Hippocampal proteomic alteration in triple transgenic mouse model of

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Hippocampal proteomic alteration in triple transgenic mouse model of Alzheimer’s disease and implication of PINK 1 regulation in donepezil treatment Xinhua Zhou, Wei Xiao, Zhiyang Su, Jiehong Cheng, Chengyou Zheng, Zaijun Zhang, Yuqiang Wang, Liang Wang, Benhong Xu, Shupen Li, Xifei Yang, and Maggie Pui Man Hoi J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.8b00818 • Publication Date (Web): 28 Nov 2018 Downloaded from http://pubs.acs.org on December 3, 2018

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Journal of Proteome Research

Hippocampal proteomic alteration in triple transgenic mouse model of Alzheimer’s disease and implication of PINK 1 regulation in donepezil treatment Xinhua Zhou 1 , Wei Xiao 2, Zhiyang Su 3, Jiehong Cheng 3, Chengyou Zheng 3, Zaijun Zhang 3, Yuqiang Wang 3, Liang Wang4, Benhong Xu5, Shupen Li6, Xifei Yang 5*, Maggie Pui Man Hoi 1* 1. State Key Laboratory of Quality Research in Chinese Medicine and Institute of Chinese Medical Sciences, University of Macau, Macau 2. College of Letters & Science, University of Wisconsin- Madison, Madison, Wisconsin, United States 3. Institute of New Drug Research and Guangdong Province Key Laboratory of Pharmacodynamic Constituents of Traditional Chinese Medicine, Jinan University College of Pharmacy; Guangzhou, China 4. Institute of Biomedical and Pharmaceutical Sciences, Guangdong University of Technology, Guangzhou, China 5. Key Laboratory of Modern Toxicology of Shenzhen, Shenzhen Center for Disease Control and Prevention, Shenzhen, China 6. State Key Laboratory of Oncogenomics, School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School, Shenzhen 518055, China Corresponding authors: *Xifei Yang: [email protected]; Key Laboratory of Modern Toxicology of Shenzhen, Shenzhen Center for Disease Control and Prevention, Shenzhen, China *Maggie Pui Man Hoi: [email protected]; State Key Laboratory of Quality Research in Chinese Medicine and Institute of Chinese Medical Sciences, University of Macau, Macau

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ABSTRACT Donepezil is a clinically approved acetylcholinesterase inhibitor (AChEI) for cognitive improvement in Alzheimer’s disease (AD). Donepezil has been used as a first-line therapeutics for the symptomatic treatment of AD, but its ability to modify disease pathology and underlying mechanisms are not clear. We investigated the protective effects and underlying mechanisms of donepezil in AD-related triple transgenic (APPSwe/PSEN1M146V/MAPTP301L) mouse model (3×Tg-AD). Mice (8-month old) were treated with donepezil (1.3 mg/kg) for 4 months and evaluated by behavioral tests for assessment of cognitive functions and the hippocampal tissues were examined by protein analysis and quantitative proteomics. Behavioral tests showed that donepezil significantly improved the cognitive capabilities of 3xTg-AD mice. The levels of soluble and insoluble amyloid beta proteins (Aβ1-40 and Aβ1-42) and senile plaques were reduced in the hippocampus. Golgi staining of the nervous tissue showed that donepezil prevented dendritic spine loss in hippocampal neurons of 3xTg-AD mice. Proteomic studies of the hippocampal tissues identified 3131 proteins with altered expression related to AD pathology, of which 262 could be significantly reversed with donepezil treatment. Bioinformatics with functional analysis and protein-protein interaction (PPI) network mapping showed that donepezil significantly elevated the protein levels of PINK 1, NFASC, MYLK2, and NRAS in the hippocampus, and modulated the biological pathways of axon guidance, mitophagy, mTOR and MAPK signaling. The substantial upregulation of PINK 1 with donepezil was further verified by western blotting. Donepezil exhibited neuroprotective effects via multiple mechanisms. In particular, PINK 1 is related to mitophagy and cellular protection from mitochondrial dysfunction, which might play important roles in AD pathogenesis and represent a potential therapeutic target.

Key words: Alzheimer’s disease (AD), donepezil, amyloid beta (Aβ), proteomics, PINK 1

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INTRODUCTION

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by progressive memory loss and cognitive impairments resulting in severe dementia. The pathogenesis of AD is complicated and poorly understood. According to the World Alzheimer Report 2018, it is estimated 50 million people worldwide are living with dementia, and the number of people affected by AD will double nearly every 20 years 1 . Over the past decade, various theories have been developed for the pathogenesis of AD, including the amyloid hypothesis, tau pathology, mitochondrial dysfunction, oxidative stress, brain inflammation and cholinergic pathology 1. Donepezil is a highly selective and reversible inhibitor of acetylcholinesterase (AChE) and is effective at improving cognition and behavior of AD patients, but the mechanism of action is not well understood 2, 3. It is generally considered that the treatment of donepezil is only symptomatic in nature, which improves cognition by inhibiting the enzymatic degradation of acetylcholine to increase its availability at cholinergic synapses, thereby enhancing cholinergic neurotransmission4. However, recent preclinical studies demonstrated that donepezil exhibited neuroprotective effects against amyloid beta (Aβ) cytotoxicity in various cellular models in vitro. 5-7. Studies with transgenic animal models showed that donepezil reduced Aβ protein levels and senile plaque deposition, and prevented synaptic loss in Tg2576 mice, and decreased mitochondrial Aβ accumulation in APP/PS1 mice5, 8, 9. The AD-related triple transgenic mouse model (3×Tg-AD) is a widely used AD animal model harboring human gene APPSwe, PSEN1M146V, and MAPTP301L mutations. Both amyloid plaques and neurofibrillary tangles are formed in the brain, and the animals develop progressive cognitive deficits resembling AD progression in humans 6 . Here, we reported the neuroprotective effects of donepezil and evaluated the underlying mechanisms in 3xTg-AD mouse model for the first time. We showed that donepezil reduced Aβ in the hippocampus, and retained neuronal dendritic spine density. We conducted detailed analysis of proteomics and bioinformatics to dissect the molecular mechanisms of donepezil for the treatment of AD. MATERIALS AND METHODS

Animals and Treatment Triple transgenic 3xTg-AD mice harboring APPSwe, PSEN1M146V, and MAPTP301L transgenes (strain: B6; 129-Psen1tm1Mpm Tg [APPSwe,tauP301L]1Lfa/Mmjax) were purchased from the Jackson Laboratory. Female 3xTg-AD mice (8-month old) or wild-type mice (WT) (strain: B6129SF2/J) were treated with donepezil (1.3 mg/kg for 4 months) or with equivalent volume of saline by intragastric administration, with 12 mice in each group. Cognitive capabilities of animals were evaluated by behavioral tests. At the end of the behavioral tests, animals were sacrificed and brain tissues were dissected and collected for histopathological and studies and proteomics analysis. All animal studies followed approved procedures by the Institutional Animal Care and Use Committee of Shenzhen Center for Disease Control and Prevention. All efforts

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were made to reduce number of animals used and minimize animal suffering. Behavioral tests Step-down passive avoidance test The step-down passive avoidance (SDA) test was used to evaluate learning and memory in rodent models of neurodegenerative disorders. The step-down apparatus consisted of a rectangular plexiglass inner box (15×15×46 cm3) with a grid floor fitted with parallel steel rods (0.3 cm in a diameter, 0.8 cm apart) (ZH-500, Anhui, China). A wooden platform (4.5 cm in a diameter) was set in the center of the grid floor. During the training session, animals were gently placed on the grid floor while electrical shock (36 V) was delivered through the grid floor for 5 min. On the following day after 24 h the animals were placed on the wooden platform while electrical shock was delivered. Retention memory was evaluated by measuring the step-down latency recorded as the duration before the animal stepped down from the platform for the first time and the number of times the animals touching the grid floor with paws 10, 11. Novel object recognition test The novel object recognition (NOR) test was used to evaluate recognition memory in rodent models. Rodents like mice explore an object (novel object) more frequently when they are exposed to it for the first time, and spend less time on an object they have seen before (familiar object). Briefly, the mouse was exposed to two identical objects (A+A) in a chamber and allowed to explore for 5 min once daily for 3 days. On day 4, one of the objects was replaced by an novel object (A +B) in the chamber, and the mouse was allowed to explore for 5 min as before with video recording and movement-tracking. Exploration was measured as sniffing or touching the object with snout with a distance ≤2 cm from the object. Discrimination index (DI) was calculated as: (Time exploring novel object - Time exploring familiar object) / (Time exploring novel object + Time exploring familiar object)*100 12, 13. Morris Water Maze Test Spatial memory was evaluated with the Morris Water Maze (MWM) test. It is a measure of the hippocampus-dependent spatial navigation and reference memory. The test was conducted in a circular tank (100 cm in diameter and 45 cm deep) filled with opaque water to a depth of 27 cm maintained at 25 ± 0.5 ℃ (XR-XM101,Shanghai,China). The test consisted of hidden platform training and a probe trial. A circular platform (8 cm in diameter and 26 cm tall) was placed at a fixed location in the tank in one of the four quadrants of the pool, submerge 1 cm beneath the water surface and was indicated by a marker emerged above water. During platform training, the mouse was placed into the water facing the wall of one of the four start positions, and its movement was tracked by computer software. The platform remained in the same position throughout the training. The ability of the mice to locate the platform was tested by conducting 4 trials at a different start location to exclude differences in vision and motivation. The animal was immediately

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removed from the pool when it found the platform. The mouse that could not find the platform within 60s was placed on the platform for an additional 15 seconds before removing from the pool. The mice were trained for 5 days. The probe trial was performed on the sixth day after the training session. During the probe trial, the platform was removed and the mice were placed in the pool from the diagonally opposite side of the platform with their head located toward the side of the tank. The mice were limited to swim freely for 2 min with movement-tracking recorded by computer software (SuperMaze+) before it was removed from the pool. 14, 15. Quantification of soluble and insoluble Aβ by using ELISA To measure the levels of soluble and insoluble Aβ, the hippocampus of mice were collected after the behavioral tests and were homogenized in RIPA with phosphatase and protease inhibitor. It was then centrifuged at 20,000 x g for 30 min at 4℃. The supernatant fraction was collected as soluble Aβ. The precipitation was resuspended in 70% formic acid, incubated on ice for 30 min and then centrifuged at 20,000 x g for 30 min at 4℃. The supernatant was diluted 1:20 in a neutralization buffer (1 mol/L tris base, 0.5 mol/L NaH2PO4), and collected as the insoluble Aβ. The levels of Aβ1-40 and Aβ1-42 were quantified by Quantikine ELISA Human Amyloid β (aa1-40/aa1-42) Immunoassay kits (R&D Systems, Minneapolis, MN, USA) following the manufacturer’s instructions 16. Immunohistochemistry Immunohistochemical staining was used to further analyze the deposition of Aβ plaques in the hippocampus. The mice were sacrificed with intraperitoneal injections with 1% pentobarbital and perfused through the aorta with 0.9% NaCl followed by phosphate bu er saline (PBS) containing 4% paraformaldehyde （PFA）. Brain tissues were dissected and post-fixed in 4% PFA for 24 h, embedded in paraffin and sectioned followed by treatment with xylene and gradual rehydration with ethanol for immunostaining. Brains sections were then incubated with primary antibody 6E10 (Biolegend, San Diego, USA) overnight at 4°C in 0.3% Triton X-100 phosphate buﬀered saline (PBS). The secondary antibody was incubated in the dark for 1 h at room temperature. The immunoreaction was measured by horseradish peroxidase-labeled antibodies for 1 h at 37 °C and visualized with the diaminobenzidine tetrachloride system17. The images were observed using a microscope (Olympus BX41, Tokyo, Japan). Golgi staining Golgi staining was performed using an FD Rapid Golgi Stain Kit (FD Neurotechnologies, Columbia, MD, USA) following the manufacturer’s instructions. Briefly, brain tissues were immersed in impregnation solution (mixing equal volumes of Solutions A and B) for 1 weeks at room temperature in the dark. Replace the impregnation solution on the next day. Transfer brain tissue into Solution C for at least 3 days at room temperature and store in the dark. Replace the Solution C at once on the next day. Brain sections (100 µm thickness) were placed on gelatin-coated

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microscope slides and rinsed in Milli-Q water 2 times. Place sections in a mixture consisting Solution D, Solution E and Milli-Q water for 10 min, then rinsed in Milli-Q water 2 times, dehydrated in 50%-100% ethanol, cleared in xylene and coverslip with Permount®. The sections were observed using a microscope (OLYMPUS BX41), and the dendrites spine of neurons in the hippocampus were analyzed. For each mouse, three section slides were randomly selected, and three segments (at least 30 µm) were randomly chosen from one neuron, and the numbers of spines per 10 µm were counted in a blinded manner using LAS AF Lit software. e Western Blot Protein samples were extracted from hippocampus tissues with lysis bu er (8 M urea in PBS, pH 8.0) with 1× phosphatase inhibitor cocktail (Thermo Fisher Scientific, USA) by ultrasonic disruption system. The protein samples were boiled with loading bu er and heated for 5 min at 96 °C, then separated on 7.5%-12% SDS–PAGE and transferred to 0.2 µmol/L PVDF membranes. Membranes were blocked for 1 h with 5% skim milk in TBST (150 mM NaCl, 10 mM Tris, 0.1% Tween-20, pH 8.0) buffer, then incubated with PINK 1 primary antibodies (ab23707,abcam) in TBST bu er overnight at 4 °C. After washing in TBST, the membranes were incubated with anti-rabbit IgG HRP secondary antibody in TBST for 1 h. Subsequently, the membranes were washed in TBST 3 times and developed using Pierce ECL Western Blotting substrate (Thermo Scientific Pierce ECL, USA). Quantitative densitometry analyses were performed using quantity one analysis software. Sample preparation and TMT labeling The processes of proteome were shown as Figure 1. Mice were sacrificed after intraperitoneal injections with 1% pentobarbital for brain collection. Then hippocampus tissues were dissected and lysed with 8 M urea homogenization buffer (8 M urea in PBS, pH 8.0, 1× protease inhibitor cocktail), and homogenized using ultrasonic disruption system. Tissue lysates were centrifuged at 12,500 RPM for 15 min at 4℃. Protein concentration was measured by Pierce BCA protein assay kit according to the manufacturer’s instructions. Protein was pooled to obtain a total of 100 µg of protein sample (6 mice in each experimental group, 16.7 µg of protein per mouse). The pooled samples were treated with 10 mM dithiothreitol (DTT) for 1 h at 55 ℃, and then incubated with 25 mM idoacetamide (IAA) for 1 h at room temperature in the dark. After that, the sample were diluted with PBS to a final concentration of 1.0 M of urea and digested with trypsin/Lys-C (1:25 w/w) (Promega, WI, USA) at 37℃ overnight. On the following day, formic acid (FA) was added to acidify the protein to achieve a final pH 1-2, followed by centrifugation at 12,000 RPM for 10 min at 4℃, and the supernatant was collected. The samples were then desalted with reversed-phase column chromatography (Oasis HLB; Waters, MC, USA) according to manufacturer’s instructions. The samples were then dried under vacuum centrifuge and dissolved in 50 µl triethylammonium bicarbonate buffer (TEAB, 200

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mM, pH8.5). Peptides were labeled with TMT reagents according to the manufacturer’s instructions (Thermo Scientific, NJ, USA). Briefly, 40µl TMT reagents (0.8 mg TMT dissolved in 41 µl of acetonitrile) was added to the peptide solution and incubated for 1 h at room temperature. Subsequently, 5 µl of 5% hydroxylamine was added to terminate labeling reaction for 15 min at room temperature. Peptides were labeled with different TMT labels: TMT-126, wild type (WT); TMT-127, 3×Tg-AD (AD); TMT-131, 3×Tg-AD + donepezil (Donepezil). The peptides solution from WT, AD and Donepezil group were mixed, then dried under vacuum centrifuge. TMT labeled peptides mixture was fractionated with high pH reversed-phase peptide fractionation kit according to the manufacturer’s instructions (Thermo Scientific, NJ, USA). Briefly, peptide were dissolved with 300 µl 0.1% FA, and loaded onto an equilibrated, high-pH, reversed-phase fractionation spin column, followed by washing with step gradient of increasing acetonitrile concentrations to elute bound peptides into eight different fractions collected by centrifugation. Each fraction is then dried in a vacuum centrifuge, then dissolved with 0.1% FA for LC-MS/MS analysis. NanoLC-ESI-MS/MS analysis The lyophilized peptides fractions were re-suspended in 2% acetonitrile containing 0.1%formic acid, and loaded on ChromXP C18 (3 µm, 120 Å) trap column. The online Chromatography seperation was performed on the Eksigent nanoLC-Ultra™ 2D System (SCIEX, Concord, ON), the trapping, desalting procedure were carried out at 4µL/min for 5 min with 100% solvent A (water / acetonitrile / formic acid (98 /2 / 0.1%)). Then, an elution gradient of 5-38% solvent B (water / acetonitrile / formic acid (2 / 98 / 0.1%)) in 60 min was used on an analytical column (75 µm x 15 cm C18- 3µm 120 Å, ChromXP, Eksigent). Data acquisition was performed with a TripleTOF 5600+ Mass Spectrometry (SCIEX, Concord, ON) fitted with a Nanospray III source (AB SCIEX, Concord, ON). Data was acquired using an ion spray voltage of 2.4 kV, curtain gas of 35 PSI, nebulizer gas of 12 PSI, and an interface heater temperature of 150 . The MS was operated with TOF-MS scans. For IDA, survey scans were acquired in 250 ms and up to 40 product ion scans (80ms) were collected if exceeding a threshold of 160 cps with a charge state of 2-5. A Rolling collision energy setting was applied to all precursor ions for collision-induced dissociation. Dynamic exclusion was set for 14 s. Database searching and protein quantification Proteins identification and relative quantification was performed by PEAKS 8.5 software (Bioinformatics Solutions, Waterloo, Canada). The raw mass spectra were searched against UniProt-Mus musculus (Mouse) database containing 51697 protein entries (released in July 2017) with the following setting parameters: parent mass error tolerance of 30 ppm and fragment mass error tolerance of 0.1 Da. Setting

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enzyme: trypsin, fixed modifications including deamidation (NQ), oxidation (M), Pyro-glu from E, Pyro-glu from Q, Acetylation (Protein N-term) and maximum variable PTM per peptide: 3；The false discovery rate (FDR) was estimated at 1.0% at psm (peptide-spectrum matches) level determined by a decoy database search as implement in PEAKS 8.5; peptide score (−10lgP) greater than 19.5 (p＜0.01), which is equivalent to a P-value of ∼1%, were regarded as confidently identified. Relative quantification of peptides and proteins were performed by the TMT 6plex method in PEAKS Q module, all TMT reporter ion spectra area of identified peptides were summed and taken as the normalized factor across different samples. Statistical analysis of differentially abundant proteins was conducted using Peaks Q algorithm, expression of peptides and proteins were considered to be significantly different between samples when the ratio ≥1.2 or ＜0.83 and protein significance score ≥10. The ratios of altered proteins in each group were set as group AD/WT, group donepezil/WT. The ratio of changed proteins by donepezil treatment was set as group donepezil/AD. Bioinformatics analysis Proteins with altered expression levels were subjected to gene ontology (GO) annotation enrichment analysis using DAVID Bioinformatics resources 6.8. The pathway analysis with altered proteins was determined using KEGG (Kyoto Encyclopedia of Genes and Genomes) path database (https://www.kegg.jp/kegg/). Heml 1.0 and Graphpad 7.0 was used for heat map analysis. For the protein-protein interaction (PPI) network analysis, we used STRING database version 10.5 (http://string-db.org/). The interaction network was mapped by Cytoscape (3.6.0). Statistical analysis All values were expressed as mean ± SEM. Statistical analysis was performed using Graphpad Prism 7.0. Data of the behavior test was analyzed using the Student’s paired t-test with the significance level of p ≤ 0.05. A standard one-way ANOVA followed by Tukey HSD test was used for other cases.

RESULTS Donepezil treatment ameliorated cognitive impairments of 3×Tg-AD mice Accumulation of Aβ and phosphorylated-tau first occur in the hippocampus, amygdala, and cortex of the brain in 3xTg-AD mouse model. We therefore evaluated the cognitive deficits associated to Alzheimer’s disease (AD) by using behavioral tests relevant to these brain regions. The step-down avoidance (SDA) test was used to evaluate amygdala-dependent learning memory, the novel object recognition (NOR) test for cortex-dependent recognition memory, and the Morris Water Maze (MWZ) test for hippocampus-dependent spatial navigation and reference memory. Results from the SDA test showed that the step-down latency of 3×Tg-AD mice (AD) was

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significantly shorter than wild type mice (WT), and donepezil treatment in AD mice significantly increased the step-down latency back to normal level (Fig. 2A). Similarly, Fig. 2B showed that the number of errors made by AD mice was much higher than WT, and donepezil reduced the number of errors. Results from NOR test showed that the recognition memory (measured as Discrimination Index, DI) of AD mice was significantly lower than WT, and donepezil could improve the memory although significance was not achieved (Fig. 2C). In MWM test, it was observed that the AD mice exhibited much longer escape latency (the time took to find the platform) than WT mice during the training period, especially from Day 3 to 5, and donepezil significantly reduced the escape latency of AD mice (Fig. 3A). During the trial period on Day 6, the platform was removed to evaluate if the mice after training would spent more time searching in the target quadrant (where the platform was placed during the training) than in the other quadrants. The trace records (Fig. 3B) showed that AD mice spent much less time in the target quadrant when compared to WT mice and donepezil-treatment AD mice. The result showed that donepezil was effective at improving the spatial memory of AD mice, as shown by the trace that the donepezil-treated AD mice were swimming above the surface of the location where the platform once was. Since the mice with spatial memory of the platform during the training period would try to search for the platform, they would swim circling around in the target quadrant, as in the case of WT mice. The AD mice took significantly much longer time to find the target quadrant (Fig. 3C) and made much less visits to the target quadrant (Fig. 3D). Both parameters could be significantly improved with the treatment of donepezil. Taken together, these results indicated that donepezil significantly improved cognitive functions in 3xTg-AD mice, especially the hippocampus-dependent memory. Donepezil treatment reduced Aβ accumulation in 3xTg-AD mice We further evaluated the effects of donepezil on the accumulation of soluble and insoluble Aβ1-40 and Aβ1-42 in the brain tissues of 3xTg-AD (AD) mice by using ELISA assay. Donepezil significantly decreased the levels of soluble and insoluble Aβ1-40 in the hippocampus (Fig. 4A). Donepezil also significantly reduced the levels of soluble and insoluble Aβ1-42 in the hippocampus (Fig. 4B). We further evaluated the effects of donepezil on reducing senile plaque deposition in the hippocampus by using immunohistochemical staining. As shown in Fig. 4C, donepezil considerably reduced the deposition of Aβ plaques in the subiculum of the hippocampus in AD mice. Donepezil treatment was also effective in reducing the expression of total amyloid precursor protein (APP) as shown in Fig. S-1A. However, donepezil had no effect on the expression of phosphorylated-tau and total tau in 3xTg-AD mice (Fig. S-1B) (Supporting Information: Fig. S-1. Protein levels of (A) APP and (B) phosphorylated-tau in WT, AD, and AD + donepezil groups). Donepezil preserved hippocampal neuron dendritic spine density in 3xTg-AD mice

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In 3xTg-AD (AD) mice, the reduction of dendritic spine density in hippocampal neurons is associated with synaptic loss and cognitive declines. We further examined the effects of donepezil on the dendritic spine density of pyramidal neurons in the hippocampal by using Golgi staining. As shown in Fig. 5, the dendritic spine density was greatly decreased in AD mice. After the treatment with donepezil, the dendritic spine density was significantly preserved. The data suggested that donepezil was able to improve AD-related cognitive declines by preserving synaptic plasticity. Donepezil altered the hippocampal proteome of 3×Tg-AD mice Analysis of the hippocampal tissues of WT, 3×Tg-AD (AD), and donepezil-treated AD mice by TMT labeling proteomic approach showed that a total of 3131 proteins exhibited alterations in expression, with at least one unique peptide (false discovery rate (FDR) < 1%). Out of the identified proteins, 262 proteins were significantly modulated by the treatment of donepezil in AD mice as shown in Fig.6A, including 161 proteins were up-regulated and 101 proteins were down-regulated (with criteria ratio ≥1.2 or ＜0.83, p＜0.01) (Table S-1) The analysis further identified 40 of those proteins with dramatic changes in levels (fold change ≥1.5 or ＜0.67) (Fig. 6B) (Table S-2). Among them, 28 proteins were up-regulated, including PINK1, NFASC, MYLK2, and RASN, which have been implicated to be highly associated with AD. (Supporting Information: Table S-1. List of differentially altered proteins by the treatment of donepezil in AD mice) To further understand the biological characterization of these differentially expressed proteins, the up-regulated proteins (161 proteins) and down-regulated proteins (101 proteins) were analyzed according to Gene Ontology (GO) terms (biological process, molecular function and cellular component) (Fig. 7). The most over-represented terms of biological process for the up-regulated proteins were small GTPase mediate signal transduction, intracellular signaling cascade, and regulation of ARF protein signal transduction. For the down-regulated proteins, the most over-represented terms of biological process were calcium ion transport, cation transport and metal ion transport. The most over-represented terms of cellular component for the up-regulated proteins were kinesin complex, cytoskeleton part and microtubule cytoskeleton. For the down-regulated proteins, the most over-represented terms of cellular component were cation channel complex, plasma membrane part and ion channel complex. The most over-represented terms of molecular function for the up-regulated proteins were purine ribonucleotide binding, ribonucleotide binding, and purine nucleotide binding. For the down-regulated proteins, the most over-represented terms of molecular function were nucleotide binding, cation channel activity and purine nucleotide binding. KEGG pathway analysis was used to present these characteristics, where for up-regulated proteins the highly enriched terms included endocytosis, tight junction in

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up-regulated proteins. For down-regulated proteins, the highly enriched terms included Huntington’s disease, arrhythmogenic right ventricular cardiomyopathy (ARVC), and cardiac muscle contraction. To further evaluate the relationships among the differentially expressed proteins induced by donepezil treatment, protein-protein interaction (PPI) networks were constructed using STRING database interpreted with Cytoscape 3.6.0. (Fig. 8). Based on the 262 differentially expressed proteins identified in AD mice treated with donepezil, more than half of the proteins were connected with each other, many of which were involved in AD pathology, such as autophagy, mTOR signaling pathway, axon guidance, oxidative phosphorylation, and MAPK pathway. The results indicated that donepezil induced neuroprotective effects in AD mice via modulating multiple signaling pathways including stress-responsive and proliferative pathways. Identification of differentially expressed proteins by Western blot analysis We further conduced Western blot assays with the samples of hippocampal tissues to further confirm results from proteomic analysis. Base on proteomic analysis including protein level fold change and PPI network references, PTEN-induced putative kinase 1 (PINK 1) was selected for further study. The proteomic result of PINK 1 expression was significantly up-regulated after donepezil treatment compared with AD mice (ratio = 2.7, -10lgP=33.27, p

Hippocampal proteomic alteration in triple transgenic mouse model of

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