FIGURE 3

FIGURE 3

Cultured oligodendrocytes, but not Schwann cells, are sensitive to specific SARM1 activators. (A) Dissociated and freshly plated P2 mouse Schwann cells cultured in DMSO or 100 μM vacor for 72 h show no cell death, judged by propidium iodide (P.I.) and DAPI nuclear staining and SOX10 immunocytochemistry. 96.4% ± 0.3 SOX10 positive cells survived after 72 h 100 μM vacor treatment (mean and SEM, n = 3: two-four mice, each from three litters producing three separate cultures, p = 0.53). Scale bar 25 μm. (B) Intracellular NAD⁺ levels do not change in Schwann cells when treated for 72 h with either 250 μM 3-AP (mean and SEM of Time zero (TZ) 0.61 ± 0.05 and 72 h 0.4 ± 1.15, n = 3, p = 0.63) or 100 μM vacor (mean and SEM of TZ 0.76 ± 0.23 and 72 h 0.46 ± 0.16, n = 3, p = 0.63) compared to control conditions (mean and SEM of H₂0: TZ 0.64 ± 0.1, 72 h 0.56 ± 0.25, n = 3, p = 0.81; mean and SEM of DMSO: TZ 0.85 ± 0.36, 72 h 0.45 ± 0.22, n = 3, p = 0.61). (C) Rat oligodendrocytes cultured in DMSO or 100 μM vacor for 72 h demonstrate substantial cell death judged by DAPI nuclear staining and SOX10 immunocytochemistry (n = 5: one rat from five litters producing five separate cultures). Scale bar 50 μm. (D) Quantification of SOX10 positive oligodendrocyte cell survival in response to 100 μM vacor treatment compared to DMSO control cultures. After six, 24 and 72 h of 100 μM vacor treatment, 47.9% ± 6.5, 21.6% ± 4.5 and 6.5% ± 1.2 of SOX10 expressing cells survived respectively (mean and SEM, n = 5, p = 0.02, p < 0.0001, p < 0.0001 respectively). (E) Cultured rat oligodendrocytes (OLs; SOX10 positive) contain substantial SARM1 protein using a polyclonal anti-SARM1 antibody generated by Y-PH. Zoomed in area (indicated by dashed box). Arrowheads indicate SOX10 positive SARM1 positive cells. Scale bars 50 μm. **P < 0.01, ****P < 0.0001.