White matter stroke comprises up to twenty-five percent of all diagnosed strokes, though up to ten times as many events go undiagnosed until accumulation leads to clinical sequelae such as vascular dementia and gait disturbances. White mater stroke leads to death of oligodendrocytes and demyelination of axons in the peri-infarct area, as well as persistent inflammation and astrogliosis. After injury, oligodendrocyte progenitor cells (OPCs) are unable to differentiate into myelinating oligodendrocytes, leaving axons chronically demyelinated. This pathology is distinct from other demyelinating diseases, but the underlying mechanism remains unknown.
This dissertation presents a series of studies that aim to elucidate genetic mechanisms of the observed OPC differentiation block, with the goal of uncovering regulators that can be manipulated to enhance OPC differentiation, and ultimately, remyelination, after injury. OPCs isolated from control as well as peri-infarct tissue at key time points after stroke are analyzed using RNA-sequencing to generate an OPC stroke-specific transcriptome. This gene expression dataset is characterized using complementary bioinformatics metrics, and comparisons to previously described transcriptomes show it is specific to OPCs and to the post-stroke context. Using quantitative metrics, ten highly differentially expressed candidate genes are selected for study to identify regulators of OPC differentiation.
First, each candidate gene is overexpressed in primary OPC cultures, and quantitative PCR is used to assess differentiation. Several candidate genes show pro-differentiation effects, but three of the most significant regulators, Chst10, C9orf9, and Csrp2, are selected for study in vivo, in a mouse model of white matter stroke. Tissue studies demonstrate that both Chst10 and Csrp2 induce significant differentiation of OPCs in post-stroke as well as stroke-naï¿½ve tissue, suggesting that these genes are involved in pathways that respond to white matter stroke, but may be applicable in oligodendrocyte biology more broadly.
A final set of experiments returns to the OPC stroke transcriptome to explore ways to apply this knowledge of OPC differentiation regulators. Small molecule regulators identified through additional transcriptome analyses are found to promote OPC differentiation both in vitro and in vivo, underscoring the power of the combined bioinformatics and cell-level approaches taken in this study.