METHOD TO RESTORE OR IMPROVE COGNITIVE FUNCTIONS

Abstract

The present invention relates to the field of memory and cognitive functions. Here the inventors show that memory stimulations induce autophagy in the mouse hippocampus, while local pharmacological and genetic modulations of hippocampal autophagy strongly influence memory acquisition. More, the inventors observe that hippocampal autophagy declines during aging and they find that restoring autophagy specifically in the hippocampus of aged mice, following autophagy inducers (such as TAT-Beclin-1), can significantly reverse age-related memory decline. Their results reveal a novel physiological role of autophagy in regulating hippocampal-dependent memory functions, and demonstrate the potential therapeutic benefits of modulating autophagy in order to prevent and/or reverse the deleterious effects of aging on cognitive function. The present invention relates to an activator of the autophagy for use in the restoration and/or improvement of cognitive functions in a subject in need thereof.

Claims

1. (canceled)

2. (canceled)

3. (canceled)

4. (canceled)

5. The method of claim 11, wherein the activator is selected from the group consisting of Earle's balanced salt solution (EBSS), Brefeldin A, Thapsigargin, Tunicamycin, Rapamycin, CCI-779, RAD001, AP23576, Small molecule enhancers rapamycin (SMER), Trehalose, L-690,330, Carbamazepine, Valproic acid sodium salt, N-Acetyl-D-sphingosine (C2-ceramide), Penitrem A, Calpastatin, Xestospongin B, Akebia saponin, Amiodarone hydrochloride, ATG13, GF 109203X synthetic, GF 109203X hydrochloride, N-Hexanoyl-D-sphingosine, MRT68921 dihydrochloride, Niclosamide, Qc1, Rottlerin, STF-62247, Tamoxifen, Temsirolimus, ULK Active, Z36 and Hydroxycitrate.

6. The method of claim 11, wherein the activator is Beclin 1.

7. The method of claim 11, wherein the activator is a peptide comprising an amino acid sequence of formula (I) (SEQ ID NO: 2):
Xaa1-N-A-T-F-Xaa2-Xaa3-Xaa4-Xaa5, wherein: Xaa1, Xaa2, Xaa3, Xaa4 and Xaa5 are each an amino acid independently selected from the group consisting of Alanine (A), Arginine (R), Asparagine (N), Aspartic acid (D), Cysteine (C), Glutamic acid (E), Glutamine (Q), Glycine (G), Histidine (H), Isoleucine (I), Leucine (L), Lysine (K), Methionine (M), Phenylalanine (F), Proline (P), Serine (S), Threonine (T), Tryptophan (W), Tyrosine (Y), Valine (V), allyl glycine (AllylGly), norleucine, norvaline, biphenylalanine (Bip), citrulline (Cit), 4-guanidinophenylalanine (Phe(Gu)), homoarginine (hArg), homolysine (hLys), 2-naphtylalanine (2-Nal), ornithine (Orn) Of and pentafluorophenylalanine.

8. The method of claim 7 wherein the activator is the peptide TAT-Beclin 1 of SEQ ID NO:37.

9. (canceled)

10. (canceled)

11. A method of i) restoring and/or improving one or more cognitive functions, ii) treating a cognitive disease, iii) preventing or reversing the deleterious effects of aging on cognitive function, and/or iv) restoring and/or improving age-related memory decline or age-related memory loss in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an activator of autophagy.

Description

FIGURES

[0128] FIG. 1: Acute modulation of hippocampal autophagy influences novel memory acquisition. A) LC3-I and LC3-II accumulation (Western blot) in 3 month-old mouse hippocampus 12 hours after stereotactic injections of pharmacological autophagy modulators: TAT-Beclin 1 or TAT-Scramble. The quantification is relative to the corresponding vehicle-treated group. B) NOR performed in 3 month-old mice after stereotactic injections with either vehicle TAT-Beclin 1 or TAT-Scramble mice. Preference index (Exploration time for the novel object/Total exploration) and discrimination index ([Exploration time for the novel object−Exploration time for the familiar object]/Total exploration) were measured for each group during the testing phase. C) CFC performed in 3 month-old mice after stereotactic injections with either vehicle, TAT-Beclin 1 or TAT-Scramble mice. Percent freezing was measured for the last minute of the training phase and the 4 min of the testing phase. All behavioral tests represented here were performed on at least two independent experiments (for each experiment: n≥5 mice per group). Data are expressed as mean±SEM. One-way ANOVA followed by Student's t test was used. *P≤0.05, **P≤0.01, ***P≤0.001, NS: not significant

[0129] FIG. 2: Maintenance of hippocampal autophagy level is necessary to counteract normal age-related memory decline and to mediate the beneficial effects of young circulating factors. A) VPS34, Beclin 1, ATGS, LC3-I and LC3-II accumulation (Western blot) and Vps34, Becn 1 and Atg5 relative expression (qPCR). B) LC3-I and LC3-II accumulation (Western blot) twelve hours after hippocampal stereotactic injections of either vehicle or TAT-Beclin 1 (1 μg/hemisphere) in 16 month- and 3 month-old mice. TAT-Beclin 1 restored LC3-II levels in old (16 months) to the level of young (3 months) mice. β-Actin was used as a loading control for each sample. C) Object location memory (OLM) task performed in 3 month- and 16-month old mice 12 hours after hippocampal stereotactic injections with either vehicle, TAT-Scramble (1 μg/hemisphere) or TAT-Beclin 1 (1 μg/hemisphere). Discrimination index ([time spent with newly located object−familiar located object]/Total exploration) was measured for each group during the testing phase. D) Novel object recognition (NOR) performed in 3 month- and 16 month-old mice 12 hours after hippocampal stereotactic injections with either vehicle, TAT-Scramble (1 μg/hemisphere) or TAT-Beclin 1 (1 μg/hemisphere). Discrimination index ([time spent with newly located object−familiar located object]/Total exploration) was measured for each group during the testing phase. E) Contextual fear conditioning (CFC) performed in 3 month- and 16 month-old mice 12 hours after hippocampal stereotactic injections with either vehicle, TAT-Scramble (1 μg/hemisphere) or TAT-Beclin 1 (1 μg/hemisphere). Percent freezing was measured over 4 min of the testing phase for each group. Discrimination index ([time spent with newly located object−familiar located object]/Total exploration) was measured for each group during the testing phase. All behavioral tests represented here were performed on at least two independent experiments (for each experiment: n≥5 mice per group). Data are expressed as mean±SEM. One-way ANOVA followed by Student's t test was used. *P≤0.05, **P≤0.01, ***P≤0.001, NS: not significant.

[0130] FIG. 3: Daily systemic injection of TAT-Beclin 1 allow to counteract normal age-related memory decline. A) Experimental protocol for behavioural test performed in mice after daily injection of TAT-Beclin or TAT-Scramble B) Contextual fear conditioning (CFC) performed in 3 month- and 16 month-old mice 14 days after daily intraperitoneal injections of either vehicle, TAT-Scramble (450 μg) or TAT-Beclin 1 (450 μg). Percent freezing was measured over 4 min of the testing phase for each group. Discrimination index ([time spent with newly located object−familiar located object]/Total exploration) was measured for each group during the testing phase. C) Novel object recognition (NOR) performed in 3 month- and 16 month-old mice 14 days after daily intraperitoneal injections of either vehicle, TAT-Scramble (450 μg) or TAT-Beclin 1 (450 μg). Preference index (Exploration time for the novel object/Total exploration) and discrimination index ([time spent with newly located object−familiar located object]/Total exploration) was measured for each group during the testing phase. All behavioral tests represented here were performed on at least two independent experiments (for each experiment: n≥5 mice per group). Data are expressed as mean±SEM. One-way ANOVA followed by Student's t test was used. *P≤0.05, **P≤0.01, ***P≤0.001, NS: not significant.

EXAMPLE

[0131] Material & Methods

[0132] Animals

[0133] All experiments were performed on C57BL/6J male mice obtained from Janvier Laboratory stock. All mice were 3 month- or 16-months old at the start of experiments. For all experiments, we used littermates as controls. Upon arrival, mice were housed at least 2 weeks before any behavioral or molecular testing. Mice were housed five animals per cage in polycarbonate cages (35.5×18×12.5 cm), under a 12 hours light/dark cycle (lights off at 8:00 pm) with ad libitum access to food and water prior to experimentation. All behavioral experiments were performed between 10 AM and 5 PM. Group sizes were determined after performing a power calculation to lead to an 80% chance of detecting a significant difference (P≤0.05). All efforts were made to minimize animal suffering and the number of animals used accordingly to the 3R's rule. In all experiments, animals were randomly assigned to treatment groups. All behavioral experiments were performed in accordance with the European Communities for Experimental animal use (2010/63/EU) and local ethical committee review procedures and protocols.

[0134] Stereotaxic Surgery

[0135] Mice were anesthetized by intra-peritoneal injection of ketamine hydrochloride (20 mg/ml BW) (1000 Virbac) and xylazine (100 mg/ml BW) (Rompun 2%; Bayer) and placed in a stereotaxic frame (900SL-KOPF). Ophthalmic eye ointment was applied to the cornea to prevent desiccation during surgery. The area around the incision was trimmed and Vetedine solution (Vétoquinol) was applied. All drugs were injected bilaterally into the dorsal hippocampi using the following coordinates (from Bregma, Paxinos and Franklin, 2008): Medio-lateral X=+/−1.4 mm, Antero-posterior Y=2.0 mm and height Z=−1.33 mm. A 1 μl volume of either AAV or drugs was injected stereotaxically over 4 min (injection speed: 0.25 μl per min) using a 10 μl Hamilton syringe (1701RN). To limit reflux along the injection track, the needle was maintained in situ for 4 min between each 1 μl injection. The skin was closed using silk suture. Behavioral tests were conducted 12 hours or 3 weeks after stereotactic injections. Mice were monitored during recovery.

[0136] Pharmacological Modulation of Autophagy

[0137] For pharmacological hippocampal induction of autophagy, we performed stereotactic injections of either vehicle (PBS), 1 μg TAT-Beclin 1 (dissolved in PBS) or 1 μg TAT-Scramble (dissolved in PBS). The Tat-Beclin 1 (peptide sequence: YGRKKRRQRRRGGTNVFNATFEIWHDGEFGT, SEQ ID NO:37), consists of 11 amino acids of the TAT protein transduction domain (PTD) at the N terminus, a GG linker to increase flexibility, and 18 amino acids derived from Beclin 1, amino acids 267-284 containing three substitutions: H275E, S279D, Q281E (Provided by Dr. C. Settembre). Control peptide, TAT-Scramble (peptide sequence: YGRKKRRQRRRGGVGNDFFINHETTGFATEW, SEQ ID NO:38), consisted of the TAT protein transduction domain, a GG linker, and a scrambled version of the C-terminal 18 amino acids from Tat-Beclin 1. For pharmacological hippocampal inhibition of autophagy formation, we performed stereotactic injections of either Spautin-1 (5 μg dissolved in DMSO/NaCl (SML0440; Sigma)) or vehicle (DMSO/NaCl (SML0440; Sigma). For pharmacological hippocampal inhibition of the late stage of the autophagic pathway, we performed stereotactic injections of either 100 ng Leupeptin (L2023-50 mg; Sigma) (dissolved in PBS), 50 μg Chloroquine (C6628-25G; Sigma) (dissolved in PBS) or vehicle (PBS). All drugs were infused in a volume of 1 μl (bilaterally) in the dorsal hippocampus (X=+/−1.4 mm, Y=2.0 mm and Z=−1.33 mm). 12 hours after injections mice were subjected to the training phase of the NOR, CFC or OLM behavioral tasks or sacrificed for brain collection.

[0138] Adeno-Associated Viruses Expressing shRNA

[0139] Adeno-associated viruses (AAV) expressing shRNA were purchased from Vector Biosystems Inc (Malvern Pa.). shRNAs specific to Beclin 1 (Becn 1) (AAV9-GFP-U6-mBECN1-shRNA) (named in the text: AAV-shRNA-Beclin 1), FiP200 (AAV9-GFP-U6-M-RB1CC1-shRNA) (named in the text: AAV-shRNA-Fip200) or scrambled non-targeting negative control (AAV-GFP-U6-scrmb-shRNA) (named in the text: AAV-Scramble) were injected in a volume of 1 μl (bilaterally), 3 weeks prior to behavioral tests or brain tissue collection. The AAV titers were between 2.8 and 4.6×10.sup.13 GC/ml.

[0140] Western Blot Analysis

[0141] Mouse hippocampi and cerebellum were dissected, snap frozen and lysed in RIPA lysis buffer (25 mM Tris HCl, pH 7.6, 150 mM NaCl, 1% NP40, 1% Na deoxycholate, 0.1% SDS and cOmplete protease inhibitors (Roche)). The samples were quantified using the Pierce 660 nm Assay, and lysates were mixed with 5.5× sample buffer (11% SDS, 1.4M saccharose, 0.5M Tris, pH 6.8, 10 mg/mL bromophenol blue and DTT 1M), loaded on a 12% SDS polyacrylamide gradient gel and subsequently transferred onto a polyvinylidene difluoride (PVDF) membrane (Biorad). The blots were blocked in Tris-buffered saline with Tween (TBST)-5% BSA and incubated with either mouse anti-β-Actin (1:5000, A-2228, Sigma), mouse anti-Beclin 1 (1:1000, 612113, BD Transduction Laboratories), rabbit anti-LC3 (1:10000, L7543, Sigma), mouse anti-ATGS (1:500, 0262-100/ATGS-7C6, Nanotools), and rabbit anti-VPS34 (1:1000; 4263, Cell Signaling). Horseradish peroxidase-conjugated secondary antibodies (anti-mouse IgG, HRP-linked antibody (7076, Cell Signaling) and anti-rabbit IgG, HRP-linked antibody (7074, Cell Signaling) and revealed using an ECL kit (Clarity Western ECL Substrate, BioRad) for protein detection. Multiple exposures were taken to select images within the dynamic range of the film (GE Healthcare Amersham Hyperfilm ECL). Selected films were scanned and quantified using BioRad Image Lab software (Version 5.2). β-Actin bands were used for normalization.

[0142] Semi-Quantitative RT-PCR

[0143] Brain tissues were immediately flash-frozen after dissection and total RNA was isolated with Trizol Reagent (Cat. No. 15596) using a homogenizer (OMNI TH). Single-strand cDNA was synthesized from total RNA (2 μg) by using SuperScript II Reverse Transcriptase (ThermoFisher Cat. No. 180640). qRT-PCR was performed using iTAQ SYBR Green (iTaq™ Universal SYBR® Green Supermix, 172-5124, BioRad). Primers were designed and used.

[0144] Primary Cultured Hippocampal Neurons

[0145] Hippocampi from E16.5 embryos were dissected in L15 cold media. Cells were dissociated chemically in Trypsine-EDTA 0.05% and mechanically by pipetting and then suspended in DMEM (Dulbecco's Modified Eagle Medium) supplemented with 10% fetal bovine serum, and 1% penicillin-streptomycin. All cell culture reagents were purchased from Thermo Fischer Scientific. The dissociated cells were plated onto poly-L-lysine-coated plates or glass coverslips for microscopic examination. 24h after plating, the media was replaced with Neurobasal medium containing B27 supplement, Glutamax and Mycozap. Half of the media was replaced twice a week and neurons were maintained in 5% CO2 and 37° C. until DIV15.

[0146] Lentiviral Infections and Transfection of Primary Hippocampal Neurons

[0147] Primary hippocampal neurons were infected (MOI5) at Day In Vitro (DIV) 1 with lentiviruses (pLKO-IPTG-3XLacO) expressing an IPTG (Isopropyl β-D-1-thiogalactopyranoside)-inducible shRNA targeting mouse Beclin-1 (Becn 1) (Sigma-Aldrich): 5′-TGC GGG AGT ATA GTG AGT TTA ATT CAA GAG ATT AAA CTC ACT ATA CTC CCG CTT TTT TC-3′ (sense, SEQ ID NO:39); 3′-TCG AGA AAA AAG CGG GAG TAT AGT GAG TTT AAT CTC TTG AAT TAA ACT CAC TAT ACT CCC GCA-5′(anti-sense, SEQ ID NO:40). The same lentiviral plasmid construct together with a plasmid expressing EGFP were also used to co-transfect neurons at DIV11 with Lipofectamine 2000 (Thermo Fischer Scientific) following manufacturer's instructions, to study dendritic spines density. Infected or transfected neurons were treated with 5 mM IPTG (Promega) for 72 hours to induce shRNA-Beclin-1 expression. Neurons were then treated at DIV15 as described below.

[0148] Neuronal Stimulation Treatment of Primary Hippocampal Neurons

[0149] For chemical long-term potentiation (cLTP), primary hippocampal neurons (DIV15) were treated as described by (Oh et al., 2005). For KCl depolarization, neurons were pretreated with neurobasal medium containing 60 nM KCL for 10 min and for bafilomycin treatment, neurons were treated with neurobasal medium containing 100 mM bafilomycin for 2 hours. After treatment, neurons were rinsed in PBS and proteins extracted in 1× Laemli buffer containing phosphatase and protease inhibitors.

[0150] Dendritic Spines Density Analysis In Vitro

[0151] Neurons were treated as described in (20) and fixed 1 h after cLTP induction in 4% PFA /4% glucose for 20 min at room temperature. The coverslips were then washed 3 times in PBS and mounted with Fluoromount™ Aqueous Mounting Medium. Lad (Millipore) detection by immunofluorescence was carried out to identify EGFP and shRNA Beclin1 co-transfected neurons. Fluorescence images of Lad and EGFP positive neurons were obtained using a Zeiss Apotome2 (40× objective). Dendritic spine density was analyzed using NeuronStudio software (Rodriguez A. et al 2008). For each neuron, spines from two distinct secondary and tertiary dendrite segments were counted. 16 neurons from 4 biological replicates were analyzed for each group. The analysis was performed blinded by two independent investigators (M.R.B and M.R).

[0152] Golgi Cox Staining and Dendritic Spines Quantification

[0153] 3 month-old treated mice were intracardially perfused with 4% PFA and the isolated brains were subjected to Golgi impregnation was using FD Rapid GolgiStain Kit (FD Neuro Technologies, Baltimore, Md.) following manufacturer's instructions. Bright field z-stacks of 100 μm sections were obtained using a Zeiss Apotome2 (40× objective). Dendritic spine density was quantified manually (ImageJ software, U.S. National Institutes of Health, Bethesda, Md., USA, http://imagej.nih.gov/ij/, 1997-2014). For each mouse group, two secondary dendrite segments from 20-30 of dentate gyrus granule cells were quantified. The analysis was performed blinded by three independent investigators (M.R.B, S.M. and M.R.).

[0154] SQSTM1/p62 Puncta Immunostaining and Quantification

[0155] Mice were deeply anesthetized with a mixture Ketamine/Xylasine and transcardially perfused with cold PBS, followed by cold 4% PFA. Brains were post-fixed overnight in 4% PFA at 4° C. 30 μm serial coronal floating sections were obtained using a vibratome. Sections were washed with PBS and blocked with 10% fetal bovine serum for 30 min at room temperature. Sections were then incubated with guinea pig anti-p62 (1:200; GP62C, Progen) overnight at 4° C. The sections were washed with PBS before and after being incubated with an Alexa Fluor-conjugated secondary antibodies (donkey anti-guinea pig IgG (Alexa Fluor 488), Life Technologies, 1:200) for 1 hour at room temperature in blocking buffer. All sections were mounted onto gelatin-subbed slides and coverslipped using Mowiol with DAPI. Images were obtained using a Zeiss Apotome.2 fluorescence microscope (20× and 40× objectives). Image analysis was performed using Zen light Zeiss LSM software. The number of cells with SQSTM1/p62 puncta were quantified on digital images with Icy software (http://icy.bioimageanalysis.org).

[0156] Electrophysiology

[0157] All recordings were carried out blind to experimental conditions. Male C57BL/6J 3 month-old mice perfused with cold artificial cerebrospinal fluid (aCSF) containing (in mM): 128 NaCl, 3 KCl, 1.25 NaH2PO4, 10 D-glucose, 24 NaHCO.sub.3, 2 CaCl2, and 2 MgCl2 (oxygenated with 95% 02 and 5% CO2, pH 7.35, 295-305 mOsm). Acute brain slices containing the CA3 were cut using a microslicer (DTK-1000, Ted Pella) in sucrose-ACSF, which was derived by fully replacing NaCl with 254 mM sucrose, and saturated by 95% 02 and 5% CO2. Slices were maintained in the holding chamber for 1 hr at 37° C. Slices were transferred into the recording chamber fitted with a constant flow rate of ACSF equilibrated with 95% O2/5% CO (2.5 ml/min) at 35° C. Glass microelectrodes (2-4 MΩ) filled with an internal solution containing (mM): 115 potassium gluconate, 20 KCl, 1.5 MgCl2, 10 phosphocreatine, 10 HEPES, 2 magnesium ATP, and 0.5 GTP (pH 7.2, 285 mOsm) were used. Cell excitability was measured with 2 s incremental steps of current injections (50, 100, 150, and 200 pA) at −60 mV holding potential. Series resistance was monitored during all recordings at the beginning and end of each recording, and data were rejected if values changed by more than 20%. All data acquisition and on-line analysis of firing rates were collected using a 700B amplifier, Digidata 1322A digitizer and pClamp 10.2 (Molecular Devices). Spontaneous excitatory postsynaptic currents (sEPSCs) were recorded in voltage clamp at a holding potential of −60 mV with series resistance of <6 MΩ, in the presence of picrotoxin (50 μM).

[0158] Novel Object Recognition Paradigm

[0159] The behavior sessions were recorded with a video camera. The testing arena consisted on two grey plastic boxes (60×40×32 cm). Mice could not contact or see each other during the exposures. The light intensity was equal in all parts of the arena (approximately 20 lx). Two different objects were used, available in triplicate. The objects were (1) a blue ceramic pot (diameter 6.5 cm, maximal height 7.5 cm) and (2) a clear, plastic funnel (diameter 8.5 cm, maximal height 8.5 cm). The objects elicited equal levels of exploration as determined in pilot experiments and training phase. Mice were transported a short distance from the holding mouse facility to the testing room in their home cages and left undisturbed for at least one hour before the beginning of the test.

[0160] The NOR paradigm consists of three phases over 3 days: a habituation phase, a training phase, and a testing phase. Mice were always placed in the center of the arena at the start of each exposure. On day 1: the habituation phase, mice were given 5 min to explore the arena, without any objects and were then taken back to their home cage or for stereotactic surgery. On day 2, the training phase (performed 12 hours after stereotactic surgery), mice were allowed to explore, for 10 min, two identical objects arranged in a symmetric opposite position from the center of the arena and were then transported to their home cage. On day 3, the testing phase, mice were given 15 minutes to explore two objects: a familiar object and a novel one, in the same arena, keeping the same object localization. The object that serves as a novel object (either a blue ceramic pot or a plastic funnel), as well as the left/right localization of the objects were counterbalanced within each group. Mice were placed in the center of the arena at the start of each exposure. Between exposures, mice were held individually in standard cages, the objects and arenas were cleaned with phagosphore, and the bedding replaced. The following behaviors were considered as exploration of the objects: sniffing, licking, or touching the object with the nose or with the front legs or directing the nose to the object at a distance ≤1 cm. Investigation was not scored if the mouse was on top of the object or completely immobile. The preference index for the novel object was calculated as (time spent exploring the new object/the total time spent exploring both objects), and the discrimination index was calculated as (time spent exploring the new object−time spent exploring the familiar object)/(total time spent exploring both objects). Behavior was scored on videos by two observers blind to treatment and the total exploration time of the objects was quantified in the training and testing phases (M.G and S.M).

[0161] Object Location Memory test

[0162] For the Object location memory task, all procedures were identical to the Novel object recognition task except that during the testing phase, rather than presenting a novel object, mice encountered both familiar objects, with one object located in a different place in the arena. The time and frequency of exploration of the novel/relocated object is measured as an index of memory. Behavior was scored on videos by two observers blind to treatment and the total exploration time of the objects was quantified in the training and testing phases (M.G and S.M).

[0163] Contextual Fear Conditioning

[0164] Mice were transported a short distance from the holding mouse facility to the testing room in their home cages and left undisturbed for at least one hour before the beginning of the test. The conditioning chambers were obtained from Bioseb (France) with internal dimensions of 25×25×25 cm. Each chamber was located inside a larger, insulated plastic cabinet that provided protection from outside light and noise (67×55×50 cm, Bioseb, France), and mice were tested individually in the conditioning boxes. Floors of the chamber consisted of 27 stainless steel bars wired to a shock generator with scrambler for the delivery of foot shock. Signal generated by the mice movements was recorded and analyzed through a high sensitivity weight transducer system. The analog signal was transmitted to the Freezing software module through the load cell unit for recording purposes and analysis of time active/time immobile (Freezing) was performed. The CFC procedure took place over two consecutive days. On day 1, mice were placed in the conditioning chamber, and received 3 foot-shocks (1 sec, 0.5 mA), which were administrated at 60, 120 and 180 sec after the animals were placed in the chamber. They were returned to their home cages 60 sec after the final shock. Contextual fear memory was assessed 24 hours after conditioning by returning the mice to the conditioning chamber and measuring freezing behavior during a 4 min retention test. Freezing was scored and analyzed automatically using Packwin 2.0 software (Bioseb, France). Freezing behavior was considered to occur if the animals froze for a period of at least two seconds. Behavior was scored by the Freezing software and analyzed by two observers blind to mouse treatment or AAV-infections (M.G and S.M).

[0165] Morris Water Maze

[0166] Animals were transported a short distance from the holding mouse facility to the testing room in their home cages and left undisturbed for at least one hour prior the first trial. Morris water maze (MWM) with an automatic tracking system was employed for assessing spatial learning and memory. The apparatus was a white circular swimming pool (diameter: 200 cm, walls: 60 cm high), which was located in a room with various distal cues. The pool was filled with water (depth: 50 cm) maintained at 22° C.±1° C., which was made opaque by the addition of a nontoxic white paint. A 12 cm round platform was hidden 1.0 cm below the water surface. The maze was virtually divided into four arbitrary, equally spaced quadrants delineated by the cardinal points north (N), east (E), south (S), and west (W). The pool is located in a brightly lit room. Extra maze geometric and high-contrast cues were mounted on the walls of the swimming pool with the ceiling providing illumination. Data was collected using a video camera fixed to the ceiling and connected to a videotracking system (Anymaze). Each daily trial consisted of four swimming trials, in which each mouse was placed in the pool facing the wall of the tank and allowing the animal to swim to the platform before 120 sec had elapsed. A trial terminated when the animal reached the platform, where it remained for 5 sec. Mice were removed and placed back in their home cages for a 5 min inter-trial interval. To prevent hypothermia, the animals were gently dried with a paper towel between and after the trials. The starting point differed at each trial, and different sequences of release points were used from day to day. Swimming time to the platform was calculated as an evaluation of performance of the mice to locate the target. At day 10, animals were given a probe trial, which consisted of letting the mouse swim in the pool for a fixed duration (120 sec), while the platform was removed but with the same distal cues on the wall. The performance in the probe trial was expressed as the time spent in the target quadrant where the platform was located during the hidden platform training. Animal movements were recorded using Anymaze to calculate parameters of the performance of mice. Behavior was scored by two observers blind to treatment (M.G and S.M).

[0167] Memory Stimulation Procedure

[0168] Animals were either exposed to CFC or MWM to induce memory stimulation adapted from (21, 25). For the CFC, we used one- or four-days for the training phase. For the one-day training, mice were placed into the conditioning chamber, received three shocks at 60, 120 and 180 sec (1 sec, 0.5 mA), and were removed 60 sec following the last shock and returned to their home cages. For the four-day training, this procedure was repeated four times but at a shock intensity of 0.25 mA. Contextual fear memory was assessed 24 hours following training by returning the mice to the same conditioning chamber and measuring the freezing behavior during a 4 min retention test. Freezing was scored and analyzed automatically using Packwin 2.0 software. Freezing behavior was considered to occur if the animal froze for at least a period of two seconds. For the MWM, the mice were subjected to the normal procedure (described above) but for 5 successive days. On the last day of stimulation, the mice were sacrificed 1 hour after the last exposition to CFC or MWM task.

[0169] Plasma Collection and Intravenously Injection

[0170] Pooled mouse blood was collected from 60 young (8 weeks) and 6 aged (16 months) mice by intracardial bleed at time of euthanasia. Plasma was prepared from blood collected with EDTA into Capiject T-MQK tubes followed by centrifugation at 1,000 g. All plasma aliquots were stored at −80° C. until use. Before administration, plasma was dialyzed using 3.5-kDa Maxi D-tube dialyzers (71508-3, Novagen) in PBS to remove EDTA. 16 month-old mice were injected with isolated plasma (100 μl per injection), by tail vein injection seven times over 24 days. Mice were then subjected to NOR, one day after the last injection.

[0171] Statistical Analysis

[0172] For all experiments, the effect of treatment was analyzed using one-way ANOVAs and with repeated-measures where appropriate. Significant ANOVAs were also analyzed using Fisher's PLSD tests where appropriate. All main effects and interactions are noted in the text or figures. All data were analyzed using GraphPad Prism v5 software. Results from data analyses are expressed as means±SEM. Alpha was set to 0.05 for all analyses. * p<0.05, ** p<0.01, *** p<0.001.

[0173] Results

[0174] To explore the role of autophagy in hippocampal neuronal function and behavior, we subjected 3 month-old mice to memory stimulation by the Morris water maze (MWM) or contextual fear conditioning (CFC). Key ATG genes and genes associated with autophagy (Becn 1, Vps34 and Atg5) were increased at both mRNA and protein levels in the hippocampus (data not shown). Moreover, there was an accumulation of the AP marker LC3-II, which together with the decrease in autophagy cargo SQSTM1/p62, suggested that memory stimulation induces autophagic flux in hippocampal neurons (data not shown). To address more directly the role of autophagy in regulating hippocampal-dependent learning and memory, we next performed hippocampal stereotactic injections of Adeno-Associated Viruses (AAV) expressing shRNA against Beclin 1 (Becn 1), a protein engaged in the initiation of autophagy (1, 2). A decrease in the conversion of LC3 (LC3-I) to the AP-associated lipidated form (LC3-II) 3 weeks after injections verified that autophagy had been hampered in the hippocampus (data not shown). The mice were then subjected to behavioral tasks assessing hippocampal-dependent memory. We found that down-regulating Beclin 1 expression impaired memory capacities as measured by the Novel object recognition (NOR) test and CFC (data not shown), two tests that require the integrity of the hippocampus (16). Next, these mice were subjected to the MWM with a hidden platform for 9 successive days, to assess the ability of mice to use spatial cues to locate a submerged platform. Hippocampal down-regulation of Beclin 1 induces a significant delay in spatial learning capacities (data not shown). This was confirmed by a probe trial MWM task 24h after the last day of acquisition that demonstrated no significant preference for the pool quadrant in AAV-shRNA Beclin 1 (data not shown). Of note, re-exposing the mice to the same context 6 days after the last acquisition day showed that Beclin 1 down-regulated mice did not consolidate memory to the same extent as control mice (data not shown). To rule out a possible autophagy-independent role for Beclin 1, we performed NOR and CFC in mice targeted for Fip200, a component of the ULK1/AGT1 complex involved in early AP formation. Fip200 down-regulation with stereotactic injections of AAV-shRNA and a decrease in autophagy were confirmed by measuring Fip200 expression and LC3-II abundance (data not shown). These mice phenocopy the decreases in memory performance observed after Beclin 1 down-regulation in NOR and CFC assays (data not shown). Taken together, these results suggest that the initiation of autophagy is required for the control of hippocampal-dependent learning and memory.

[0175] To determine whether autophagy is required for the adaptive response of hippocampal neurons to novel memory stimulation, we acutely modulated autophagy by performing single hippocampal stereotactic injections of inhibitors or inducers of autophagy in 3 month-old WT mice (data not shown). Induction or inhibition of autophagy 12h after acute pharmacological injections was confirmed by measuring LC3-II abundance and SQSTM1/p62 levels in the hippocampus (data not shown). We then subjected them 12 hours later to the training (acquisition) phase of NOR and CFC (data not shown). Acute injections of the Spautin-1 (specific autophagy inhibitor-1) (data not shown), interfering with AP formation, or blocking the late stage of the autophagic pathway by injecting chloroquine or leupeptin (data not shown), all led to a significant decrease in memory performance during the testing phase of both NOR and CFC (data not shown). Conversely, an acute induction of autophagy following hippocampal injections of the TAT-Beclin 1 peptide (17), an inducer of AP formation, during the training phase (FIGS. 1A, B and C), enhanced learning/memory capacities in both tests. Hence, these data indicate that hippocampal autophagy is necessary for the control of novel memory acquisition.

[0176] Novel memory acquisition relies on the adaptive response of hippocampal neurons to stimuli, characterized by lasting changes in neuronal morphology, synaptic activity and neurotransmission (5, 10, 18). Long-term potentiation (LTP) is one of the key cellular mechanisms underlying learning and memory (18, 19). Increased synaptic strength following LTP induction results in the rapid formation of new dendritic spines, small actin-rich protrusions extending from dendrites that house excitatory synapses, along with phosphorylation of post-synaptic AMPA glutamate receptors and CAMKII (calcium/calmodulin-dependent protein kinase II) (5, 18). We observed that chemical long-term potentiation (cLTP), which induces long-lasting synaptic plasticity, or KCL depolarization, increases autophagy in mature primary hippocampal neurons, as determined by measuring LC3-II level (data not shown). Therefore, we investigated whether down-regulation of autophagy may influence neuronal plasticity of hippocampal neurons in response to cLTP (20) or memory stimulation induced by MWM (21), which promotes formation of novel dendritic spines in granular neurons of the hippocampal dentate gyrus. In vitro, we performed acute knockdown of Beclin 1 in primary hippocampal neurons by infection with lentiviruses expressing an IPTG inducible-shRNA against Beclin 1. Treatment with IPTG induced a robust decrease in Beclin 1 protein levels (data not shown). Neuronal plasticity induction by cLTP significantly increased novel dendritic spine formation, and phosphorylation of GluA1 receptors (pGluA1) in Ser831 and CAMKII (pCaMKII) in the control group, whereas no significant induction of either novel dendritic spines or synaptic molecular strength in response to cLTP treatment was observed after down-regulation of Beclin 1 (data not shown). This observation suggests that autophagy is required to mediate the adaptability of hippocampal neurons to respond to a long-lasting stimulation. To confirm this observation in vivo, 3 month-old mice, locally injected with either AAV-shRNA Beclin 1 or AAV-shRNA Scramble, were subjected to MWM for 5 consecutive days (22). After Golgi staining, we observed a significant induction of dendritic spine formation in hippocampal granular neurons of control mice (AAV-Scramble-injected), but not in those of the AAV-shRNA Beclin 1-injected mice (data not shown). Accordingly, electrophysiological analyses showed a significant decrease in neuronal excitability (data not shown) and sESPC frequency (data not shown) in hippocampal neurons of the CA3 after local down-regulation of Beclin 1. These data indicate that autophagy in hippocampal neurons is required for an adaptive neuronal response to novel memory stimulations.

[0177] ATG protein levels and autophagic flux are reduced during normal brain aging (3). In agreement with these data, we observed a decrease in Beclin 1, VPS34 and ATG5 at both protein and mRNA levels, and a decrease in LC3-II accumulation together with an increase in SQSTM1/p62 in the hippocampi of 16 month-old WT mice (FIG. 2A). We next tested whether the decrease in hippocampal autophagy during aging may contribute to age-related cognitive decline by subjecting 3 or 16 month-old WT mice to NOR, CFC and Object location memory (OLM) tests after local stereotactic injections of either vehicle, TAT-Scramble or TAT-Beclin 1. We observed that increasing autophagy levels in 16 month-old animals was sufficient to rescue age-related memory deficits and to restore memory to levels seen in 3 month-old mice (FIGS. 2A, 2B, 2C, 2D, 2E, 3A, 3B and 3C). As various environmental factors, such as young circulating factors, can protect against age-related hippocampal-dependent memory and adult neurogenesis decline (23, 24), we hypothesized that hippocampal autophagy could mediate, at least in part, the recently described beneficial effects of young plasma in aged mice on hippocampal-dependent memory. We found that 16 month-old mice receiving injections of plasma collected from 8 week-old mice had increased LC3-II levels, indicating increased autophagy (data not shown) and corrected age-related memory deficits (data not shown). This beneficial effect was not observed when plasma from 2 month-old mice was injected in 16 month-old animals in which Beclin 1 was down-regulated (data not shown).

[0178] Our study describes for the first time an important role for autophagy in the brain, for the regulation of hippocampal-dependent memory. It demonstrates that neuronal autophagy has an unprecedented, functionally critical role in the brain rather than only protecting it from energy shortage or degradation of mis-folded proteins. Indeed, hippocampal autophagy is required to mediate the activity-dependent changes of neurons in response to memory stimulation and environmental stimuli. Moreover, our findings demonstrate that restoring autophagy activity (via the TAT-beclin for example) level in old hippocampus could reverse age-related memory deficits. In that respect, our results may have important therapeutic implications to prevent the decline and/or extend resilience of mental health during aging.

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