Animals

APPswe/PSEN1dE9 (APP/PS1) mice of B6C3 hybrid background (RRID: MMRRC_034829-JAX, from stock number 004462) and 5XFAD mice were originated from Jackson Laboratory (USA). APP/PS1 maintained as hemizygotes by crossing transgenic mice to B6C3 F1 mice. Genotypes were determined by PCR using the following primers – APP/PS1f − 5’ AAT AGA GAA CGG CAG GAG CA 3’; APP/PS1r − 5’ GCC ATG AGG GCA CTA ATC AT 3’. All mice were maintained under 12:12-h light-dark cycle (lights on at 8:00 AM) and has ad libitum access to food and water. Animal care and handling were performed according to the directives of the Animal Care and Use Committee and institutional guidelines of KIST (Seoul 02792, South Korea). Immunohistochemistry and behavioral tests were performed on virus-injected mice, for which both sexes of 10- to 13-month-old transgenic mice and wild-type littermates were used.

Chemicals

We used several autophagy inhibitors: chloroquine (CQ) (Sigma, C6628, USA), a lysosome fusion inhibitor; epoxysuccinyl-leucylamide(3-methyl-butane ethyl ester)(E64D) (Sigma, E8640, USA), a cysteine protease inhibitor; Pepstatin A (P) (Sigma, 77,170, USA), an aspartate protease inhibitor; 3-methyladenine (3-MA), a PI3-kinase inhibitor (Sigma, 189,490, USA). KDS2010, an ROS scavenger and reversible MAO-B inhibitor was obtained from Institute of Basic Science (IBS, Daejeon, South Korea).

Cell culture

Primary cortical astrocytes were prepared from postnatal day (P) P0–P3 C57BL/6 mice as described [23]. The cerebral cortex was dissected free of adherent meninges, minced and dissociated into single-cell suspension by trituration. Dissociated cells were plated onto plates coated with 0.1 mg/ml poly-D-lysine (Sigma, USA). Cells were grown in DMEM (Invitrogen, USA) supplemented with 25 mM of glucose, 10% heat-inactivated horse serum, 10% heat-inactivated FBS, 2 mM of glutamine and 1,000 Uml− 1 penicillin-streptomycin. Cultures were maintained at 37 °C in a humidified 5% CO2 incubator. On the third day of culture, cells were vigorously washed with repeated pipetting and the medium was replaced to get rid of debris and other floating cell types. Human astrocyte cell line was purchased from Applied Biological Materials Inc. company (Immortalized Human Astrocytes, fetal-SV40; Cat. No. T0280; Canada). The human astrocyte cell line was maintained in DMEM supplemented with 25 mM of glucose, 10% heat-inactivated FBS, 2 mM of glutamine and 1,000 Uml− 1 penicillin-streptomycin. The passages of astrocytes between 10 and 20 were used for in vitro experiments.

Preparation of oligomeric form of Aβ42

Amyloid-beta 1–42 (Aβ; DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA; Abcam, USA) was prepared as previously described [24]. Aβ was dissolved in dimethyl sulfoxide (DMSO) at 10 mM and further diluted to 1 mM in PBS and incubated at 37 °C for 1 week and stored at -80 °C for further use as an oligomeric form of Aβ (Aβ oligomer). The monomeric form of Aβ (Aβ monomer) was prepared using same sequence without oligomerization process.

Immunocytochemistry

For immunostaining in cultured cells, the cells were rinsed with PBS twice and fixed with 4% PFA for 10 min at room temperature. Cells were then washed with PBS twice and permeabilized through incubation in 0.1% Triton X-100 in PBS for 15 min at room temperature. Nonspecific binding was blocked through incubation in 3% bovine serum albumin (Sigma, USA), normal goat serum (Abcam, USA) or normal donkey serum (Abcam, USA) in PBS with 0.1% Triton X-100 for 2 h at room temperature. Afterwards, cells were incubated with the following antibodies for 24 h: anti-LC3B antibody (1:500, Novus bio, USA); anti-SQSTM1 antibody (1:500, Abcam, USA), anti-glial fibrillary acidic protein (GFAP; 1:500, Abcam, USA), anti-calcium-binding protein B (S100B; 1:500, Synaptic systems, Germany), anti-microtubule associated protein 2 (MAP2; 1:500, Abcam, USA) and anti-Neuronal nuclear antigen (NeuN; 1:400, Abcam, USA). After secondary antibody reaction, samples were washed 3 times with PBS and examined under a confocal microscope.

Immunohistochemistry

Mice were deeply anesthetized with 2% avertin (20 mg/ml, 20 µl/mouse weight (g); intraperitoneal injection) and perfused with 0.9% saline followed by ice-cold 4% paraformaldehyde (PFA). Excised brains were postfixed overnight at 4 °C in 4% PFA and dehydrated in 15% and 30% sucrose for 48 h. Coronal hippocampal sections were cut at 30 μm in a cryostat and stored in storage solution at 4 °C. Sections from storage solution were washed in PBS and incubated for 1 h in a blocking solution (0.3% Triton X-100, 2% normal donkey serum in 0.1 M of PBS). Primary antibodies in blocking solution were immunostained on a shaker at 4 °C overnight. After washing in PBS 3 times, sections were incubated with corresponding fluorescent secondary antibodies for 1 h at room temperature and then washed with PBS at 3 times. The nuclei were counter stained with 4′,6-diamidino-2-phenylindole (DAPI) was adding in solution (1:5,000; Abcam, USA) during the second washing step. Finally, sections were mounted with fluorescent mounting medium (Dako, USA) and dried at room temperature. A series of fluorescent images were obtained with a Nikon A1 confocal microscope (Nikon, Japan) with 26 μm Z stack images in 2 μm steps were processed for Sholl analysis using the NIS-Elements software (Ver. 4.5, Nikon, Japan) and ImageJ software (Ver. 1.52s, NIH, USA). Primary antibodies were diluted to the following amounts: anti-LC3B (1:500, Abcam, USA), anti-SQSTM1 (1:200, MBL, USA), anti-AT8 (1:200, Abcam, USA), anti-GFAP (1:500, Abcam, USA), and anti-NeuN (1:400, Millipore, USA). Secondary antibodies were diluted 1:500 in the blocking solution for 2 h at room temperature.

Human brain samples

Neuropathological examination of postmortem brain samples from normal subjects, (NeuroPathologically and Clinically Diagnosed AD)-MCI (NPCAD) and severe AD (SAD) patients was determined using procedures previously established by the Boston University Alzheimer’s Disease Center (BUADC) [25]. Next of kin provided informed consent for participation and brain donation. Institutional review board approval for ethical permission was obtained through the BUADC center. This study was reviewed by the Institutional Review Board of the Boston University School of Medicine and was approved for exemption because it only included tissues collected from post-mortem subjects not classified as human subjects. The study was performed in accordance with institutional regulatory guidelines and principles of human subject protection in the Declaration of Helsinki. The sample information is listed in Supplementary Table S3.

Double chromogenic staining for human brain tissuesFirst staining

Paraffin-embedded human postmortem brain tissues were sectioned in a coronal plane at 10 μm. BLOXALL® Blocking solution (SP-600, Vector Laboratories, USA) was used to block endogenous alkaline phosphatase. Hippocampal tissue sections were blocked with 2.5% normal horse serum (S-2000, Vector Laboratories, USA) for 1 h and then incubated with GFAP antibody (1:400 dilution) (AB5541, Millipore, USA) for 24 h. After washing three times with PBS, tissue slides were processed with Vector ABC Kit (PK-4000, Vector Laboratories, USA). The GFAP immunoreactive signals were developed with DAB chromogen (D7304, Thermo Fisher Scientific, USA).

Second staining

Tissue slides stained with GFAP were incubated with LC3B antibody (1:200 dilution) (ab192890, Abcam, USA) for 24 h. After reaction with secondary antibodies, sections were incubated with ImmPRESS-AP anti-rabbit IgG (Aalkaline phosphatase) polymer detection reagent (MP-5401, Vector Laboratories, USA) for 2 h at room temperature. A Vector Blue alkaline phosphatase substrate kit (SK-5300, Vector Laboratories, USA) was used to develop LC3B signals. Double-stained tissue slides were gradually processed back to Histo-clear (HS-200, National Diagnostics, USA) through an increasing ethanol gradient [70%, 80%, 90%, 95%, and 100% (1 time)] and subsequently mounted. The chromogenic signals of GFAP (brown) and LC3B (blue) were examined under a light microscopy (BX63, Olympus, Japan) equipped with high definition (1920 × 1200 pixel) digital camera (DP74) (Olympus, Japan).

3D reconstruction of microscopic image

Images were analyzed using a Spinning Disk Confocal microscope (IX2-DSU, Olympus, Japan) that has z-stack modulation stage. The 40x objective images were acquired z-stack in a 0.25 μm steps and deconvolved using Cell Sense software (Olympus, Japan). The deconvolution images were applied to the process of 3D reconstruction with IMARIS software (Ver. 13, Oxford Instrument, UK). The clipping plane mode was used to clearly visualize the localization of Aβ plaques and astrocytes.

Illumina Hiseq library preparation and sequencing

Sample libraries were prepared by Ultra RNA Library Prep Kit (#E7530, NEBNEXT, USA), Multiplex Oligos for Illumina (#E7335, NEBNEXT, USA) and poly(A) mRNA Magnetic Isolation Module (#E74900, NEBNEXT, USA) according to the manufacturer’s instructions. Full details of the library preparation and sequencing protocol are provided on the website (https://international.neb.com/products/e7530-nebnext-ultra-rna-library-prep-kit-for-illumina#Product%20Information). The Agilent 2100 Bioanalyzer (Agilent Technologies, USA) and the associated High Sensitivity DNA kit (Agilent Technologies, USA) were used to determine quality and concentration of the libraries. Sample libraries for sequencing were prepared by the HiSeq Reagent Kit Preparation Guide (Illumina, USA) as described previously [26]. Briefly, the combined sample library was diluted to 2 nM, denatured with 0.2 N fresh NaOH, diluted to 20 pM by addition of Illumina HT1 buffer. The library (600 µl) was loaded with read 1, read 2 and index sequencing primers on a 150-cycle (2 × 75 paired ends) reagent cartridge (HiSeq Reagent kit, Illumina, USA), and run on a HiSeq NEXT generation high-throughput sequencer (Illumina, USA). After the 2 × 75 bp Illumina HiSeq paired-end sequencing run, the data were base-called and reads with the same barcode were collected and assigned to a sample on the instrument, which generated Illumina FASTQ files.

Quantitative real time-PCR (qPCR)

Total RNA was isolated from cells and brain tissues using a commercial extraction system (Qiagen, USA). 1 µg total RNA has been used for cDNA preparation with iScript cDNA Synthesis Kit (Bio-Rad, USA) according to manufacturer’s protocols. cDNA from each sample was amplified by real-time PCR using iQ SYBR Green Supermix (Bio-Rad, USA). RNA quantities were normalized using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. PCR cycling conditions were denaturation for 3 min at 95°C; then 40 cycles of amplification for 15 sec at 95°C, 15 sec at 60°C, 20 sec at 70°C; followed with 30 sec at 72°C. For melt curve data collection has been used 33 cycles, 6 sec each, with the temperature increased from 60°C to 92°C (increase set point temperature after cycle 2 by 1°C). The PCR primer for LC3B were as following: forward, 5’-ATC CCG GTG ATA ATA GAA CG -3’ and reverse, 5’-GAA GAA GGC CTG ATT AGC AT -3’; The PCR primer for SQSTM1 were as following: forward, 5’-ATG ACT GGA CCC ATC TGT CT-3’ and reverse, 5’-TCA TCA GAG AAG CCC ATG GA-3’, The PCR primer for Beclin1 (BECN1) were as following: forward, 5’-AGG TAC CGA CTT GTT CCC TA-3’ and reverse, 5’-TCC ATC CTG TAC GGA AGA CA-3’. The PCR primer for GAPDH were as following: forward, 5’- GAA ATC CCA TCA CCA TCT TCC-3’ and reverse, 5’- GAG GCT GTT GTC ATA CTT CTC-3’.

Western blot analysis

Western blot analyses were performed as described previously. The transferred blots were incubated with the following primary antibodies at 4 °C for 24 h: anti-LC3B (1:2000, Abcam, USA); anti-SQSTM1(1:2000, Abcam, USA); anti-ACTB/β-actin (1:2000, ACTB; Abcam, USA) and anti-TUBB3/tubulin (1:2000, TUBB3; Abcam, USA). After washing 3 times with Tris-buffered saline with 0.05% Tween 20), the blots were incubated with the appropriate secondary antibodies conjugated to horseradish peroxidase (HRP) by anti-rabbit HRP (Amersham Pharmacia, USA) at room temperature for 2 h. Then, the blots were developed by Immobilon Western ECL solution (Merck Millipore, USA) and immunoreactive bands were visualized using an Image Station 4000MM (#745,280; Kodak, Japan). ACTB or TUBB3 was used as the loading control.

Cell viability assay and cell death assay

Cell viability of primary astrocyte cells were assessed using a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2 H-tetazolium inner salt (MTS) assay, which is based on a tetrazolium compound, MTS, and an electron acceptor agent, phenazine methosulfate (PMS; Promega, USA). Briefly, cells were seeded into a 96-well plate at a density of 3 × 104 cells per well in 200 µl of medium, stabilized for growth, and then treated with various concentrations of Aβ oligomers and autophagy inhibitors in 100 µl of medium. After 24 h of incubation at 37 °C, 20 µl of MTS/PMS mixture solution was added to the culture medium, and cells were further incubated for 3 ∼#x2009;4 hr at 37 °C. Finally, absorption readings were performed at 490 nm using a spectrophotometry. Astrocyte cells were seeded in a 96-well plate at 1 × 104 cells per well. After overnight, the Aβ oligomers were added to the wells with 1 µg/ml. After 24 h, inhibitors added to the wells with E/P. After indicated times, the cellular viability was determined by Cell Titer-Glo reagent (#G7572, Promega, USA). Dose-response curve was fitted using Graphpad prism 8.0 software (GraphPad Software, USA). All assays were performed in triplicate, and standard deviation (SD) was determined from three independent experiments. Apoptosis and necrosis of astrocyte cells was quantified by Annexin V and EthD-III based quantification assay kit (#30,065, Biotium, USA). Astrocyte cells were treated with Aβ oligomers and autophagy inhibitors for 24 h, followed by incubation with a staining solution at room temperature for 15 min. Live cell imaging was obtained by Image ExFluorer (Live Cell Instrument, Korea). The intensity and quantification of cell death signals were analyzed with ImageJ software (Fiji; NIH, USA).

Live cell imaging in astrocyte culture with MitoTracker and MitoSOX

MitoTracker-green (Thermo Scientific, USA) staining was performed to measure the mitochondrial morphology in astrocyte cells as previously described [27]. Mito-Sox (Thermo Scientific, USA) staining was performed to measure the oxidative stress in astrocyte cells. MitoTracker-green (0.1 µM) and Mito-Sox (2.5 µM) staining for 30 min at 37 °C incubator prior to acquire images. Live cell imaging was obtained by Image ExFluorer (Live Cell Instrument, Korea). The intensity of mitochondria oxidative stress and mitochondria morphology were analyzed by ImageJ software (NIH, USA).

DCF-DA assay

Intracellular ROS levels were detected using cell-permeable non-florescent probe 2′,7′-Dichlorofluorescin diacetate (#D6883, DCFDA; Sigma, USA). DCF-DA is de-esterified into its fluorescent form after action of intracellular esterases and oxidation by reactive oxygen species within the cell [28]. Primary astrocyte culture was seeded onto 48-well plates (Corning, USA) and treated with Aβ oligomer (1 µM) in the presence or absence of KDS2010 (100 nM), a ROS scavenger and reversible MAO-B inhibitor, or CQ (20 µM) for 1 day. Then, cells were washed twice with Hanks Buffered Salt Solution (#LB-003-002, HBSS; Welgene, USA) and incubated with 30 µM DCF-DA in Hanks′ Balanced Salt solution (HBSS) at room temperature for 30 min in the dark. The DCFDA was replaced with HBSS and fluorescence was measured using SpectraMax iD5 Multi-Mode Microplate Reader (Excitation 485 nm and emission 538 nm, Molecular Devices, USA).

Amplex Red assay

Extracellular ROS levels were detected using Amplex Red reagent after collecting the media from cell culture treated with Aβ oligomer (1 µM) in the presence or absence of KDS2010 (100 nM) or CQ (20 µM) for 1 day.

Metabolite analysis

For metabolite analysis, aspartate, ornithine, 15N-ornithine, arginine, 15N-arginine, citrulline, 15N-citrulline, glutamate, putrescine, 15N-putrescine, GABA and 15N-GABA were analyzed. The system used for the analyses was an Exion LC AD UPLC coupled with an MS/MS (Triple Quad 4500 System, AB Sciex LLC, Framingham, USA) using an Acquity® UPLC BEH C18 column (1.7 μm, 2.1 mm x 75 mm, Waters, USA) at 50℃, controlled by Analyst 1.6.2 software (AB Sciex LP, Ontario, Canada). 70% methanol (100 µl, with internal standard d5-glutamine at a final concentration of 1 µM) was added to the astrocyte sample pellets and vortexed for 30 s. Cells were lysed by three consecutive freeze/thaw cycles using liquid nitrogen, and the lysate was centrifuged for 10 min at 20,817 x g (14,000 rpm) at 4℃. The supernatant (5 µl) from each sample was used for DNA normalization. DNA concentrations were analyzed using a Nano-MD UV-Vis spectrophotometer (Scinco, Seoul, Korea). 40 µl of the supernatant from each sample was evaporated to dryness at 37℃ under a gentle stream of nitrogen. Phenylisothiocyanate (PITC) derivatization was performed by adding 50 µl of a mixture of 19:19:19:3 ethanol:water pyridine:PITC (v/v) and the mixture was vortexed for 30 s and shaken for 20 min. Then the mixture was evaporated to dryness at 37℃ under a gentle stream of nitrogen. The residue was reconstituted by adding 50 µl of the mobile phase A (0.2% formic acid in deionized water): B (0.2% formic acid in acetonitrile) = 5:5 solvent and vortexing for 30 s. The initial chromatographic conditions were 100% solvent A at a flow rate of 0.4 mL·min− 1. After 0.9 min at 15% B, solvent B was set to 15% over the next 4.1 min, solvent B was set to 70% over the next 5 min, solvent B was set to 100% over the next 0.5 min, and these conditions were retained for an additional 2 min. The system was then returned to the initial conditions over the next 0.5 min. The system was re-equilibrated for the next 2.5 min in the initial conditions. The total running time was 15 min. All samples were maintained at 4℃ during the analysis, and the injection volume was 5 µl. The MS analysis was performed using ESI in positive mode. The ion spray voltage and vaporizer temperature were 5.5 kV and 500℃, respectively. The curtain gas was kept at 45 psi, and the collision gas was maintained at 9 psi. The nebulizer gas was 60 psi, while the turbo gas flow rate was 70 psi. The metabolites were detected selectively using their unique multiple reaction monitoring (MRM) pairs. The following MRM mode (Q1 / Q3) was selected: arginine (m/z 310.000 / 217.000), 15N-arginine(m/z 311.000 / 218.000), ornithine (m/z 403.200 / 310.200), 15N-ornithine (m/z 404.000 / 311.200), citrulline (m/z 311.200 / 113.100), 15N-citrulline(m/z 313.200 / 114.100), glutamate (m/z 283.200 / 130.200), aspartate (m/z 269.200 / 116.200), putrescine (m/z 359.200 / 266.100), 15N-putrescine(m/z 360.200 / 267.100), GABA (m/z 238.875/ 87.103), 15N-GABA(m/z 239.875 / 87.103). As to monitor specific parent-to-product transitions, the standard calibration curve for each metabolite was used for absolute quantification.

Electron microscopy of primary astrocyte culture

In order to fix human astrocytes, media was replaced with pH 7.2-4 buffered 2.5% glutaraldehyde for 1 h. And cells were collected and embedded in epon resin. Subsequently, 70 nm sections were obtained using an ultra-microtome and stained with uranyl acetate and lead citrate. Cell sections were finally analyzed using an 80 kV transmission electron microscope. To quantify the autophagic compartments, size and number were measured autophagic vesicles which were defined by dark and circular shape vesicles in cytoplasmic region [29]. For immune-gold labelling, astrocyte culture cells were fixed and incubated with anti-LC3B (NB100-2220, Novus, USA) antibody in blocking solution (0.5% Ttiton-X100 with 5% NGS in PBS) and proceed with goat anti-rabbit-gold particle antibody (G3779, 10 nm, Sigma, USA, ).

Viral plasmids and virus production

For gene silencing in astrocytes of mouse hippocampus in vivo, we generated adeno-associated virus (AAV)-CREon shRNAs for LC3B (GenBank NM_026160.5) and SQSTM1 (GenBank NM_011018.3) by applying a newly developed pAAV-R-CREon vector that turns on shRNA in a GFAP promoter (pGFAP)-driven CRE-dependent manner through AAV-pGFAP-CRE as previously described [30]. In order to generate an astrocyte-specific viral vector, we subcloned a 681 bp of human GFAP promoter, gfaABC1D, to AAV2 vector [31]. It is well established that the 681 bp gfaABC1D promoter sequence possess higher transcriptional activity in an astrocyte-specific manner similar the 2.2 kb gfa2 promoter [31]. Otherwise, to induce astrocyte-specific cell death and observe the degree of Aβ deposition in the hippocampus of APP/PS1 mice., we applied AAV-LoxP-active Caspase 3 and AAV-GFAP-Cre together [32]. LC3B shRNA sequence, 5’-GCAGCTTCCTGTTCTGGATAA-3’; SQSTM1 shRNA sequence, 5’-ACTGGACCCATCTGTCTTCAA-3’. On the other hand, to determine the gain of LC3B function in astrocytes, we inserted the cDNA of human LC3B (GenBank NM_022818.5) into AAV-pGFAP overexpression system. To generate high-titer AAV at concentrations ranging from 1 × 109 to 1011 plaque-forming units per milliliter (pfu/ml), desired plasmids and pRC5, along with the pHelper plasmids, were transiently introduced into HEK293TN cells. Following a 72 h incubation, cell lysates were subjected to benzonase treatment (50 units/ml; Sigma, USA). Then, viral particles were purified and concentrated using a heparin column (GE Healthcare, Sweden) in conjunction with a 100k filtering tube (Millipore, USA). Quantitation of viral titers was measured by qPCR method.

Stereotaxic injection

Mice were anesthetized with 2% avertin (200 mg/kg; Sigma, USA) in saline (20 µl/g; saline volume/mouse body weight) and placed in a stereotaxic frame (Stoelting Co, USA). AAV-R-CREon-LC3B or SQSTM1 shRNA and AAV-pGFAP-CRE overexpression viruses were co-injected using a stereotaxic micro-injector (Stoelting Co. USA). Control groups were injected with AAV-scramble shRNA. AAV containing solution (2 µl) was injected into the molecular layer of dentate gyrus (DG) in dorsal hippocampus; anterior-posterior (AP): -2.0 mm, medial lateral (ML): ±1.5 mm, dorsal ventral (DV): -1.85 mm to bregma using a micro-syringe pump (Micro 4, WPI, USA) with a 33-gauge needle (WPI, USA) (0.1 µl/min). Neuropathological experiments were performed at 3 weeks after injection. Mice were housed on a 12:12 h light-dark cycle and maintained at 18 ∼ 23 °C with humidity between 40 and 60% in pathogen-free facilities at Korea Institute of Science and Technology.

Behavior testsNovel object recognition (NOR) and novel object place recognition (NOPR)

NOR and NOPR tasks were performed in a white open field box (40 × 40 × 40 cm) with slight modifications from the procedures. Two types of objects were different in shape, color and texture. One of them was a yellow regular tetrahedron, made of acryl. The other one was a black and red color sphere, made of urethane. The objects were fixed to the ground of the box, not to be moved by mice. Sniffing objects was considered as the explorative action of mouse. NOR test was composed of 3 steps such as habituation, training and test, and given once per day. During the habituation step, mouse was placed in an open field box for 10 min without objects. Then, during 2 times of training period, two identical objects were presented to the mouse for 10 min. At 2 h after the last training, one of the familiar objects was replaced with a novel object and presented to the mouse for 10 min for the test. Procedures for NOPR task were similar to NOR task except that one of the objects was moved to a different location for the test. The test was video-recorded with encoding software (Ethovision XT, Noldus, USA) and the results including total number of arm entries and alternation behavior were analyzed later.

Quantification and statistical analysis

Cell culture preparation and mice with the required genotypes were randomly assigned to the experimental groups and treated in the same way. All quantitation analyses were done blindly. Numbers and individual dots refer to individual samples (individual cells, separate batches of cultured cells or animals) unless clarified otherwise in figure legends. N represents number of animals used for the experiment, while n refers to number of cells or culture batches. Data distribution was assumed to be normal but this was not formally tested. Data are presented as the mean ± SEM. For behavioral analysis, Ethovision XT (Noldus, USA) was used. For image analysis, ImageJ software (NIH, USA) and IMARIS software (Oxford instrument, UK) was used. All statistical analyses were performed using Prism v.8.4.3 (GraphPad Software, USA); an unpaired, two-tailed Student’s t-test was used to compare two groups. A one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test was used to compare more than two groups as indicated in the figure legends. A two-way ANOVA with Tukey’s multiple comparisons test was used to compare groups having two independent variables. Statistical differences were considered significant when p < 0.05 and the significance was set at *, p < 0.5 and **, p < 0.01, which are indicated in the figures or figure legends. No statistical methods were used to predetermine sample sizes but sample sizes are similar to those reported in previous publications. The number of experimental samples, mean, SEM and additional statistics values are listed in Supplementary Tables S1 and S2.