In addition, a link between inhibition of lipid synthesis in transgenic mice overexpressing lamin B1 in oligodendrocytes and demyelination has recently been established by Rolyan et al
In addition, a link between inhibition of lipid synthesis in transgenic mice overexpressing lamin B1 in oligodendrocytes and demyelination has recently been established by Rolyan et al. of their CEP-32496 activity may affect myelin formation and integrity The role of sterol regulatory element-binding proteins (SREBPs), transcription factors regulating lipid synthesis, was investigated during oligodendrocyte differentiation cholesterol synthesis. This suggests SREBPs activity during oligodendrocyte maturation is required for myelin formation and integrity. Preventing SREBP maturation inhibited process growth from differentiating oligodendrocytes. This effect was accompanied by a reduction in the myelin protein MBP, whereas the immature oligodendrocyte marker GalC and PLP, another major myelin protein, were not affected. Rabbit Polyclonal to Lyl-1 We also observed a dramatic down-regulation of genes for both fatty acid and cholesterol biosynthesis following inhibition of SREBP processing, which correlated with a decline in SREBP mRNA expression. Furthermore, protein levels of fatty acid synthase (FASN), acetyl-CoA carboxylase (ACC) and HMGCR were dramatically reduced when SREBP maturation was prevented, and this effect correlated with decreased intracellular cholesterol synthesis. Finally, addition of cholesterol to the cultures at the onset of differentiation prevented the inhibition of process growth in maturing oligodendrocytes. Our data demonstrate that this control of lipid synthesis, notably cholesterol, via the SREBP pathway is essential for proper oligodendrocyte differentiation and myelin membrane production. Materials and Methods Dissociated Cerebral Cortex Oligodendrocyte Cultures All experiments were performed following the guidelines set forth by The Childrens CEP-32496 Hospital of Philadelphia Institutional Animal Care and Use Committee (IACUC). Cerebral cortices were isolated from newborn (males and females) CD1 mice (Charles River Laboratories, Malvern, PA, USA) with modifications from previously described protocols (Feigenson et al. 2009; Jensen et al. 2015) Briefly, the tissue was minced, triturated in 0.3% trypsin (Life Technologies) at 37C before 10% fetal bovine serum was added, and centrifuged at 200 g for 4 min. The supernatant was removed, Neurobasal medium (NBM; Life Technologies) supplemented with B27 made up of 0.5 mM L-glutamine and 5000 U/ml Penicillin/Streptomycin was added, and the tissue dissociated by trituration. Cells were plated at 4 105 cells/ml on poly-D-lysine (PDL; 10 g/ml; Sigma-Aldrich)-coated 100 mm cell culture dishes. After 24 hrs, the culture medium was replaced with growth medium (NBM/B27/L-glutamine/Penicillin/Streptomycin) made up of 10 ng/ml basic fibroblast growth factor (bFGF; R&D Systems, Minneapolis, MN) and 2 ng/ml platelet-derived growth factor alpha (PDGF). Cells were produced at 37C in 5% CO2 and were fed growth medium every 2 days until they reached approximately 80% confluence. Enriched oligodendrocyte cultures were generated using a altered washdown procedure (Feigenson et al. 2009). Cells were either plated at 0.8 105 cells/ml on PDL-coated glass coverslips in 24-well culture plates or at 1 105 cells/ml on PDL-coated 100 mm cell culture dishes. Cells were produced CEP-32496 until they reached approximately 70% confluence, and then were switched to differentiation medium (DM). To differentiate OPCs into mature oligodendrocytes, growth medium was replaced with DM, consisting of DMEM/F12 supplemented with N2 (Life Technologies), made up of 2 mM L-glutamine, 30% D-glucose, 0.4 g/ml L-Thyroxine (T4; Sigma-Aldrich), and 10 ng/ml biotin (Sigma-Aldrich). Drug Treatments OPCs were placed into DM with or without S1P inhibitor PF-429242 (1C3 M; Hay et al. 2007) for various periods of time. For reversibility experiments, cultures placed in DM were exposed to the S1P inhibitor for 3 days. Then, both S1P inhibitor-treated and control cultures were washed with fresh DM and incubated for an additional 1 CEP-32496 day before analysis. For rescue experiments, OPC cultures were switched to DM made up of 1 M water-soluble cholesterol (Sigma-Aldrich) in the presence or absence of 1 M S1P inhibitor, and were allowed to differentiate for 3 days. Cell Viability Cell survival was determined by incubating with 15 g/ml fluorescein diacetate and 4.5 g/ml propidium iodide (15 min, 37C). Cells.