Investigating the Function of MCL-1 During Oligodendrocyte Development

Abstract

Oligodendrocytes are the myelinating cells of the central nervous system. Regulation of the early stages of oligodendrocyte development is critical to the function of the cell. Specifically, myelin sheath formation is an energetically demanding event that requires precision, as alterations may lead to dysmyelination. Fatty acid β-oxidation has been shown to be critical for the function of oligodendrocytes. We previously showed that MCL-1, a well-characterized anti-apoptotic protein, is required for the development of oligodendrocytes in vivo. Further, MCL-1 regulates long-chain fatty acid β-oxidation in cancer cells through its interaction with Acyl-CoA Synthetase Long-Chain Family Member 1 (ACSL1), an enzyme responsible for the conversion of free long-chain fatty acids into fatty acyl-CoA esters. Here, we developed novel tools to investigate the function of MCL-1 during oligodendrogenesis. First, we optimized an in vitro system to isolate human stem cell-derived oligodendrocyte progenitor cells (OPCs) and used a motor neuron-oligodendrocyte co-culture system to investigate early stages of myelination. Secondly, we modified CRISPRoff and sgRNA Sleeping Beauty transposon vectors that depend on tetracycline-controlled transcriptional activation to silence the expression of MCL-1, an embryonic lethal gene, in early development of oligodendrocytes. Lastly, to expand our toolkit and modulate activity of MCL-1 in OPCs, we pharmacologically inhibited MCL-1 and elucidated a non-apoptotic function of the protein at this developmental stage. We demonstrate that the mitochondrial network changes in human oligodendrocyte development resemble those reported in mouse tissue. Our findings also point to MCL-1 as a critical factor essential at the OPC stage for proper oligodendrocyte morphogenesis.