Reagents and mice
The AAV9-GFP and AAV9-GFP-(GR)100 viruses were a kind gift from Dr. Leonard Petrucelli. Unless specified otherwise, cell culture reagents were obtained from ThermoFisher Scientific (Waltham, MA) and antibodies were obtained from Abcam (Cambridge, MA). C57BL/6J WT mice (#000664) and Nlrp3−/− mice (#021302) were obtained from The Jackson Laboratory (Bar Harbor, ME) and socially housed on a 12:12-h light/dark cycle with lights on at 07:00 h in a temperature-controlled (20 ± 2°C) vivarium. Mice were given food and water ad libitum and were bred to obtain mouse pups used in our studies. All procedures were approved by the Institutional Animal Care and Use Committee of the Icahn School of Medicine at Mount Sinai.
C9orf72 experimental model
Upon confirmation of pregnancy, WT and Nlrp3−/− dames were separated and monitored daily for litters. After the identification of a milk spot to confirm that newborn (p0) pups were being nursed, the pups were injected with AAV9-GFP or AAV9-GFP-(GR)100 by the ICV method [13]. Pups were cryoanesthetized by placing them in a padded 15 mL tube partially submerged in a slurry of water and crushed ice. Upon confirmation of anesthesia by toe-pinch, we identified the point of injection, approximately two-fifths of the distance between bregma and each eye. A Hamilton syringe (#7634-01; Reno, NV) with a 32 gauge needle (10 mm long, Hamilton #7803-04) was loaded with 4 µL of the AAV vector and placed 2 mm deep, orthogonal to the mouse’s head at the point of injection. 2 µL of the AAV vector was injected at a rate of 1 µL min into each lateral ventricle [14]. Upon completion of injections, each pup was rapidly returned to physiological temperatures by placing on a warming pad. The litters were returned to dames upon completion of injection for the entire litter and monitored for acceptance. Mice were weaned at p30 by sex. For confirmation of diffusion prior to studies, a small number of mice were injected with Trypan Blue and sacrificed two hours later for dissection of the brain and examination by stereoscopic microscope.
Behavioral Testing
After reaching 3 months of age, mice were tested through a battery of behavioral tests. All behavioral testing took place during the light phase of the day. On all days of behavioral testing, mice were acclimated to an anteroom directly adjacent to the behavioral testing room for 30 min. On Day One, mice were tested in the Open Field Test for basal anxiety and general locomotor impairments [15]. The Open Field apparatus consisted of a 40cm x 40cm x 40cm Plexiglas box with opaque white walls, situated within a dimly-lit room (200 lux). Mice were placed in the center of the apparatus and were allowed to freely explore for 10 minutes before being returned to their home cage. On Day Three through Five, mice were tested in a Hanging Wire Test for muscular impairments [16]. The Hanging Wire apparatus consisted of a 2 mm-thick wire suspended 35 cm over a layer of corn cob bedding, situated in a brightly lit room (500 lux). Mice were lifted from their home cage by the base of their tail and were placed near the wire until they grasped it with their forelimbs. The number of falls over a 2 min period was recorded. Falls in which the mice hung from the wire from their hindlimbs were excluded from the number of falls. At the end of the behavioral trial, mice were returned to their home cages. On Days Seven through Nine, mice were tested in a Contextual and Cued Memory Test in two Contexts. Context A was a 30cm x 24cm x 21cm conditioning chamber (Med Associates, Fairfax VT) within a room with white walls and bright lighting. Context A had a bare metal grid floor, bare grey walls, bright lighting, and background fan noise. Context A was cleaned with a 0.5% hydrogen peroxide solution (Virox, Oakville Canada) between each trial. Context B was a chamber of the same dimensions within a room with dim lighting. The chamber had a white plastic floor, curved white plastic walls, dim lighting, no background fan noise, and was scented with 0.25% benzaldehyde in 70% ethanol. Context B was cleaned with a 70% ethanol solution between each trial. On Day Seven, mice were allowed to explore Context A for 4 min. At 180s, white noise (85 dB) played for 30 s and was co-terminated with a footshock (2 s, 0.75 mA). After the 4 min trial, the mouse was returned to its home cage. On Day Eight, mice were allowed to explore Context A for 4 min in the absence of white noise and footshock (Context Recall). On Day Nine, mice were allowed to explore Context B in the constant presence of white noise (85 dB, Cue Recall). For all tasks, the behavior was analyzed and recorded with Any-Maze v6.0 (Stoelting, Wood Dale IL).
Immunohistochemistry
After completion of behavioral studies, a subset of mice was deeply anesthetized with ketamine/xylazine (100 mg/kg + 10 mg/kg, intraperitoneally), and then perfused transcardially with cold sterile PBS followed by 4% PFA in PBS. Brains were removed, drop-fixed in 4% PFA overnight, then washed once with cold PBS and stored in PBS. Tissue sections (50 µm thick) were taken with a vibratome (Leica; Wetzlar, Germany) and were stored in PBS with 0.02% sodium azide (w/v). Sections were washed in PBS followed by 10 min permeabilization in 0.1% Triton X-100 in PBS (PBST). Sections were then incubated in blocking solution (5% normal goat serum in PBST) for 1.5 hr. The sections were washed three times with PBST and incubated with primary antibodies diluted in blocking solution: 1:500 Rabbit anti-Iba1 (ab178846, Abcam) and 1:250 chicken anti-GFP (A10262, ThermoFisher) overnight at 4°C. Post incubation, the sections were washed three times in PBST and incubated with secondary antibodies diluted in blocking solution for two hours at room temperature (RT): Goat anti-Rabbit conjugated Alexa Fluor 568 (Abcam #175471, 1:500) and 1:500 Goat anti-Chicken Alexa Fluor 488 (A11039, Thermofisher). The sections were washed three times in PBST and incubated in 1µM DAPI solution (#ab228549, Abcam) for 5 min. The sections were washed twice in PBS and mounted on slides using ProLong Diamond Antifade Mountant (P36970, ThermoFisher).
Stereology And Cell Morphology
Assessment of neuronal loss was assessed by GFP-expressing neurons count in the in the layers IV and V of the primary somatosensory (S1) cortex. Similarly, microglia density was determined in the same layers IV and V of the S1 cortical region by MBF Stereo Investigator (Williston VT). The number of sections required for each region was determined by an initial pilot study which found that six sections were needed for the S1 cortical region, each spaced equidistantly for both GFP-expressing neurons and IBA-1 immunopositive microglia. A widefield microscope (AxioImager M2/Z2, Carl Zeiss, Oberkochen Germany) along with MBF Stereo Investigator were used to visualize each section and to determine the number of microglia in each region. Using the Optical Fractionator workflow, the region of interest was traced onto each section based on the mouse brain atlas by Paxinos and Franklin. This was followed by a systematically random grid overlay consisting of squares measuring 300 µm by 300 µm, covering the region of interest. From each of these squares, an unbiased sampling region (100 µm by 100 µm) was used to manually count microglia somata in the area. Once each section was completed, the software ran an algorithm to estimate the number of microglia in the region. In parallel for IBA-1 immunopositive microglia, the Cavalieri estimator in the software was used to determine the volume of each region. The microglia count and volume were then used to determine the microglia density in the S1 cortical region. For assessment of cell morphology, images of coronal sections were taken on a Zeiss LSM880 Airyscan confocal microscope (Oberkochen, Germany) using an X20/0.8 NA air immersion objective controlled by Zeiss Zen Black software. For 3D analysis, z-stack images were obtained by capturing an image every 0.7µm covering the entire 50µm-thick section. Images were deconvoluted using AutoQuant X3.1 (Media Cybernetics, Rockville MD) and 3D analysis was performed using Imaris 9.1.2 (Bit Plane Inc, Concord MA) using the surface tool to reconstruct the soma and the filaments tool to reconstruct the branches.
Western Blotting
After completion of behavioral trials, a subset of mice was sacrificed and brain regions were immediately frozen on dry ice. Cerebral cortex was lysed with 1X Cell Lysis Buffer supplemented with 1 mM PMSF (Cell Signaling #8553S, Danvers MA) and Protease Inhibitor Cocktail (Sigma-Aldrich #11873580001). Tissue lysates were separated by electrophoresis and then transferred to a nitrocellulose membrane. Membranes were blocked for 1 h at RT, followed by treatment with primary antibodies (anti-caspase-1 antibody, 20B-0042; anti-IL-1β antibody 6243S; anti-α-tubulin antibody, T9026) at 4°C for overnight. Each membrane was then treated with a secondary antibody conjugated with horseradish peroxidase (anti-rabbit-or anti-mouse IgG -HRP-antibodies) for 1h at RT. Each band was detected using chemiluminescence detection kit (32106; Thermo Fisher Scientific) and data was analyzed using image J software to measure relative protein expression.
Cortical Microglia Cultures
Cortices from 1–3 day-old C57BL/6J mouse pups were isolated, digested, and seeded at a density of 8 cortices per 24-well plate (12ml total volume). Every three days, the medium (DMEM + 10% FBS + 1% penicillin-streptomycin) was replenished. After 3 weeks, mixed glial cultures reached confluence and were isolated by mild trypsinization as previously described [17]. Briefly, cells were washed with culture medium without FBS and treated with a mixture of trypsin (0.25% without EDTA) and DMEM-F12 medium in a 1:3 ratio. After 40 min incubation, mixed glial cells detached and left a layer of microglia attached to the bottom of the culture dish. Pure microglia were isolated by 15 min incubation with trypsin (0.05% with EDTA) at 37°C followed by gentle shaking. Cells were counted and seeded in 24-well plates at a density of 7.5 x 104 cells/well.
Cortical Neuron Cultures
Cortices from 3-day-old C57BL/6J mouse pups were isolated and finely diced in ice-cold HBSS. Cortices were then incubated in 10X Trypsin Solution (Sigma #59427C) with DNase I (Sigma #D4513) for 15 min at 37°C with period inversion. Cells were then spun at 200 g for 5 min, and the pellet was triturated with a serological pipette and strained in a 40 µm cell strainer (BD Falcon #352340). Centrifugation followed by trituration was repeated once, and then the pellet was spun down and resuspended in Neurobasal medium supplemented with 0.25% GlutaMAX, 2% B-27, 10% Fetal Bovine Serum, and 1% Penicillin/Streptomycin at a density of 5 x 105 cell/mL. Two hours later, the medium was changed to Neurobasal medium supplemented with 0.25% GlutaMAX, 2% B-27, and 1% Penicillin/Streptomycin, and half the medium was replenished every 3–4 days afterward. Three days later, cells were infected with 2 x 1010 vg/mL AAV. Expression was confirmed by fluorescence microscopy. Cell supernatant and lysates were collected for assessment of LDH release and CXCL10 production (R&D Systems #DY466).
Analysis Of Cytokines
IL-1β in microglia culture supernatant was measured with Mouse IL-1β /IL-1F2 DuoSet ELISA Kit (R&D Systems DY401, Minneapolis MN) according to the manufacturer’s instructions. CXCL10 was measured in neuronal culture supernatant with Mouse CXCL10 DuoSet ELISA (R&D Systems DY466-05, Minneapolis MN) according to the manufacturer’s instructions. Primary microglia were stimulated with recombinant Mouse CXCL10 protein (R&D Systems 466-CR- 608 050/CF, Minneapolis MN) and incubated with Mouse CXCL10 antibody (R&D Systems AF-466- 609 NA, Minneapolis MN). TNF-α and IL-1β were measured in the supernatant by ELISA with Mouse TNF-α Quantikine ELISA Kit (R&D Systems MTA00B, Minneapolis MN) and Mouse IL-1β/IL-1F2 DuoSet ELISA Kit according to manufacturer’s instructions.
Gene Expression And Rnaseq
Total RNA was isolated using the RNeasy Minikit (QIAGEN #74106; Hilden, Germany) and precipitated by the ethanol/sodium acetate method. RNA concentration and quality were initially measured using a Nanodrop 2000 (Thermofisher). Secondary analysis of concentration and quality was conducted by the Genomics CoRE Facility at the Icahn School of Medicine at Mount Sinai using Qubit RNA BR Assay Kit (ThermoFisher #Q10211). Library construction and RNA sequencing were performed by Novogene (Durham, NC). RNAseq data were processed using the NGS-Data-Charmer pipeline. Briefly, adaptors and low-quality bases were trimmed from reads, which were then aligned to the mm10 genome using HISAT2 (version 2.2.1). Read counts in the mm10 GENCODE annotation (version M22) were generated using FeatureCounts DESeq2 (version 1.24.0, R version 3.6.1) was then used to calculate differential expression from the read counts. The R package ‘biomaRt’ (version 2.40.5) was used to translate ensembl ids into common gene symbols.
Gene Ontology
Mouse genes involved in inflammatory processes were extracted from the Mouse Genome Informatics group database (MGI). MGI mouse genes with the GO term “Inflammatory response‟ were extracted. Ingenuity Pathway Analysis was applied to all differential genes identified in the previous analysis.
Quantitative Reverse Transcription Pcr
For qPCR analysis, gene expression was measured in 4 replicates by Power UP SYBR Green Master Mix (ThermoFisher #A25778) using an ABI PRISM 7900HT Sequence Detection System. Hypoxanthine phosphoribosyl transferase (Hprt) expression level was used as an internal control and data was normalized using the 2−ΔΔCt method [18]. Levels of target gene mRNA were expressed relative to those of GFP + Veh mice for in vivo studies. Primers used in this study were designed using Primer-BLAST software [19] and are listed in Table S1.
Tissue Processing
Four mice from each group (GR100 and GFP) were processed for sequencing in cerebral cortex (CTX) per mouse. After assessing sample quality, one FC sample was excluded due to poor quality, and all tissue samples from a single mouse were removed due to sample misclassification.
Statistical Analysis
All figure values are presented as the mean and standard error of the mean (s.e.m.). Statistical tests are indicated in the figure legends. A confidence interval of 95% was used for all analyses. In all studies, outliers (> 2 SD from the mean) were excluded. All statistical analysis was performed using GraphPad Prism 9 software (GraphPad Software, San Diego CA). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns not significant. Statistically insignificant trends are indicated by p-value.