Deficit of RACK1 contributes to the spatial memory impairment via upregulating BECLIN1 to induce autophagy

    Deficit of RACK1 contributes to the spatial memory impairment via upregulating BECLIN1 to induce autophagy Jiejun Zhu, Xu Chen, Yun Song, Yuanyuan Zhang, Liming Zhou, Lihong Wan PII: DOI: Reference:

S0024-3205(16)30064-9 doi: 10.1016/j.lfs.2016.02.014 LFS 14699

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Life Sciences

Received date: Revised date: Accepted date:

3 August 2015 14 December 2015 5 February 2016

Please cite this article as: Zhu Jiejun, Chen Xu, Song Yun, Zhang Yuanyuan, Zhou Liming, Wan Lihong, Deficit of RACK1 contributes to the spatial memory impairment via upregulating BECLIN1 to induce autophagy, Life Sciences (2016), doi: 10.1016/j.lfs.2016.02.014

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Title page

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Deficit of RACK1 contribute to the spatial memory impairment via up regulating BECLIN1 to induce autophagy

Department of Pharmacology, West China School of Preclinical and Forensic Medicine,

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Jiejun Zhu1,2, Xu Chen3, Yun Song4, Yuanyuan Zhang1,2, Liming Zhou1,2, Lihong Wan1,2*

Sichuan University, Chengdu, Sichuan 610041, PR China

Sichuan University “985 project -- Science and Technology Innovation Platform for Novel

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Drug Development”, Sichuan University, Chengdu, Sichuan 610041, PR China Department of Psychiatry, Shandong Mental Health Center, Shandong University, Jinan,

Department of Neurology, Qianfoshan Hospital Affiliated to Shandong University, Jinan,

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Shandong 250014, PR China

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Shandong 250014, PR China

Correspondence should be addressed to

Professor Lihong Wan, Department of Pharmacology, 3-17 Renmin South Road, West China School of Preclinical and Forensic Medicine, Chengdu, Sichuan 610041, P.R. China E‑mail: [email protected] Tel: 86-28-85501278

E-mail: JiejunZhu, [email protected]; Xu Chen,[email protected];

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Yun Song, [email protected];

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Yuanyuan Zhang, [email protected]; Liming Zhou, [email protected];

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Lihong Wan, [email protected]

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Abstract

Aims: Deficiency of activated C kinase1 (RACK1) in the brain of aging animal and Alzheimer

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diseases’ was characterized by cognitive dementia and spatial memory impairment. However,

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the correlation between the RACK1 and spatial memory impairment and the mechanism

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involved in it remains unknown.

Main methods: Spatial memory impairment was performed in mice by lateral ventricle injection of Aβ25-35 (n=16, 10 µl) and intraperitoneal injection of scopolamine (n=16, 10 ml/kg). After the Morris water maze (MWM) which was performed to determine the ability of learning and memory in mice, expression of RACK1 was tested and the damage of hippocampus was confirmed by histopathology test. ShRACK1 was then used to decrease the level of RACK1 in hippocampus to test the ability of learning and memory and histopathology changes in hippocampus. To look into the mechanism of RACK1 on spatial memory impairment, we further measured the expression of autophagy proteins BECLIN1 and LC3-II/I in hippocampus of all mice. Key findings: Both the Aβ25-35, scopolamine impaired the spatial memory in mice (for escape latency, P=0.0004, P<0.0001) and severely damaged hippocampal DG neurons (P=0.012,

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P=0.014). The expression of RACK1 was significantly decreased which was concomitant with

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elevated BECLIN1 and LC3-II/I (P<0.001). Suppression of RACK1 by ShRACK1 plasmid (shGnb2l1) significantly impaired the spatial memory in mice, damaged hippocampal DG

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neurons (P=0.013), and increased the proteins of BECLIN1 and LC3-II/I (P<0.005).

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Significance: It demonstrated that the deficit of RACK1 in hippocampus impairs the ability of

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learning and memory in mice via up regulating autophagy.

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Key words: Learning and memory; RACK1; BECLIN1; LC3

Introduction

Alzheimer's disease (AD) is a progressive, neurodegenerative disorder accompanied with the impairment of learning and memory, especially spatial memory impairment (Palmer 2002). It has been estimated that AD patients in 2050 will be 13.8 million in the United States, almost 3-fold compared with the number in 2000 (Hebert et al., 2003; Hebert et al., 2013). Accompanied with the evident increase of AD patients is the huge economic burden (Costa et al., 2013). Despite the distinct evidence of fibrillar amyloid-β (Aβ) protein, neurofibrillary tangles (NFTs) and loss of cholinergic neuron in hippocampus and cortex (Salthouse 2003; Mohapel et al., 2005; Reddy and

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McWeeney 2006), mechanism applicable for clinical target remains deficient. Current studies

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indicated a possibility of activated C kinase1 (RACK1) protein may play an important role in the spatial memory impairment, and thus a promising target for AD treatment (Battaini et al., 1997;

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Liu et al., 2011; McCahill et al., 2002).

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RACK1, a scaffolding/anchoring protein with 7 WD repeats, was first characterized as an intracellular receptor involved in anchoring activated Protein Kinase C (PKCs) to relevant

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subcellular compartments (Mochly-Rosen et al., 1991). It is ubiquitously expressed in the brain,

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especially at higher levels in memory related brain area, such as hippocampus, cortex, and cerebellum (Ashique et al., 2006). As a shuttling protein, RACK1 has been observed to form the

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protein complex via interacting intraneuronally with AchE-R and PKCβII (Birikhet al., 2003).

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Simultaneously, it has been reported that expressions of RACK1 were significantly decreased in

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the brain of aging animals and AD patients (Battainiet al., 1999; Van der Zeeet al., 2004; Pascaleet al., 1996). Our previous study also indicated that the increase of RACK1 in hippocampus by administration of compound danshen tablet can obviously protect the mice from the spatial memory impairment induced by Aβ25-35. (Teng et al., 2014). Taken together, these findings suggested that RACK1 may play a role in spatial memory impairment and in the regulation of signaling events required to protect neurons. However, the exact association and potential mechanism of RACK1 on spatial memory impairment have not yet been elucidated. Currently, evidence suggested that endogenous RACK1 partially co-localized with early autophagic structures and involved in autophagosome formation in Drosophila (Erdi et al., 2012). Simultaneously, it has been indicated that autophagy plays an important and dual role in central neural system (CNS). Abundant studies reported that excess autophagic activity in brain promoted neuronal cell death and implicated in the pathological changes of dementia and

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cognitive deficits (Liu et al., 2014; Chen et al., 2014). On the other hand, it was also shown that

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inhibition of autophagy induces degeneration of neuronal cells in central nervous system (Hara et al., 2006; Komatsu et al., 2006; Li et al., 2013). So we hypothesized that RACK1 plays

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important roles in spatial memory impairment of AD through regulating autophagy, and may

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serve as a therapeutic target of AD.

Besides i.c.v administration of Aβ25-35, i.p administration of scopolamine, a muscarinic receptor

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antagonist, is another common used method in AD study (Bajo et al., 2015). To elucidate the

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potential effects and mechanism of RACK1 on spatial memory impairment, we evaluated learning and memory in mice using the Morris water maze test, and assessed autophagy markers

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Animals

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Materials and Methods

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(BECLIN1 and LC3-II/I) in mice exposed to Aβ25-35, scopolamine and ShRACK1.

Male Kunming mice, 8-10 weeks old, weighing 38-42 g, were obtained from Chengdu Dashuo Experimental Animal Co., Ltd, and group-housed in standard conditions with laboratory rodent chow and tap water ad libitum. The holding room was under standard conditions of ambient temperature (23±2°C), humidity (55±5%), and with a 12-h light/dark cycle (lights on at 8:00 AM) throughout the whole study. Animal experiments were approved by the Committee on the Ethics of Animal Experiments of Sichuan University. Aged Aβ25-35 Peptide Preparation Amyloid β-protein fragment 25-35 (Aβ25-35) was purchased from Beijing Biosynthesis Biotechnology Co, LTD (Beijing, China). The Aβ25-35 was dissolved in sterile saline at a concentration of 2 g/L and incubated at 37°C for 7 days to allow for fibril formation as described previously (Teng et al., 2014).

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Plasmid

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The plasmid of shRACK1 (shGnb2l1) and the control plasmid (vehicle) were synthesized by Shanghai GenePharma Co., Ltd. The sequences of shRACK1 (shGnb2l1) are 5′-

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CACCGCCCACTTCGTTAGTGATGTTGTTCAAGAGACAACATCACTAACGAAGTGGGT

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TTTTTG (forward) and 5′-

CAAAAAACCCACTTCGTTAGTGATGTTGTCTCTTGAACAACATCACTAACGAAGTGG

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GCGGTG-3′ (reverse). The sequences of vehicle (control plasmid) are 5-

TGGATCCACT-3′ (forward) and 5′-

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CACCGTTCTCCGAACGTGTCACGTCAAGAGATTACGTGACACGTTCGGAGAATTTTT

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AGTGGATCCAAAAAATTCTCCGAACGTGTCACGTAATCTCTTGACGTGACACGTTCG

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GAGAACGGTG -3′ (reverse). Before injection, plasmid DNA or vehicle was diluted in 10%

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glucose to a stock concentration of 0.609 mg/ml. In vivo-jet PEI® (Polyplus-transfection SA, France) was diluted to 0.1M concentration in 10% glucose and added to the DNA solution. Final concentration of shRACK1 (shGnb2l1) or vehicle for injection is 0.28 µg/ul. Morris water maze (MWM) The MT-200 Morris water maze system was purchased from Chengdu TME Technology Co, Ltd, (Chengdu, China), and used to assess the spatial learning and memory abilities in mice before and after the animal treatment. The swimming pool (100 cm in diameter, 50 cm in height) filled with water at 12 cm depth was divided into four quadrants. The water, kept at a steady 25±2℃, was made opaque with white nontoxic dye. The quadrant housing the escape platform (10 cm diameter) which was fixed in a permanent position 2 cm under the water surface during the course of the MWM, was defined as the goal quadrant.

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The MWM includes two part i.e. place navigation trial and spatial probe trial. In place navigation

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trial, the mice were dropped into water facing the poll wall and allowed to swim until it finds the hidden platform in 60 s. If the mouse cannot find the platform in 60 s, it was gently guided to the

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platform and recorded the escape latency as 60 s. With or without the help of experimenters,

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mice were allowed to rest at the platform for 10s. The place navigation trial was conducted for three trials daily, with an inter-trial interval of 15 min. The escape latency record was calculated

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as average of 3times for each day. A spatial probe trial was performed the day after the last place

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navigation trial. The platform was moved away and the mouse was dropped into water from the opposite position of the platform and was allowed to swim freely for 60 s. During the probe trial,

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the number of platform crossings and the time spent in the goal quadrant were recorded.

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Animal treatment

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By performing the place navigation trial for four consecutive days and a spatial probe trial after that, we screened 80 mice with the ability to find the platform in 60 s and healthy postures from 104 mice. These mice were randomly divided into six groups: the Aβ25-35 control group (n=8, saline i.c.v, 10 µl per mouse, 7 days before MWM), the Aβ25-35 group (n=16, 2 mg/ml Aβ25-35 i.c.v, 10 µl per mouse, 7 days before MWM), the scopolamine control group (n=8, saline i.p, 10 ml/kg, 30 min before trail during the MWM), scopolamine group (n=16, 0.1 mg/ml scopolamine i.p, 10 ml/kg, 30 min before trail during the MWM ), vehicle group (n=16, vehicle plasmid i.c.v, 10 µl per mouse) and shRACK1 group (n=16, ShRACK1 plasmid i.c.v, 10 µl per mouse). Mice in the Aβ25-35 control group and the Aβ25-35 group were anaesthetized with 3%chloral hydrate (0.13 ml/10g) and subjected to lateral ventricle injection of 0.9% saline 10µl and 2 mg/ml Aβ25-35 10 µl respectively. The injection was performed for only once and mice were recovered for 1 week before the behavioral assessment. Mice in vehicle group and ShRACK1

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group were treated the same way the Aβ25-35 group did except that these mice were injected 4 µl

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either vehicle or shRACK1 (0.28 µg/ul) and the injection procedure was performed for 3 times with the interval of 2 days. Mice in the scopolamine control and scopolamine group were

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administrated saline or 1mg/kg scopolamine (10 ml/kg) i.p 30 min before the trail for

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consecutive 5 days respectively. Histopathology

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After the probe trial, all mice were allocated into two subgroups randomly and sacrificed.

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The brain tissues were removed immediately. Brains in one subgroup of each group were fixed in 4% paraformaldehyde for histopathology assay. The hippocampus of the mice in

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the other subgroup were rapidly removed, snap frozen in liquid nitrogen, and stored at

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−80°C for further use. Coronal sections (4 µm) through the brain were embedded in

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paraffin, and stained with hematoxylin and eosin (H&E) for microscopic evaluation of neuronal damage. Three sections from each hippocampus were used for the morphometric analysis. Light-microscope images were photographed, and the total pyramidal cell numbers per millimeter in the hippocampus and in the subregions dentate gyrus (DG) were measured on the photographs, and then averaged to a single value. Data were calculated as average of six slices for each group. Real Time-PCR Total RNA (1µg), obtained from the hippocampus using E.Z.N.A.® Total DNA/RNA/Protein Kit (Omega Bio-TekInc, Norcross, GA), was subjected to reverse transcription with a Revert Aid First Strand cDNA Synthesis Kit (Thermo scientific Inc, MA, USA) according to instructions of manufacturers. Realtime RT-PCR was performed with CFX96TM Real-Time PCR Detection System, and SYBR@Premix Ex TaqTM II (Tli RnaseH Plus) (Takaro Bio Inc, Tokyo, Japan).

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Primers for gapdh were used in control reactions. The primers sequences and conditions were

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listed in Table1. Western blot analysis for RACK1, BECLIN1 and LC3II/I level

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The total protein of hipocampus was extracted by E.Z.N.A.® Total DNA/RNA/Protein

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Kit (Omega Bio-Tek Inc, Norcross, GA). Protein concentrations were determined using the BCA protein assay kit (Pierce Biotechnology, Rockford, IL). Equal amounts of

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proteins(40 µg) were loaded to 15% SDS-PAGE gels, and separated by electrophoresis

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(80V, 200 min), and then transferred to polyvinylidenedifluoride (PVDF) membranes (0.45 nm) (Millipore, Billerica, MA). Then the membrane was incubated separately with

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mouse monoclonal anti-RACK1 antibody(Santa Cruz Biotechnology, Santa Cruz, CA),

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rabbit polyclonal anti-LC3-I/II antibody(Sigma-Aldrich, St Louis, MO), or mouse

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monoclonal anti-β-actin antibody(ZSGB-BIO, Beijing, CHN) as internal control for 3h at room temperatures, or with rabbit monoclonal anti-Beclin1 antibody (Cell Signaling Technology, MA, USA) as internal control for 5h at room temperature. Membranes were washed with three times with Tris buffered saline with Tween-20 (TBST), and incubated with horseradish peroxidase-conjugated goat anti-rabbit (ZSGB-BIO, Beijing, CHN) or goat anti-mouse (ZSGB-BIO, Beijing, CHN) IgG for 1h at room temperature. All proteins were detected using Western Lightning TMC hemiluminescence Reagent Plus, and results were quantified by scanning densitometry using the Kodak IS4400CF image analysis system and the corresponding software (Eastman Kodak, Rochester, NY). Statistical analysis Relationship between escape latency and RACK1 level in hippocampus was assessed using Pearson correlations. Simultaneously, the Pearson’s r correlation index was also used to ascertain

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the degree of correlation between RACK1 and Beclin1 and LC3-II/I protein in hippocampus in

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mice. The mean escape latency were analyzed by one-way ANOVA with a repeated-measure factor of sessions (number of days) followed by the least significant difference testing. Linear

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regression was applied to assess the correlation of two variables by χ2 test. The significance of

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difference was evaluated by ANOVA using SPSS softwarev.13.0. The statistical significances of the other data were analyzed using ANOVA followed by least significant difference testing.

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Difference of P<0.05 were considered statistically significant.

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Results

Aβ25-35 impaired the spatial memory, decreased expression of RACK1, and

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increased expressions of BECLIN1 and LC3-II/I

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ANOVA for repeated measures was conducted after Mauchly's test of sphericity (F(1)=3.77,

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P=0.0049) in escape latency. Escape latency of Aβ25-35 group from day 1 to 5 was significantly increased compared with that of control group in the spatial navigation trail (F(1, 60)=24.23, P=0.0004, Fig 1A). In the probe trail, mice of Aβ25-35 group took less times crossing the location of the platform, compared to the control group (F(1, 14)=65.68, P=0.0001, Fig 1B). Moreover, the time spent in the target quadrant of the Aβ25-35 group was significantly shorter than that of the control group (F(1, 14)=29.99, P<0.0001, Fig 1C). Histopathological findings revealed a significantly reduced number of neurons, especially in DG, with much more nuclear pyknosis, nucleolus loss, and triangulated neuronal body than that of sham group (P=0.0102; P=0.0124, respectively, Figure 1E and 1D). The realtime RT-PCR and Western blot (WB) showed a significant reduction on the mRNA and protein expressions of RACK1 in hippocampus of mice of Aβ25-35 group (P<0.05, Fig 1F and 1G), as compared to control mice. Additional analyses revealed a negative correlation between

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the escape latency and RACK1 protein in hippocampus of mice of Aβ25-35 group (r2=0.753,

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P<0.01, Fig 1H). Using WB, we further examined the expression of BECLIN1 and LC3-II/I. We observed that the

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protein levels of Beclin11 and LC3-II/I in hippocampus of mice of Aβ25-35 group were markedly

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increased (P<0.05, Fig 1I and 1J) than that of control mice. Protein levels of BECLIN1 and LC3II/I were both observed to be negatively correlated with RACK1 protein level (BECLIN1:

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r2=0.688 and LC3-II/I: r2=0.753, P<0.01, respectively, Fig 1K and 1L).

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Scopolamine also impaired the spatial memory, decreased expression of RACK1, and increased expressions of BECLIN1 and LC3-II/I

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Similar results were obtained in scopolamine induced mice. There were significant differences in

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escape latency between groups in the spatial navigation trail between scopolamine control group

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and scopolamine group (F(1, 36)=7.68, P=0.0088, Fig 2A) from day 2 to 5. In the probe test, mice of the scopolamine group took less times crossing the position of the platform, compared to the control group (F(1, 14)=9.06, P=0.0094, Fig 2B). Moreover, the time spent in the target quadrant of mice of the scopolamine group was significantly shorter than that in control group (F(1, 14)=11.65,

P=0.0042, Fig 2C). It was observed that the number of neurons reduced, especially in

DG, with much more nuclear pyknosis, nucleolus loss, and triangulated neuronal body in mice of the scopolamine group than that of sham group (P=0.0175; P=0.0148, respectively, Figure 2D and 2E). The mRNA and protein expression of RACK1 in hippocampus of mice of the scopolamine group were also significantly compared to that of control group (P<0.05, Fig 2F and 2G). A negative correlation (r2=0.636, P<0.01) between the escape latency and RACK1 protein in hippocampus of mice of the scopolamine group was revealed (Fig 2H). Moreover, scopolamine increased the protein levels of BECLIN1 and LC3-II/I in hippocampus (P<0.05, Fig

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2I and 2J), which were negative correlated with RACK1 protein level (BECLIN1: r2=0.463 and

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LC3-II/I: r2=0.533, P<0.01, respectively, Fig 2K and 2L). ShRACK1 impairs spatial memory in mice via enhances the expression of BECLIN1

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and LC3-II/I in hippocampus

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To confirm the role of RACK1 in spatial memory impairment, the mice were transfected with ShRACK1 plasmid and control ShRNA (vehicle). Realtime RT-PCR and WB analyses

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confirmed the significantly inhibition of the mRNA and protein levels of RACK1 (P<0.05, Fig

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3A and 3B). There were significant differences in escape latency between groups in the spatial navigation trail between shRNA control group and ShRACK1 group (F(1, 70)=6.119, P=0.04, Fig

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3D) from day 3 to 5. In the probe test, mice of the ShRACK1 group took less times crossing the

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position of the platform, compared to the control group (F(1, 28)=40.52, P<0.0001, Fig 3E).

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Moreover, the time spent in the target quadrant of mice of the ShRACK1 group was significantly shorter than that in control group (F(1, 28)=13.09, P=0.0028, Fig 3F). A severe neuronal degeneration of mice of the ShRACK1 group was also observed in hippocampus, especially in DG (P=0.0005; P=0.014, respectively, Fig 3G and 3H). In addition, the protein expression of BECLIN1 and LC3-II/I in hippocampus were significantly higher in mice of the ShRACK1 group, compared with control mice (P<0.05, Fig 3C). Discussion The major finding of the present study was that deficiency of RACK1 protein in hippocampus was implicated with the spatial memory impairment, and the elevated BECLIN1 and LC3-II/I levels in two memory impaired animal models (induced by Aβ25-35 or scopolamine). Down regulation of RACK1 by ShRNA can activate autophagy related proteins (BECLIN1 and LC3II/I) to injury the neuron in DG, and finally resulted in the spatial memory impairment.

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A great deal of evidence indicated that excessive accumulation of the Aβ peptide and deficiency

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of the brain cholinergic system were the leading causes of memory impairment (Reddy and McWeeney2006; Han et al., 2013; Lee et al., 2014). To comprehensively evaluate the

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relationship of RACK1 deficiency with memory impairment, we choose two AD mice models

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(induced by Aβ25-35 or scopolamine), and analyze the association of RACK1 level and autophagy related proteins. By intracerebroventricular infusion the active fragment of Aβ protein, Aβ25-35,

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we induced spatial learning and memory deficits in mice, as we previously reported (Teng et al.,

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2014). Since RACK1 was deficient in the brain of AD patients (Battainiand Pascale2005), we speculated that Aβ25-35 induced spatial memory impairment might be associated with a decrease

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in the expression of RACK1 in hippocampus. Our results demonstrated that both RACK1 mRNA

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and protein levels were significantly decreased in Aβ25-35 induced AD mice, which was

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consistent with previous reports (Battainiet al., 1999; Van der Zeeet al., 2004; Pascaleet al., 1996; Teng et al., 2014). Moreover, we found that RACK1 protein level was positively correlated with memory function, with lower expressers showing worse memory. Autophagy is an evolutionarily conserved lysosome-dependent degradation process that has been implicated in age-associated diseases (Nixon RA., 2013). However, the role of autophagy in central nervous system remains controversial. To elucidate the precise role of autophagy in Aβ2535

induced dementia mice, we determined expressions of two autophagy markers (BECLIN1 and

LC3-I/II) in hippocampus of AD mice. BECLIN1, a Bcl-2-homology (BH)-3 domain only protein, plays important role in the autophagosome formation and autolysosome fusion, which is located in neurons and astrocytes (Liang et al.,1998) in the cerebral cortex, hippocampus and cerebellum (Yue et al., 2002) in the adult mammal brain. Up-regulation of Beclin1 indicated increases of autophagy (Lu et al., 2011). The microtubule-associated protein 1 light chain 3

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(LC3) is another marker of autophagy, which exists as native cytosolic form of light chain 3-I

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and membrane-bound lapidated light chain 3-II (Kanno et al., 2011). The transformation of LC3I to LC3-II is regarded as an important process of auto-phagosome formation (Klionsky et al.,

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2007). And the LC3-II/I ratio is associated with the number of autophagic vacuoles (Alonso et

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al., 2007). It was demonstrated that the BECLIN1 and LC3-II/I ratio was greater in the hippocampus of Aβ25-35 induced dementia mice than in the sham-operated mice. Interestingly,

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both of BECLIN1 level and LC3-II/I ratio were negatively correlated with RACK1 protein level

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in hippocampus. Our results indicated that activated autophagy accompanied with loss of RACK1 in the hippocampus of dementia mice was involved in the Aβ25-35 induced memory

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impairment.

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Our results on two murine AD models confirmed that autophagy was activated in the

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hippocampus of AD mice, which was associated with RACK1 protein level. Therefore, we speculated that both Aβ25-35 and scopolamine down-regulated RACK1 expression, leads to activation of autophagy, and damage of DG neuron, which resulted in learning and memory impairment.

To confirm our hypothesis, we treated the AD mice with shRNA specific for RACK1. Our data showed that there was a 30% decline in mRNA expression and 70% decline in protein level for the RACK1 in hippocampus after treatment with shRack1. In addition, reducing RACK1 expression by shRack1 markedly enhanced BECLIN1 level and LC3-II/I ratio in the hippocampus of AD mice. We also observed extended escape latency, and delayed in learning and serious neuronal damage in DG, suggesting that RACK1 may play a role primarily in learning memory via inhibiting autophagy.

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However, there are some limitations in this experiment. This is a preliminary study about the

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relationship of RACK1 and spatial memory, in future study, we will further demonstrate the potential cellular and molecular substrates might be involved in this process.

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Conclusions

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Our study demonstrated that the deficit of RACK1 in hippocampus impairs the ability of learning and memory in mice via up regulating autophagy. Altering the RACK1 signaling pathway and its

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effects on autophagy may represent a new target against memory impairment.

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Acknowledgements

This study was supported by the National Nature Science Foundation of China (No. 81100989 to

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Lihong Wan).

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Conflict of Interest

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The authors declare that there are no conflicts of interest. Authors Contribution

Lihong Wan was responsible for the study concept and design. Jiejun Zhu conducted behavioural and molecular biology procedures. Xu Chen and Yun Song performed the statistical analyses. Lihong Wan undertook the manuscript preparation. Yuanyuan Zhang and Liming Zhou revised the manuscript. All authors critically reviewed content and approved final version for publication. References Alonso, S., Pethe, K., Russell, D.G., Purdy, G.E., 2007. Lysosomal killing of Mycobacterium mediated by ubiquitin-derived peptides is enhancedby autophagy. Proc. Natl. Acad. Sci. U. S. A. 104, 6031-6036.

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Figure legend

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Fig.1 Aβ25-35 induced spatial memory impairment, neuronal damage, the decreased expression of RACK1 and the increased expression of autophagy protein BECLIN1 and

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LC3II/I in mice.

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(A) Aβ25-35 reduced the escape latency in mice in the place navigation trial of the Morris water

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maze (F(1, 60)=24.23,P=0.0004); (B) Aβ25-35 decreased the number of platform crossings in mice in the probe trial(F(1, 14)=65.68, P=0.0001); (C) Aβ25-35 attenuated the time spent in goal quadrant in mice in the probe trial (F(1, 14)=29.99, P<0.0001); (D) Large numbers of damaged neurons were seen in the DG of the hippocampus in Aβ25-35 treated mice (a and b, control group; c and d, Aβ25-35 group). HE staining of hippocampus (a and c: Magnification×100) and pyramidal cells in hippocampal DG region (b and d: Magnification×400), the arrowheads represents the nuclear pyknosis; (E) Counts of the total number of cells in the hippocampus and DG (P=0.0102; P=0.0124). (F) The mRNA levels of RACK1 were reduced in hippocampus of Aβ25-35 treated mice (P=0.0357); (G) The protein levels of RACK1 in hippocampus analyzed with western blotting (upper panel); Lower intensity of the RACK1/actin band in hippocampus of Aβ25-35 treated mice (lower panel) (P=0.0475); (H) Negative correlation of RACK1 protein levels with escape latency (r2=0.753, P<0.05); (I) The protein levels of BECLIN1 and LC3-II/I in

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hippocampus analyzed with Western blotting; (J) Higher intensity of the BECLIN1/actin and LC3-II/I/actin band in hippocampus of Aβ25-35 treated mice (P=0.009; P=0.03); (K) Negative

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correlation of expressions between Beclin1 and RACK1 protein (r2=0.688, P<0.05); (L)

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Negative correlation between LC3-II/I ratio and RACK1 protein level (r2=0.753, P<0.05). Data

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are expressed by the mean ± sem, *P<0.05 compared with control group. Fig.2 Scopolamine coursed spatial memory impairment, neuronal damage, the decreased

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expression of RACK1 and the increased expression of autophagy protein BECLIN1 and

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LC3II/I in mice.

(A) Longer time of escape latency in the scopolamine treated mice in the place navigation trial of

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the Morris water maze (F(1, 36)=7.68, P=0.0088); (B) Less numbers of platform crossings in the

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scopolamine treated mice in the probe trail (F(1, 14)=9.06, P=0.0094); (C) Less time spent in the

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scopolamine treated mice in the probe trail (F(1, 14)=11.65, P=0.0042); (D) Large numbers of damaged neurons were seen in the DG of the hippocampus in scopolamine treated mice (a and b, control group; c and d, scopolamine group); HE staining of hippocampus (a and c: Magnification×100) and pyramidal cells in hippocampal DG region (b and d: Magnification×400), the arrowheads represents the nuclear pyknosis; (E) Counts of the total number of cells in the hippocampus and DG (P=0.0175; P=0.0148). (F) The scopolamine treated mice showed a reduced mRNA level of RACK1 in hippocampus (P=0.0387); (G) The protein levels of RACK1 in hippocampus analyzed with western blotting (upper panel); The scopolamine treated mice showed a lower intensity of the RACK1/actin band compared with the control groups (lower panel) (P=0.039); (H) Negative correlation of RACK1 protein levels with the escape latency (r2=0.636, P<0.05); (I) The protein levels of BECLIN1 and LC3-II/I in hippocampus analyzed with Western blotting; (J) The scopolamine treated mice showed a higher

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intensity of the BECLIN1/actin and LC3-II/I/actin band compared with the control groups (P=0.03; P=0.005); (K) Negative correlation of expressions between BECLIN1 and RACK1

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protein(r2=0.463, P<0.05); (L) Negative correlation between LC3-II/I ratio and RACK1 protein

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level (r2=0.533, P<0.05). Data are expressed by the mean ± sem, *P<0.05 compared with control

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group.

Fig.3 RACK1 negatively regulate autophagy related protein level in hippocampus involves

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in spatial memory impairment.

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(A)Treated with ShRACK1 plasmid decreased the mRNA level of RACK1 in hippocampus (P=0.01); (B) The protein levels of RACK1 analyzed with Western blotting (upper panel);

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Treated with ShRACK1 plasmid reduced the protein level of RACK1 in hippocampus (lower

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panel) (P=0.003); (C)The protein levels of BECLIN1 and LC3-II/I in hippocampus analyzed

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with Western blotting (upper panel); Treated with ShRACK1 plasmid augmented the intensity of the BECLIN1/actin and LC3-II/I/actin band (P=0.0003; P=0.05) (lower panel); (D)Treated with ShRACK1 plasmid attenuated escape latency in spatial navigation trials of the Morris water maze (F(1, 70)=6.119, P=0.04); (E) Treated with ShRACK1 plasmid lessened the number of platform crossings in probe trials of the Morris water maze (F(1, 28)=40.52, P<0.0001); (F) Treated with ShRACK1 cut down the time spent in goal quadrant (F(1, 28)=13.09, P=0.0028); (G) Larges number of damaged neurons were seen in the DG of the hippocampus in ShRACK1 treated mice (a and b, control plasmid group; c and d, ShRACK1 group); HE staining of hipocamppus (a and c: Magnification×100) and pyramidal cells in hippocampal DG region (b and d: Magnification×400), the arrowheads represents the nuclear pyknosis (d); (H) Counts of the total number of cells in the hippocampus and DG (P=0.0005; P=0.014). Data are expressed the mean ± sem, *P<0.05 compared with control group.

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Table1 Primer sequence and condition for Real Time-PCR Forward（5’to 3’

Reverse (5’ to

）

3’)

PCR product Condition

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Gene

length (bp)

GGGTGGTCCA 5s, 60°C or 55°C

gadph GGGTTTCTTA

186

30s) for 39 cycles,

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GCGACTTCA

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GGTTGTCTCCT

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95°C 30s， (95°C

GGATCTCAATG

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melt curve.

TTGCTGCTGG

Gnb2l1

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TGCTGATAAC

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AAGGCAAGC

95°C 30s, (95°C 5s, 60°C 30s) for 183 39 cycles, melt curve.