse TrkB-Fc chimera protein, which can sequester endogenous96BDNF, was purchased from R&D Systems (MN, USA). It was dissolved in 0.9%97saline (1.0 mg/ml). For TrkB-Fc solution infusions, obturators were removed and98 ### Page 5 5injectors were placed into the guide cannulas, with the injector tips extended 1.0 mm99beyond the guide cannula. One day before testing, mice were habituated to handling,100and injectors were passed through the cannula (without infusion). The mice received101two infusions, administered 7 days apart. Each infusion volume was 1 μl per side,102delivered at a rate of 0.25 μl/min. Bilateral microinfusions were performed through10326-gauge injection cannulas connected to a 10 μl microsyringe mounted on a104microinfusion pump (Harvard Apparatus). To better control drug diffusion, the 1 μl105volume was divided into four injections of 0.25 μl each. For each injection, the106infusion rate was 2 nl/s, followed by an additional 2-minute diffusion period. After107each infusion, animals were returned to their home cages.108109Behavioral tests110Cognitive behaviors in mice were assessed with three widely used paradigms:111Morris water maze (MWM) test, Y-maze task and novel object recognition (NOR) test.112Mice were identified by earmarks and randomly assigned to experimental groups113using a table of random numbers. Each testing squad was counterbalanced to ensure114equal representation of all groups. The experimental procedures were conducted in a115blinded manner. The order and time interval of the behavioral tests were as described116in the figures. Immediately after each test, mice were returned to their home cages,117and a 24-hour interval was maintained between each behavioral procedure.118119MWM test: The water maze task was conducted over nine consecutive days to120 ### Page 6 6assess spatial memory[2]. A circular pool (diameter: 120 cm) made of black plastic was121used. Water temperature was maintained at 20±1℃ with a bath heater prior to each122session. Swim paths were recorded and analyzed using a computerized video-tracking123system (AXIS-90 Target/2; Neuroscience).124On days 1-2 of training, a cylindrical dark-colored platform (7 cm in diameter)125was placed 0.5 cm above the water surface. On days 3-8, the platform was moved to126the opposite quadrant and submerged 1 cm below the surface. Mice were given 90127seconds to locate the platform. Latency to reach the visible or hidden platform and the128swim distance were recorded. If a mouse failed to find the platform within 90 seconds,129it was guided to the platform and the trial was terminated.130Each mouse was released into the pool from one of four quadrants in a random131order, with its head facing the wall. Four trials per day were conducted with a13230-minute intertrial interval for six consecutive days. On day 9, a probe trial was133performed in which the platform was removed, and the percentage of time spent in134each quadrant was measured.135Y-maze task: The Y-maze task was performed 24 hours after the probe trial[9].136The Y-maze was constructed from black-painted wood. Each arm measured 40 cm in137length, 13 cm in height, 3 cm wide at the bottom, and 10 cm wide at the top, and the138three arms converged at equal angles. Each mouse was placed at the end of one arm139and allowed to explore freely for an 8-minute session.140Arm entries were recorded visually, and an entry was counted when all four paws141of the mouse had fully entered an arm. Spontaneous alternation was defined as142 ### Page 7 7consecutive entries into three different arms in overlapping triplet sequences. The143percentage alternation was calculated as the ratio of actual alternations to the total144possible alternation (defined as the total number of arm entries minus 2).145NOR test: The experiments were conducted in a white Plexiglas apparatus146containing four compartments, each measuring 40 × 40 × 40 cm. The protocol was147performed as previously described with minor modification[10]. The test was148conducted over three days.149On Day 1, animals were habituated by allowing them to explore the apparatus150freely for 6 min. On Day 2, animals were exposed to two identical objects placed151along the centerline of the compartment for 6 minutes, and this session was repeated152three times. On Day 3, one of the familiar objects was replaced with a novel object of153a different shape but similar size, and animals were again allowed to explore for 6154minutes. Exploration time for each object on Day 3 was recorded manually, and the155difference between the exploration time of the novel object and the familiar object156was used as the measure of memory performance. Experiment and control groups157were tested simultaneously to minimize environmental variability. The apparatus was158cleaned with ethanol between trials to eliminate odor cues.159160Reverse Transcription-Polymerase Chain Reaction (RT-PCR)161Real-time RT-PCR was performed as described previously[11]. Total RNA was162isolated from the hippocampus with TRIzol reagent (Invitrogen, Camarillo, CA) and163reversetranscribed into cDNA using a Prime Script RT reagent kit (Takara, Dalian,164 ### Page 8 8China) for quantitative PCR (ABI Step One Plus, Foster City, CA) in the presence of165a fluorescent dye (SYBR Green I; Takara, Dalian, China). The relative expression of166genes was determined using the 2−ΔΔct method with normalization to Gapdh. The167sequences of primers are as follows:168Hdac1: forward: 5’-CCATCCTGGAACTGCTAAAG-3’;169reverse: 5’-ACCCGGTCTGTAGTATAGAAG-3’;170Hdac2: forward: 5’-GCTTGCCATCCTCGAATTA-3’;171reverse: 5’-CATCACGCGATTGTTGT-3’;172Hdac3: forward: 5’-CCCGCATCGAGAATCAGAAC-3’;173reverse: 5’-TCAAAGATTGTCTGGCGGATCT-3’;174Gapdh: forward: 5’-TGGGTGTGAACCACGAG-3’;175reverse: 5’-AAGTTGTCATGGATGACCTT-3’.176Bdnf p1:forward: 5’-TGATCATCACTCACGACCACG-3’ ;177reverse: 5’-CAGCCTCTCTGAGCCAGTTACG-3’;178Bdnf p4:forward: 5’-GCGCGGAATTCTGATTCTGGTAAT-3’ ;179reverse: 5’-GAGAGGGCTCCACGCTGCCTTGACG-3’;180181Western blot analysis182The samples of the dorsal hippocampus (dHPC) were collected as described183previously[12]. Briefly, brains were rapidly removed and blocked on ice into coronal184sections. Serial hippocampal sections (200 μm) were then prepared using a vibratome185(Leica) in a bath of ice-cold PBS. The dHPC was dissected from the first five186 ### Page 9 9hippocampal sections under a dissecting microscope and immediately placed into187pre-chilled RIPA lysis buffer (containing 150 mM NaCl, 1 mM EDTA-Na, 1% NP-40,1880.02% sodium azide, 0.1% SDS, 0.5% sodium deoxycholate, 1%PMSF, 1‰ aprotinin,1891‰ leupeptin, and 0.5‰ pepstatin A). The samples containing equivalent amounts of190protein (20 μg) were applied to 10-12% (10% for HDAC2 and Flag; 12% for BDNF,191Ac-H4 and H4) acrylamide denaturing gels (SDS-PAGE). The separated proteins192were transferred onto polyvinylidene difluoride membranes overnight at 4 ℃.193Blotting membranes were incubated with blocking solution (5% nonfat dried milk194powder dissolved in TBST buffer (pH 7.5, 10 mM Tris-HCl, 150 mM NaCl, and 0.1%195Tween 20)) for 1 hour at room temperature, washed three times, and then were196incubated with rabbit anti-HDAC2 (1:4000; Abcam, #32117), or rabbit anti-BDNF197(1:1000; Thermo Fisher Scientific, #PA585730), or rabbit anti-Ac-H4 (1:1000; Abcam,198#ab51997), or rabbit anti-H4 (1:1000; Abcam, #ab10158), or mouse anti-Flag (1:1000;199Smart LifeSciences, #SLAB01C), in TBST overnight at 4 °C. Internal control was200carried out using mouse anti-GAPDH (1:10000, Proteintech, #60004-1-Ig) or rabbit201anti-GAPDH (1:10000, Proteintech, #81640-5-RR). After several washes with TBST202buffer, the membranes were incubated for 2 hours with horseradish peroxidase-linked203secondary antibody (1:10000). The membranes were then processed with enhanced204chemiluminescence western blotting detection reagents (Pierce). Western blot bands205were scanned and analyzed with the image analysis software package (Image J; NIH206Image, Bethesda, MD, USA).207208 ### Page 10 10Chromatin Immunoprecipitation (ChIP)209ChIP assays were performed using a ChIP assay kit (EMD Millipore). Briefly,210DNA and its associated proteins in the dHPC tissue were chemically cross-linked by211the addition of 37% formaldehyde (1% final concentration) for 15 minutes at 37°C.212The cross-link reaction was then quenched with glycine (0.125 mol/L final213concentration) for 5 minutes. DNA was sheared to fragments between 200 and 1000214base pairs using a Branson Digital Sonifier 450, applying twenty-five 20-s pulses at21545% maximum amplitude with 50-s intervals between pulses. Cell debris was216removed by centrifugation, and the supernatants were diluted with ChIP dilution217buffer. A portion of the diluted supernatant was reserved as the input control. The218remaining lysate was precleared by incubation with Protein A-Sepharose beads for 30219minutes at 4°C, followed by overnight immunoprecipitation with an antibody against220acetylated histone H4 (acetyl-K5, ab51997, Abcam). Immune complexes were221collected by incubation with Protein A-Sepharose beads for 1 hour at 4 °C. The222bead-antibody complexes were then subjected to a series of sequential washes. Bound223complexes were eluted from the beads by vortexing in an elution buffer containing2241% SDS and 0.1 mol/L NaHCO3, and crosslinking was reversed by incubation225overnight at 65 °C. Purified DNA samples were normalized and subjected to real-time226PCR for 45 cycles using primer pairs specific for 150-250 bp segments corresponding227to mouse gene promoter regions (upstream of the start codon, near the first exon).228Real-time PCR products were further analyzed by electrophoresis on 2% agarose gel.229230 ### Page 11 11BrdU Immunohistochemistry and cell counting231Mice were perfused transcardially with 200 ml of 0.05 M sodium phosphate (pH2327.4) containing 0.8% NaCl, followed by 300 ml of 4% paraformaldehyde in 0.05 M233sodium phosphate (pH 7.4, containing 0.8% NaCl). BrdU staining was performed as234described previously[6]. Brains were removed and post-fixed overnight in the same235solution. Serial dHPC sections (40 μm) were made on an oscillating tissue slicer in a236bath of physiological saline. For immunohistochemistry, sections were washed 5237times in PBS (5 × 10 min) and then blocked in 10% donkey serum containing 0.1%238Triton-100 at room temperature. One hours later, sections were incubated with mouse239polyclonal anti-BrdU (Millipore, #MAB4072; 1:200) or rabbit monoclonal anti-DCX240(1:400, abcam), or rabbit monoclonal anti-NeuN (1:400, abcam) overnight at 4 °C.241Next day, the sections were washed for 5 times in PBS (5 × 10 min) and then242incubated with the fluorescent secondary antibody solution in dark at room243temperature for 2 h, followed by 5 washes in PBS (5 × 10 min).244Alexa-fluor-conjugated secondary antibodies included: Alexa 488 (donkey anti-rabbit2451:400, Jackson Immuno Research Labs, 711-545-152), CyTM3 (donkey anti-mouse2461:400, Jackson Immuno Research Labs, 715-165-150). Then, sections were stained to247label nuclei with Hoechst (1:400, Sigma) for 10 min, followed by 5 times washes in248PBS (5 × 10 min). Finally, sections were mounted on slides with Fluoromount-G249(Southern Biotech). Immunofluorescence images were acquired using a confocal250microscope (Nikon).251One experimenter coded all slides from the experiments before quantitative252 ### Page 12 12analysis. Proliferating cells, detected by their BrdU-positive nuclei, were counted by253another experimenter who was unaware of the experimental conditions of each254sample. Brains were serially sectioned coronally at 40 μm throughout the dHPC.255Sections from AP -1.5 mm to AP -2.3 mm were taken from each brain. Each mouse256brain produced 20 slices and every fourth section throughout the dHPC was processed257for cell counting. In order to obtain a homogeneous representation of the dHPC, no258more than 2 sections were lost during the sectioning of a single brain. The fluorescent259images were acquired at 40 × for BrdU+DCX+cells and BrdU+NeuN+cells counting.260The images were analyzed with UTHSCSA ImageTool 3.0 software. The values from261all sampled sections of each animal were finally averaged as the final value of a single262brain.263264Electrophysiology265A total of 80 transverse acute hippocampal slices (400 μm thick) from 20 adult266male Wistar rats (5-7 weeks old) were used for electrophysiological experiments.267Briefly, the animals were decapitated after anesthetization using CO2. The brains were268quickly removed and cooled in 4 ℃ ACSF that contained the following (in269millimolar): 124 NaCl, 3.7 KCl, 1.0 MgSO4 .7H2O, 2.5 CaCl2, 1.2 KH2PO4, 24.6270NaHCO3 and 10 D-glucose, equilibrated with 95% O2-5% CO2 (carbogen; total271consumption 16 L/hr), and acute hippocampal slices were prepared from the right272hippocampus using a manual tissue chopper.273Hippocampal slices were then transferred to the interface brain slice chamber274 ### Page 13 13(Scientific Systems Design, Canada) and incubated for three hours at 32 ℃ with275ACSF before the electrophysiology studies. Slices were treated with 200 nM Aβ1-42276oligomers (Anaspec Inc., USA) in a similar manner described in our previous reports277[3,7] at a flow rate of 1 ml/min of ACSF and 16 L/hr of carbogen for three hours during278the incubation time. The entire process of animal dissection, hippocampal slice279preparation and placement of slices on the chamber was done within approximately280five minutes to ensure that hippocampal slices were in optimal condition for281electrophysiology studies[13]. The signals were amplified by a differential amplifier282(Model 1,700; AM Systems, USA), digitized using a CED 1,401 analog-to-digital283converter (Cambridge Electronic Design, UK), and monitored online.284After the pre-incubation period, a synaptic input-output curve (afferent285stimulation vs. fEPSP slope) was generated. Test stimulation intensity was adjusted to286elicit fEPSP slope of 40% of the maximal slope response for both synaptic inputs S1287and S2. Theta burst stimulation-LTP (TBS-LTP) was induced using a protocol288consisting of 50 bursts (each burst with four stimuli) at an interstimulus interval of 10289ms. The 50 bursts were applied over a period of 20 s at 5 Hz[14]. In all experiments, a290stable baseline was recorded for at least 30 min using four 0.2-Hz biphasic291constant-current pulses (0.1 ms per polarity) at each time point.292293Statistical analysis294Appropriate parametric statistics were utilized to test our hypothesis. The sample295size was predetermined by analyzing pre-experimental data with PASS (power296 ### Page 14 14analysis and sample size) software (http://powerandsamplesize.com/) using a297significance level of α = 0.05 with 90% power to detect statistical differences. For298animal studies, the number of animals for each group was predetermined according to299numbers reported in published studies or our prior experiment. Investigators were300blind to the treatment group when assessing the outcome. GraphPad Prism 8 software301was used to make graphs and analyze statistical data. Two-way repeated ANOVA302followed by Sidak post hoc test for behavior tests, two-way ANOVA followed by303Tukey’s post hoc test for comparisons of multi-groups, unpaired two-tailed Student’s304t-test for comparisons between two groups. All data are presented as means ± SEM. p305< 0.05 was set as statistical significance.306307Supplementary references3081. Chen T, Zhang B, Li G, Chen L, Chen L. Simvastatin enhances NMDA receptor309GluN2B expression and phosphorylation of GluN2B and GluN2A through increased310histone acetylation and Src signaling in hippocampal CA1 neurons.311Neuropharmacology. 2016;107:411-421.3122. Cai C, Wang L, Li S, Lou S, Luo JL, Fu DY, Chen T. Ras Inhibitor Lonafarnib313Rescues Structural and Functional Impairments of Synapses of Aβ1-42 Mice via314α7nAChR-Dependent BDNF Upregulation. J Neurosci. 2022;42(31):6090-6107.3153. Sharma M, Dierkes T, Sajikumar S. Epigenetic regulation by G9a/GLP complex316ameliorates amyloid-beta 1-42 induced deficits in long-term plasticity and synaptic317tagging/capture in hippocampal pyramidal neurons. Aging Cell. 2017; 16(5):3181062-1072.3194. Bian XL, Qin C, Cai CY, Zhou Y, Tao Y, Lin YH, Wu HY, Chang L, Luo CX, Zhu320DY. Anterior Cingulate Cortex to Ventral Hippocampus Circuit Mediates Contextual321Fear Generalization. J Neurosci. 2019; 39(29):5728-5739.3225. Mann DM, Ponieman D, Montori VM, Arciniega J, McGinn T. The Statin Choice323decision aid in primary care: a randomized trial. Patient Educ Couns. 2010; 80(1):324138-40.3256. Cai CY, Wu HY, Luo CX, Zhu DY, Zhang Y, Zhou QG, Zhang J. Extracellular326regulated protein kinaseis critical for the role of 5-HT1a receptor in modulating nNOS327expression and anxiety-related behaviors. Behav Brain Res. 2019; 357-358:88-97.328 ### Page 15 157. Baby N, Alagappan N, Dheen ST, Sajikumar S. MicroRNA-134-5p inhibition329rescues long-term plasticity and synaptic tagging/capture in an Aβ(1-42)-induced330model of Alzheimer's disease. Aging Cell. 2020; 19(1):e13046.3318. Mans RA, McMahon LL, Li L. Simvastatin-mediated enhancement of long-term332potentiation is driven by farnesyl-pyrophosphate depletion and inhibition of333farnesylation. Neuroscience. 2012;202:1-9.3349. Zhao L, Chen T, Wang C, Li G, Zhi W, Yin J, Wan Q, Chen L. Atorvastatin in335improvement of cognitive impairments caused by amyloid β in mice: involvement of336inflammatory reaction. BMC Neurol. 2016;16:18.33710. Mutlu-Burnaz O, Yulug B, Oncul M, Celik E, Atasoy NS, Cankaya S, Hanoglu L,338Velioglu HA. Chemogenetic inhibition of MCH neurons does not alter memory339performance in mice. Biomed Pharmacother. 2022; 155:113771.34011. Yang C, Ni HY, Yin JJ, Zhou T, Gu QX, Chen TT, Cai CY. Atorvastatin341ameliorates depressive behaviors via regulation of α7nAChR expression by342PI3K/Akt-BDNF pathway in mice. Biochem Biophys Res Commun. 2022;593:57-64.34312. Powell KL, Ng C, O'Brien TJ, Xu SH, Williams DA, Foote SJ, Reid CA.344Decreases in HCN mRNA expression in the hippocampus after kindling and status345epilepticus in adult rats. Epilepsia. 2008;49(10):1686-95.34613. Shetty MS, Sharma M, Hui NS, Dasgupta A, Gopinadhan S, Sajikumar S.347Investigation of Synaptic Tagging/Capture and Cross-capture using Acute348Hippocampal Slices from Rodents. J Vis Exp. 2015; (103):53008.34914. Navakkode S, Gaunt JR, Pavon MV, Bansal VA, Abraham RP, Chong YS, Ch'ng350TH, Sajikumar S. Sex-specific accelerated decay in time/activity-dependent plasticity351and associative memory in an animal model of Alzheimer's disease. Aging Cell. 2021;35220(12): e13502.353 ### Page 16 16Supplementary figures and figure legends354355Supplementary Figure S1. Interfering with HDAC2 expression attenuates356Aβ1-42-induced cognitive impairment. (A) The flow chart for the experiment shown357in (B-F). (B) Representative coronal section and Western blots showing effective358expression of AAV-U6-HDAC2 RNAi-eGFP in the dHPC. (C) Latency to find the359visible platform and hidden platform in the MWM test. (D) The percentage of time360(%) spent in the target quadrant in the probe trial test in MWM. (E) The alternation361rate (%) in the Y-maze task. (F) Discrimination index of the exploratory behavior in362the NOR test. n = 11 per group for all behavioral tests (MWM, Y-maze, and NOR). *p363< 0.05, **p < 0.01, and ***p < 0.001. “ns” means no significance.364 ### Page 17 17365Supplementary Figure S2. HDAC2 knockdown rules out non-specific effects on366motor activity. (A) The average swimming speed in the MWM test. (B) The total arm367entries in the Y-maze task. n = 11 per group for all behavioral tests (MWM, Y-maze).368 ### Page 18 18369Supplementary Figure S3. HDAC2 overexpression in the dHPC causes cognitive370dysfunction in control mice. (A) The flow chart of the experiment. (B) Latency to371find the visible platform and hidden platform in the MWM test. (C) The average372swimming speed in the MWM test. (D) The percentage of time (%) spent in the target373quadrant in the probe trial test in MWM. (E) The alternation rate (%) in the Y-maze374task. (F) The total arm entries in the Y-maze task. (G) Discrimination index of the375exploratory behavior in the NOR test. n = 10 per group for all behavioral tests (MWM,376Y-maze, and NOR). n = 10 per group for all behavioral tests (MWM, Y-maze, and377NOR). *p < 0.05, **p < 0.01, and ***p < 0.001. “ns” means no significance.378 ### Page 19 19379Supplementary Figure S4. HDAC2 overexpression in the dHPC does not affect380swimming speed or total arm entries in Aβ-treated mice. (A) The average381swimming speed in the MWM test. (B) The total arm entries in the Y-maze task. n =38210 per group for all behavioral tests (MWM, Y-maze).383 ### Page 20 20384Supplementary Figure S5. SV treatment may enhance neurogenesis in the dHPC385to improve cognitive behaviors. (A) The representative images showing the386expression of DCX and BrdU in the DG of the dHPC after SV treatment. (B)387Quantification of the number of BrdU+DCX+ cells in the DG of the dHPC after SV388treatment. n = 5 per group. (C) The representative images showing the expression of389NeuN and BrdU in the DG of the dHPC after SV treatment. (D) Quantification of the390number of BrdU+NeuN+ cells in the DG of the dHPC after SV treatment. n = 5 per391group. *p < 0.05, **p < 0.01 and ***p < 0.001. “ns” means no significance.392 ### Page 21 21393Supplementary Figure S6. Uncropped Western blot images corresponding to all394figures in the main text.395