Fig 2: Anti-MOG antibodies are necessary for EAE sera to induce aB-crystallin upregulation in primary oligodendrocyte cultures. (a) ELISA of anti-MOG antibody titers demonstrates efficacy of anti-MOG antibody depletion. (b–d) EAE serum, but not that depleted of anti-MOG antibodies, is able to immunolabel unfixed oligodendrocytes (red, anti-IgG; green, olig2). (e–g) Oligodendrocyte cultures exposed to 1% sera for 90 min followed by overnight recovery were immunolabeled with antibodies against CNPase (red) and aB-crystallin (cryaB, green). (h) Western blot with quantification showing upregulation of aB-crystallin following 90 min exposure to 1% EAE serum, but not with EAE serum depleted of anti-MOG antibodies. (i) Reactive oxygen species were quantified in oligodendrocyte cultures using CellROX assay. Cells exposed to H2O2 had increased ROX positivity, but not cells exposed to 1% serum. Overnight exposure of cells to 10% serum or H2O2 served as positive controls. (j) Oligodendrocyte toxicity was assessed using a fixable LIVE/DEAD assay demonstrating increased cell death upon exposure to H2O2, and also EAE serum when exposed overnight. (k) Lentiviral delivery of shRNA against aB-crystallin successfully reduced aB-crystallin expression in oligodendrocyte cultures compared to delivery of a scrambled control (SCR) shRNA sequence. All cultures were exposed to 10% EAE serum for 90 min followed by overnight recovery. (l) Oligodendrocytes treated with shRNA targeting aB-crystallin had increased cell death upon 90 min exposure to 1% EAE sera compared to those treated with scrambled shRNA, or exposure to sham sera. Dashed line indicates the untreated control levels (as shown in panel j). Scale bar = 50 µm; *p < .05, **p < .01, ANOVA

Fig 3: Ras inhibition inhibits oligodendroglial cell morphological differentiation. (A,B) FBD-102b cells were treated with vehicle or Lanafamib (5 µM) and were allowed to be differentiated for 0 or 3 days. Differentiation efficiencies were divided into three categories and depicted in graphs (**, p < 0.01; n = 5 fields). The number of branches in cells is also counted and shown (**, p < 0.01; 50 cells). (C,D) Cells at 3 days following the induction of differentiation were collected, lysed, and immunoblotted with an antibody against PLP1, CNPase, SOX10, or actin. Immunoreactive band intensities were also compared to be depicted in graphs (**, p < 0.01; n = 3 blots).

Fig 4: Postnatally induced heterotopia do not develop aberrant axons or myelination. (a) Confocal images of an immunolabeled coronal section from P30 mouse cortex, showing no changes in myelination (CNPase) or axonal pathfinding (NF-H) associated with a P0-induced heterotopion (NeuN; arrowheads), compared with its contralateral control region (left). (b) In contrast, neuronal heterotopia induced in utero at E15 (NeuN; arrowheads) show dramatic changes in local layer I myelin (CNPase) expression and patterning. (c) Quantifications of CNPase expression, showing no significant differences in the myelin or oligodendrocyte cell body densities (n = 6 animals) between postnatally induced heterotopia and control regions. Each point represents the P0-induced layer I heterotopion or control region from one animal, with horizontal lines and error bars denoting mean and SEM, respectively. Wilcoxon matched-pairs signed rank nonparametric test was used to determine significance; NS, no significance. NF-H, neurofilament heavy chain. Descriptive statistics are given in Supplementary Table 1. Scale bars, 100 µm (a, b).

Fig 5: Knockdown of ACE2 by gRNA promotes oligodendroglial cell morphological differentiation. (A,B) FBD-102b cells were transfected with the plasmids encoding ACE2 gRNA with CasRx or control plasmids and were allowed to be differentiated for 0 or 3 days. Differentiation efficiencies were divided into three categories and depicted in graphs (**, p < 0.01; n = 5 fields). The number of branches in cells is also counted and shown (**, p < 0.01; 50 cells). (C,D) Cells at 3 days following the induction of differentiation were collected, lysed, and immunoblotted with an antibody against PLP1, CNPase, SOX10, or actin. Band intensities were also compared to be depicted in graphs (**, p < 0.01 and *, p < 0.05; n = 3 blots).