Fig 2: The NAA pathway affects metabolic pathways, lipid synthesis and energy metabolism.Mature iBACs with stable overexpression of Nat8l (Nat8l o/e) or control cells were used for the analysis. (A) Analysis of Nat8l mRNA expression. (B) Analysis of Asp-NAT, ASPA and ACSS2 protein expression (n = 3). (C) Aspa activity was determined by fluorometric detection of aspartate (n = 3). (D) Transcriptomics of 250 genes up-regulated by Nat8l o/e were submitted to DAVID functional annotation using KEGG pathways. Count shows the number of mapped genes. Benjamini-Hochberg's correction was used for adjusting p-value for multiple testing. (E) NAA concentration in iBACs analyzed with HPLC/HRMS (n = 3). (F–G) 13C-labeled metabolite concentrations measured by NMR after cultivating iBACs for indicated time points in the presence of 10 mM 13C-glucose. Metabolite concentration was normalized to internal scaling factor (n = 5). (H–I) Metabolites measured by HPLC/HRMS (n = 3). (J–K) 13C metabolite concentrations measured by NMR after cultivating iBACs for indicated time points in the presence of 10 mM 13C-glucose. Metabolite concentration was normalized to internal scaling factor (n = 5). (L) Fold change of measured amino acids by HPLC/HRMS. Significantly reduced and increased amino acids are depicted in yellow and red, respectively (n = 3). (M) Fold change of BCAA measured by NMR (n = 3). If not otherwise mentioned, data are shown as mean ± SD. Statistical significance was calculated using two-tailed Student's t-test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig 3: Effect of OPC ablation on other neural cell types(A) Immunolabeling of GFP and CC1 on coronal sections of the rostral corpus callosum of ablated and non-ablated control mice.(B) Mean densities of CC1+GFP+ cells in the rostral forebrain (gray and white matter combined). OPC ablation did not change mature OL density at the assessed time points.(C) Densities of ASPA+ OLs in the corpus callosum remained similar in OPC-ablated versus control mice.(D) Generation of newborn OLs (ASPA+EdU+ cells) after infusion was transiently reduced in OPC-ablated mice. Two-way ANOVA revealed overall effects of time (p < 0.0001) and treatment (p < 0.01).(E) Black-Gold II-stained coronal sections of the rostral corpus callosum of vehicle- or AraC-infused mice at 0, 10, and 20 dppr.(F) Mean Black-Gold II intensity across the entire corpus callosum.(G) Analysis of ALDH1L1+ astrocyte density in the rostral forebrain revealed an overall effect of OPC ablation (p = 0.0038) and a significant increase in the number of activated astrocytes was observed at the end of AraC infusion (*p = 0.0153).(H) GFAP immunohistochemistry revealed minimal effect of OPC ablation on astrocyte morphology in the cerebral cortex.(I) Densities of Iba1+ cells in the rostral corpus callosum of Pdgfra+:DTA+ mice that received no treatment (gray bar), TAM alone (blue bars), or either TAM + vehicle (black bars) or TAM + AraC (magenta bars) and were assessed at 0, 10 or 20 dppr.(J) Iba1 immunohistochemistry revealed that OPC ablation transiently reduced microglial process complexity in the cerebral cortex.(K) Densities of NeuN+ cells in the rostral cerebral cortex revealed no effect of OPC ablation on cortical neuron density.Data represent mean ± SEM. Unpaired two-tailed Student’s t test (I) and two-way ANOVA with Sidak’s multiple-comparisons test (B–D, F, G, and K); *p < 0.05 and ***p < 0.001. CC, corpus callosum; LV, lateral ventricle. Scale bars, 200 µm (A), 150 µm (E), 25 µm (H), and 20 µm (J). See also Figure S3.

Fig 4: Combined treatment of Sephin1 and BZA accelerates remyelination.GFAP-tTA;TRE-IFN-? mice were released from Dox at W0 and given cuprizone chow for 5 weeks. Treatment with vehicle, Sephin1, BZA or combined BZA and Sephin1 was started at W3, and the corpus callosum was harvested and processed at W0 and after 3 weeks of remyelination (W8). (A) Immunofluorescent staining for ASPA and DAPI in the corpus callosum at W8 in the presence of IFN-?. Scale bar = 100 µm. (B) Representative EM images of axons in the corpus callosum at W8 in the presence of IFN-?. Scale bar = 1 µm. Quantifications of the number of cells positive for ASPA (C), percentage of remyelinated axons (D) and g-ratios of axons (E) in the corpus callosum areas at W0 and W8 in the presence of IFN-?. Two males and two females in each treatment group and four males at W0. Data are presented as the mean ± SEM (n = 4 mice/group). *p<0.05, **p<0.01, ****p<0.0001. Significance based on ANOVA.Figure 8—source data 1.The number of ASAP+ cells, myelinated axons and g-ratio of various treatments.

Fig 5: Oligodendrocyte and OPC number are increased in the MNTB independent of tonotopy in Fmr1 KO mice compared to B6. Representative images for OLs (yellow/magenta ASPA/sox-10 marker, arrowhead) and OPCs (magenta sox-10 marker, arrowhead without ASPA (yellow) staining) in Fmr1 KO (A) and B6 (B) mice. Scale bar is 100 µm. Box plots show data from each individual measurement for Fmr1 KO (left) and B6 (right) for each animal (as indicated by different colors within the boxplots) for the total, medial, center, and lateral MNTB for OLs (C). N is 7 Fmr1 KO animals and 6 B6 animals. Box plots show data from each individual measurement for Fmr1 KO (left) and B6 (right) for each animal (as indicated by different colors within the boxplots) for the total, medial, center, and lateral MNTB (D) for OPCs. N is 4 Fmr1 KO animals and 5 B6 animals. *p < 0.05.