Animals
We generated PtenY68H/+ mice on a C57BL/6J (Jackson Laboratory, Bar Harbor, Milwaukee) background by introducing one missense mutation into exon three of the mouse Pten gene, specifically Pten c.202 T>C, via standard cre-lox methodology (Fig. 1A). This mutation targets the sequence analogous to the ATP-binding motif B found in human PTEN [33,34]. Mice were backcrossed onto C57BL/6J (Jackson Laboratory) inbred strain for five generations to reduce the impact of genetic heterogeneity on the results. Genotyping was performed on genomic DNA from clipped toes per the Jackson Laboratory protocol using modified PCR primers: Y68H F1, 5′-GTTTCACAGCTGGTTGGAAGG -3′, and Y68H R1, 5′-TGTACCCAGCTCACAGACTTCC -3′. Mice were maintained on a 14:10 light: dark cycle with access to food and water ad libitum. The room temperature was maintained between 18 and 26°C. Animals were euthanized via CO2 asphyxiation followed by cervical dislocation. All experiments were conducted under protocols approved by the Institutional Animal Care and Use Committee (IACUC) at Cleveland Clinic. Additionally, for all experiments described, we utilized only male mice for our experiments
Western blot analysis
Cortical regions of the brain were isolated, snap-frozen, and stored at -80°C. Tissue was thawed on ice and lysed in RIPA buffer (10 mM Tris-Cl [pH 8], 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl), containing phosphatase inhibitor #2 (Sigma, St. Louis, Missouri, #P5726-5ML), phosphatase inhibitor #3 (Sigma, #P0044-5ML), and protease inhibitor (Sigma, #P8345-5ML). Lysates were quantified for protein concentration using bicinchoninic acid assay (BCA) assay, equalized to a concentration of 1 μg/μl of protein per sample, and finally 20 μg of protein was loaded to a 4-15% gradient polyacrylamide gel for SDS-PAGE separation. The separated proteins were transferred to a nitrocellulose membrane and blocked overnight at 4°C in 3% bovine serum albumin (BSA) in 1X Tris-buffered saline, containing 0.2% Tween-20 (TBST). Membranes were then washed with TBST and incubated with experiment-specific primary antibodies diluted in bovine serum albumin (BSA) overnight at 4°C. The following antibodies were used: PTEN (1:5000, #ABM-2025, Cascade Bioscience, Winchester, Massachusetts), IBA1 (1:500, #019-19741, Wako, Bellwood, Virginia), MBP (1:1000, MAB386, EMD Millipore, Burlington, Massachusetts), PLP (1:1000, ab28486, Abcam, Cambridge, Massachusetts), GAPDH (1:5000, 2118L, Cell Signaling, Danvers, Massachusetts), HSP90 (1:1000, 4874, Cell Signaling), Lamin A/C (1:1000, 2032, Cell Signaling), Beta-actin (1:5000, AM4302, Thermo-Fisher, Waltham, Massachusetts), phospho-AKT Ser473 (1:1000, 9271, Cell Signaling), AKT (1:1000, 4691, Cell Signaling), phospho-ERK1/2 (1:1000, 9101, Cell Signaling), ERK1/2 (1:1000, 9102, Cell Signaling), phosph-S6 (1:1000, 4858S, Cell Signaling), and C1q (1:500, ab71940, Abcam). We removed the primary antibody solution and performed three washes, 10 minutes per wash, with TBST. Blots were probed with goat anti-mouse secondary antibody IRDye800CW (1:20,000, #213965, LI-COR, Lincoln, Nebraska) or goat anti-rabbit IRDye680 (1:20,000, #213971, LI-COR) diluted in BSA, for two hours at room temperature. The membranes were washed three times, 10 minutes each in TBST, and imaged using the Odyssey CLx imaging system (LI-COR). Using ImageJ (National Institute of Health, Bethesda, Maryland, 1995), we performed densitometry analysis on these images to quantify protein expression.
Behavior Testing
To assess changes in social behavior, we employed the three-chamber sociability test according to a previously reported protocol [23,35]. Mice were placed in a center chamber for five minutes and then returned to their original cage. Next, the assessment consisted of a 10-minute trial, where the test mouse was returned to the central chamber and given a choice between two identical containers, one chamber containing a mouse, and the other an empty chamber. In order to measure preference for social novelty, the 10-minute trial was repeated two days later with a familiar mouse in one chamber and a novel mouse in the other. Time spent in each chamber and time spent in close contact with the containers were recorded and quantified using Noldus EthoVision software (Wageningen, Netherlands).
To assess repetitive behavior, we administered the marble burying test to our mice per a previously published protocol [36]. This trial is performed by placing 20 marbles atop clean bedding material and placing the trial mouse into the case for a 30-minute session. Upon completion of the trial the number of buried and non-buried marbles was scored.
Primary microglia cell culture
Mixed glia were obtained by trypsinization of postnatal day 2 (P2) cortices followed by plating on poly-D-lysine coated T-75 culture flasks. Mixed glia cultures, were maintained in DMEM (Lerner Research Institute Media Core, Cleveland, OH) with 10% FBS and 1% Penicillin and Streptomycin (Pen/Strep). Once the mixed glia cultures reached confluency at approximately DIV 10, they were agitated for one hour at 170 RPM. At this point, the supernatant was removed and spun down at 1200 RMP in order to isolate primary microglia. Isolated microglia were resuspended in DMEM with 10% FBS and 1% Pen/Strep and seeded on poly-D-lysine coated glass cover slips subsequently used for immunofluorescence staining and phagocytic assays at DIV 3 post-shaking.
Phagocytosis assay
We plated primary microglia at a density of 1 x 105 in a 12-well dish with PDL-coated coverslips for 48 hours in a 37°C cell incubator with 5% CO2 and 100% humidity. Next, we blocked 1 μm fluorescent beads (Sigma-Aldrich, #L1030) in FBS for one hour at 37°C at a ratio of 1:5 v/v. Florescent beads were diluted with DMEM to reach a final concentration of 0.01% (v/v). Microglial culture media was replaced with 250 μl DMEM containing beads, and incubated for one hour at 37°C in a cell incubator. Cultures were washed thoroughly five times with ice-cold PBS (Lerner Research Institute Media Core) and fixed in ice-cold methanol prior to immunofluorescent staining for Iba1 (1:500, #019-19741, Wako).
Immunofluorescence staining of brain tissue
Mice were euthanized and perfused with approximately 50 ml of 1X PBS. Brain tissue was then extracted and fixed in 4% PFA (pH = 7) for 24 hours at 4°C. Brains were then washed three times with PBS and cryoprotected in 30% sucrose dissolved in PBS for 94 hours at 4°C. Frozen brain sections were cut coronally to a width 10 μm on a cryostat and mounted on polarized glass slides (Fisherbrand Superfrost Plus microscope slides, #12-550-15, Fisher Scientific, Waltham, MA). OCT was removed by washing slides in PBS for 10 minutes and tissue was permeabilized with 3% Triton-X dissolved in PBS for 10 minutes. Slides were next washed three times for five minutes each in PBS and probed with experiment specific primary antibodies: Iba1 (1:500, #019-19741, Wako), Plp (1:1000, ab28486, Abcam), NeuN (1:250, MAB377, EMD Millipore), Olig2 (1:250, ab9610, Abcam), S100b (1:200, ab52642, Abcam), Gfap (1:250, sc-33673, Santa Cruz), Oxt (1:250, ab212193, Abcam), Pten (1:5000, #ABM-2025, Cascade Bioscience) and incubated overnight at 4°C. The following day, slides were washed with PBS for three times five minutes each. This was followed by incubation with secondary antibody for two hours: goat anti-mouse Alexa Fluor 568 (1:2000, #A11031, Thermo-Fisher) and goat anti-rabbit Alexa Fluor 488 (1:2000, #A11008, Thermo-Fisher). Post incubation, slides were washed and mounted in Vectashield medium with DAPI (Vector Laboratories, Burlingame, CA), coverslipped, and sealed with nail polish.
In vitro immunofluorescence staining
We cultured primary microglia on poly-D-lysine (PDL)-coated cover slips until DIV 14. Microglia were washed with ice-cold PBS and fixed in ice-cold methanol for two minutes. This was followed by three washes for five minutes each with ice-cold PBS. We then permeabilized the microglia with 0.03% Triton X-100 dissolved in PBS for four minutes. Next, cells were blocked with 10% normal goat serum for one hour at room temperature, followed by incubation with primary antibody Iba1 (1:500, #019-19741, Wako) diluted in 10% normal goat serum in PBS. Cells were then incubated in primary antibody overnight at 4°C. The following day cells were washed with PBS three times for five minutes and secondary was added, goat anti-mouse Alexa Fluor 568 secondary antibody (1:2000, #A11031, Thermo-Fisher) diluted in 10% normal goat serum in PBS. The cells were incubated in secondary antibody for two hours at room temperature, washed with PBS three times for five minutes, and coverslipped with Vectashield medium with DAPI (Vector Laboratories).
Immunofluorescence quantification
We captured images of brain sections and primary microglia as confocal images using a Leica TCS-SP8-AOBS inverted confocal microscope (Leica Microsystems, GmbH, Wetzlar, Germany). Brain sections and microglia cultures were imaged with a minimum of N = 3 biological replicates. ImageJ software was used to measure area and intensity of the stain and calculated integrated density of brain images. Additionally, ImageJ was used to measure area of stain per microglia in vivo to assess morphological changes.
Transcriptomic data analysis
We isolated total RNA from the cortex of eight PtenY68H/+ mice and seven Pten+/+ mice. Aliquots of roughly 60 ng/μL total RNA (average RIN score = 9.1; Additional file 1: Table S1) were prepared (TruSeq Stranded Total RNA – RiboZero Gold, Illumina, San Diego, CA) and then sequenced using an Illumina NOVA-Seq. The resulting Fastq sequences were subject to standard processing and quality control (QC) evaluation, using MultiQC v1.9 (https://multiqc.info/). Then, we performed an alignment to a the mouse reference genome (mm10) using Spliced Transcripts Alignment to a Reference (STAR) 2.7.5 (https://github.com/alexdobin/STAR) [37–39] and repeated a quality control evaluation using MultiQC v1.9. One Pten+/+ sample and three PtenY68H/+ samples were discarded due to a high proportion of repetitive sequences and generally poor alignment statistics (Additional file 1: Fig S1). Additionally, we used Salmon 1.8.0 (https://bioconductor.org/packages/release/workflows/html/rnaseqDTU.html) as an alternative method to count reads mapping to a present index of known cDNA transcripts. Subsequently, we performed DeSeq2 1.28.1 on STAR-aligned counts and Salmon-produced counts to assess differential expression (DE). These two methods were used to ensure concordance between both approaches. Genes experiencing DE were analyzed in RStudio 1.2.5001 using R 4.0.0 to construct volcano plots and heatmaps. Generally, a p-value (P < 0.05), fold change (Log2(Fold Change) ≥ 1.0 or (Log2(Fold Change) ≤ -1.0), and count (RPKM > 10) thresholds were used for these analyses. In order to assess the biological impact of the DE results, we used STRING (https://string-db.org/) and Ingenuity Pathway Analysis (Qiagen, Redwood City, California) software.
Statistical analysis
We analyzed normally distributed data using a one-way analysis of variance (ANOVA) or Student’s t-test, where appropriate (GraphPad Prism 8). After performing a one-way ANOVA (F), we performed a post-hoc Tukey-Kramer analysis. When data were not normally distributed, we performed non-parametric analyses including Mann-Whitney U and Kruskall-Wallis tests (H), where appropriate (Graph Pad Prism 8). P-values that are less than 0.05 were considered statistically significant.