Fig 2: mTOR is necessary for normal cholesterol biosynthesis in oligodendroglia(A and B) Top five pathways dysregulated in mTOR cKO spinal cord (A) and brain (B). IPA was done on genes differentially expressed in all mTOR cKO cells compared with all control cells; the most significantly altered pathways are shown. Blue, downregulated genes; orange, upregulated genes.(C) Inhibiting mTOR in vitro reduces mRNA expression of cholesterol biosynthesis genes. Expression of Hmgcr, Hmgcs1, and Fdps by qRT-PCR in primary rat OPCs differentiated for 3 days in the presence or absence of 10 nM rapamycin to inhibit mTOR is shown. Expression was normalized to Gapdh as a housekeeping gene and then normalized to control. Data represent six replicates over two independent OPC preps. Values are expressed as the mean ± SD.(D and E) Cholesterol biosynthesis enzymes are reduced in mTOR cKO spinal cord (D) and brain (E), with a greater impact in the spinal cord. Representative western blots show expression of HMGCR, HMGCS1, FDPS, and ACAT2 in control and mTOR cKO spinal cord (D) and brain (E) O4+ cells. HMGCR, HMGCS1, and FDPS expression was normalized first to β-actin and then to control. ACAT2 expression was normalized first to β-tubulin and then to control. Graphs present data from three animals/genotype/CNS region. Values are expressed as the mean ± SEM.(F, G, and H) mTOR cKO O4+ cells have lower cholesterol content. Total cholesterol measured in isolated O4+ cells from P10 spinal cords (F), P14 brains (G), and P18 brains and spinal cords (H) and normalized to cell number. Graphs present data from three animals/genotype/CNS region. Values are expressed as the mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.

Fig 3: Deficits in cholesterol biosynthesis persist at 8 weeks of age, myelin protein expression is downregulated, and brain oligodendroglia undergo apoptosis(A and B) Cholesterol biosynthesis enzymes are downregulated at 8 weeks in mTOR cKO corpus callosum. Representative western blots showing expression of HMGCR and FDPS (A) and HMGCS1 (B) in control and mTOR cKO microdissected corpus collosum. Graphs present data from three control and five mTOR cKO animals.(C) Representative western blots showing expression of MBP and PLP in control and mTOR cKO microdissected corpus collosum at 8 weeks of age. Graph presents data from four control and four mTOR cKO animals. For (A), (B), and (C), control and mTOR cKO samples were run on the same western blot. Expression was normalized first to β-tubulin or actin and then to control. Values are expressed as the mean ± SEM.(D) MOG expression is dysregulated in mTOR cKO corpus callosum. Representative images of MOG staining show area of positivity in corpus callosum. Scale bar, 500 μm. Graph presents data from four control and five mTOR cKO animals. All values are expressed as the mean ± SEM.(E) Representative images of SOX10 (blue, oligodendroglial marker) and cleaved caspase-3 (red, marker of apoptosis) immunostaining from control and mTOR cKO corpus callosum at 7–8 weeks of age. Scale bar, 50 μm. Arrows indicate cleaved caspase-3+/SOX10+ cells. Graphs present data from six animals/genotype. All values are expressed as the mean ± SEM. *p < 0.05; **p < 0.01.

Fig 4: Quantitative assessment of myelin-related protein densities at 16 days of age. (A) Western blot analysis of MBP, PLP1, HMGCS1, MAG, and MOG protein (blue font) expression in tissue isolated from the PFCWM, CC-Genu, CC-Body, and CC-Splenium of control (C) and germ-free (GF) animals at P16. Loading controls: β-Actin for MBP, GAPDH for PLP1, HMGCS1, MAG, and MOG. (B) Quantification of protein expression normalized to respective loading controls. Individual male and female animals are marked by orange triangles and circles, respectively. Data expressed as mean ± SEM, n = 3 animals/group. No significant differences determined by unpaired student’s t-test or Mann-Whitney U test. PLP1, proteolipid protein; HMGCS1, Hydroxymethylglutaryl-CoA synthase; MAG, myelin-associated glycoprotein; MOG, myelin oligodendrocyte glycoprotein.

Fig 5: Membrane proteins associated with cholesterol biosynthesis in the cerebellum of Mecp2-null mice. Protein levels of (a) SREBP2 and (b) HMGCS1 were measured from the cerebellum of Mecp2-/Y and their wild-type littermate mice at 7 weeks of age. Mecp2-/Y (n = 6, triangles) and wild-type mice (n = 6, circles). Graph bars represent means ± SEM. Two-tailed unpaired Student’s t-tests were used to compare the data, and no differences were observed.