GoKawiilHuman tissue sample collection and compliance
Single-nucleus capture and RNA-seq analysis of age- and sex-matched human control (9) and MS (12) cortices was performed previously2. For this study, human post-mortem brain samples with MS grey matter pathology were sourced from the UK Multiple Sclerosis Tissue Bank at Imperial College London. Ethical approval for the use of these samples was granted by the National Research Ethics Committee in the UK (08/MRE09/31). In total, nine snap-frozen brain blocks (four from patients with MS and five from individuals without diagnosed neurological disease) were analysed using immunohistochemistry. For CUX2 and ATF4 expression analyses, the 25-year-old sample was obtained from University of California, San Francisco’s Pediatric Neuropathology Research Laboratory and collected in accordance with guidelines established by the Committee on Human Research (UCSF) and approved by the Institutional Review Board. Participant details are provided in Supplementary Table 1.
Primary rat glial cultures
Fresh Wistar/Han postnatal day 5 rat heads were obtained from Charles River Laboratory (UK) in ice-cold Hibernate-A medium (Thermo Fisher Scientific, A1247501). Whole brains were extracted, homogenized and subjected to centrifugation (3 min, 100g at room temperature). The supernatant was removed and the tissue was resuspended in a dissociation solution (34 U ml−1 papain (Lorne, LS003126) and 20 μg ml−1 DNase type I (Millipore, 10104159001) in Hibernate-A medium) that had been heat-activated at 37 °C and shaken for 20–30 min at 55 rpm and 37 °C. The tissue was centrifuged for 5 min at 200g, the supernatant was aspirated and then the tissue was resuspended in a cold neutralizing solution (Hibernate A medium supplemented with 1× B27 (Thermo Fisher Scientific, 17504044) and 2 mM sodium pyruvate (Sigma-Aldrich, S8636)). Tissue was triturated using a serological pipette ten times and, after 1 min rest, the cell suspension (supernatant) was transferred through a 70-μm cell strainer into isotonic Percoll (90%, Sigma-Aldrich, GE17-5445-01). The remaining tissue aggregates were subjected to a further three rounds of trituration and 1 min resting, and the supernatant was transferred into isotonic Percoll. The cell suspensions were brought to a final Percoll concentration of 22.5% by adding DMEM/F12 with HEPES (Thermo Fisher Scientific, 11-039-021) and centrifuged for 20 min at 800g. The supernatant was removed, and the brain cell pellet (bottom 3 ml) was resuspended in HBSS without calcium and magnesium (Thermo Fisher Scientific, 14170112), then centrifuged for a further 5 min at 300g; the supernatant was next removed and the cells were resuspended in 1 ml of red blood cell lysing buffer (Sigma-Aldrich, R7757). The suspension was incubated for 90–120 s before washing with HBSS without calcium and magnesium. The cells were centrifuged for 5 min at 300g before resuspending in 500 μl sorting solution (1× PBS supplemented with 2 mM EDTA (Thermo Fisher Scientific, 15575020), 2 mM sodium pyruvate, 0.5% BSA and 25 μg ml−1 insulin (Sigma Aldrich, I9278)). The cells were counted and 2 μl of CD11b/c rat microbeads (Miltenyi Biotec, 130-105-634) was added per 1 million cells and incubated for 15 min at 4 °C with gentle agitation on a tube roller. A further 8 ml of sorting solution was added before the cell/bead mix was centrifuged for 5 min at 300g. The supernatant was removed, and cells were resuspended in 2 ml sorting solution. Cells were added to an LS column (Miltenyi Biotec, 130-042-401) fitted to a QuadroMACS Separator (Miltenyi Biotec, 130-091-051) stand that had been pre-wet with sorting solution. The column was washed three times with 2.5 ml cold sorting solution. Microglia were collected from the column by disassociating the column from the stand, adding 2 ml microglial medium (DMEM/F12 supplemented with 60 μg ml−1 N-acetyl cysteine (Sigma-Aldrich, A9165), 10 μg ml−1 insulin, 1 mM sodium pyruvate, 1× SATO (100× SATO: 1.61 mg ml−1 putrescine dihydrochloride (Sigma-Aldrich, P5780), 4 µg ml−1 sodium selenite (Sigma-Aldrich, S526110), 60 µg ml−1 progesterone (Sigma-Aldrich, P8783), 41.25 mg ml−1 BSA (Sigma-Aldrich, A4919), 5 mg ml−1 apo-transferrin (Sigma-Aldrich, T1147) in DMEM/F12 (Gibco, 11039021)) before using a plunger to gently release the cells. Microglia were counted and seeded at 400,000 cells per well on 12-well plates precoated with 5 μg ml−1 poly-d-lysine (Sigma-Aldrich, P6407). To obtain astrocytes, the cell flow-through was further sorted to remove oligodendroglia. Flow-through suspensions were centrifuged for 5 min at 300g and the supernatant was aspirated. Cells were resuspended in 500 μl of sorting solution before adding 1.7 μl of mouse IgM anti-A2B5 antibody (about 1:250, Sigma-Aldrich, MAB312, AB-94709) and incubated for 25 min at 4 °C with gentle agitation. The cell suspension was diluted with sorting solution and centrifuged for 5 min at 300g. The supernatant was aspirated, and the pellet was resuspended in 160 μl of sorting solution with 40 μl of rat anti-mouse IgM microbeads (Miltenyi Biotec, 130-047-302) and further incubated for 15 min at 4 °C, with gentle agitation at the 5- and 10-min mark. The cells were sorted through the LS column as described above for microglia but the flow-through was collected for further astroglial isolation. The solution was centrifuged for 5 min at 300g and then resuspended in astrocyte medium (DMEM/F12 without L-glutamine (Thermo Fisher Scientific, 21331020) supplemented with 1 mM sodium pyruvate, 60 μg ml−1 N-acetyl cysteine, 1× N2 (Thermo Fisher Scientific, 17502-001), 1× B27 (Gibco, 12587-010), 1% penicillin–streptomycin, 10 μg ml−1 insulin and 10 ng ml−1 human recombinant HB-EGF (Peprotech, 100-47-100UG)). The cell solution was added to a flask coated with 5 μg ml−1 poly-d-lysine and glia were allowed to attach for 1 h at 37 °C. After 1 h, the unattached cells (predominantly containing neurons and endothelia) were washed away, and fresh astrocyte medium was provided. After 24 h, astrocytes were passaged by first washing with PBS minus calcium and magnesium chloride (Sigma-Aldrich, D8537) before replacing with trypsin-EDTA (0.05%; Gibco, 25300054) at 37 °C for 5 min. Astrocyte medium was added and astroglia were collected and centrifuged at 300g for 5 min. Astrocytes were resuspended in astrocyte medium and plated at 300,000 cells per well of 12-well plates. Cells were then subjected to rat IFNγ (Thermo Fisher Scientific, 400-20) before collection.
Demyelination and neuroinflammation models
ROSA26-eGFP-DTA mice52 were bred to the PLP/creERT transgenic mice53 to generate the PLP/creERT;ROSA26-eGFP-DTA (DTA) mice, as previously described17,18. All mice were on the C57BL/6J background. PLP/creERT;ROSA26-eGFP-DTA (DTA) mice and ROSA26-eGFP-DTA littermates (control; aged 5–7 weeks) were injected intraperitoneally with 0.8 mg of 4-hydroxytamoxifen (Hello Bio, HB6040) per day, either for 4 consecutive days (males) or 3 consecutive days (females). Brain tissue was collected from the DTA and control animals at the indicated timepoints after tamoxifen injection. Equal numbers of male and female mice were used for experiments, which were conducted in compliance with Northwestern University’s Animal Care and Use Committee (IACUC) guidelines.
Myrffl/fl were crossed to Sox10creERT mice to generate Myrf-cKO mice as previously described20. All of the mice were on a mixed C57BL/6N and C57BL/6J background and creERT-negative littermates served as non-demyelinated controls. Eight-week-old mice were injected intraperitoneally with 100 mg per kg tamoxifen (T5648, Sigma-Aldrich) dissolved in corn oil (C8267, Sigma-Aldrich) for 5 days. These experiments were conducted in compliance with the Institutional Animal Care and Use Committee of OHSU.
Ectopic IFNγ expression model
Gfap-tTA mice on the C57BL/6J background were crossed with TRE-Ifng mice on the C57BL/6J background to generate Gfap-tTA;TRE-Ifng double-transgenic mice, as described previously54,55. To suppress IFNγ transcription, doxycycline (200 ppm) was administered in the diet (Envigo) from conception. Mice were maintained on doxycycline throughout development, and doxycycline repression was lifted at 8 weeks of age. Brain tissue was collected from both double-transgenic and single-transgenic control mice at 12 months after doxycycline withdrawal. These experiments were conducted in compliance with Northwestern University’s Animal Care and Use Committee (IACUC) guidelines.
KO and cKO lines
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