Dissertation Defense: George Melchor
Candidate: George S. Melchor, Jr.
Major: Neuroscience
Advisor: Jeff Huang, Ph.D.
Title: A View from Within: Insights in Oligodendrocyte Lineage Cells and Demyelinated Lesion Microenvironment Dynamics Utilizing Ribotag and Single Nuclei RNA Sequencing
Multiple sclerosis (MS) is a central nervous system (CNS) disease, in which immune cells target and destroy oligodendrocyte lineage cells (OLCs) and the myelin they produce, causing multiple, focal demyelinated lesions. Myelin replacement can occur naturally via the recruitment of oligodendrocyte precursor cells (OPCs) in a process known as remyelination. However, this process is incomplete in MS, becomes less efficient with chronic disease, and ultimately fails despite disease modifying drugs that prevent the infiltration of immune cells into the CNS. In order to understand why remyelination fails in MS, it is necessary to examine the intrinsic molecular signals involved in the progression of OLCs, as well as how they interact with other cell populations within demyelinated lesion microenvironments as remyelination progresses. In this dissertation, I utilized the ribosome tagging strategy, RiboTag, to fate map OLCs during development, adulthood, and post-demyelinating injury. Three distinct RiboTag lines were created utilizing Cre or inducible Cre-ERT recombinase controlled by the Cspg4 (NG2), Pdgfra, or Plp promoter. Using the Cspg4(NG2)Cre:Rpl22HA(RiboTag) line, I observed Rpl22HA expression in both OLCs and neurons during development, and in OLCs, microglia, and astrocytes following demyelinating injury. These results suggest the Cspg4 promoter is not specific to OLCs, and instead, is differentially activated in multiple CNS cell populations during development and under demyelinating injury. Using the inducible PdgfraCreERT:Rpl22HA line, I observed Rpl22HA labeling after tamoxifen administration specifically in OLCs in the homeostatic CNS and following demyelinating injury. Similarly, the inducible PlpCreERT:Rpl22HA line enabled Rpl22HA labeling after tamoxifen administration specifically in mature oligodendrocytes (OLs) in the homeostatic CNS and post-demyelinating injury. However, in all three lines, the Cre-mediated activation of Rpl22HA exhibited high sensitivity and labeled OLCs both throughout the CNS and inside the demyelinating lesion, making further investigations into the genetic complexities of OLCs involved in remyelination challenging. To characterize the gene expression profile of all cell types involved in the remyelination process, I conducted transcriptomic bioinformatic analysis following single nucleus RNA-sequencing (snRNAseq) of lysolecithin-induced demyelinated lesions at time points corresponding to different stages of remyelination. Through my analyses, I established an understanding of the cell populations within a regenerating lesion microenvironment at the molecular level and observed detailed gene expression changes among major cell populations during remyelination. My findings confirmed previous research on the interactions between OLCs and other major cell types such as microglia, astrocytes, vascular and mesenchymal cells within the lesion microenvironment, while generating new insights on cell population shifts and dynamics within a remyelinating lesion. My results further suggest that OLC progression is mediated through a complex set of intrinsic transcriptional changes within recruited OPCs themselves, as well as extrinsic interactions with activated peripheral immune cells and CNS resident cells that are initiated at different stages of remyelination. I further investigated the potential to assess therapeutic interventions via snRNAseq through the analysis of transcriptomic changes in recruited OPCs and the lesion microenvironment with the pharmacological inhibition of LAT-1 (Slc7a5), a neutral amino acid transporter, upregulated in myeloid cells following demyelination. Through my analyses, I found that pharmacological inhibition of LAT-1 increased pro-regenerative myeloid cell profiles and increased remyelination outcomes. These results suggest that pharmacological inhibition of LAT-1 (Slc7a5) may enhance remyelination by creating a more favorable lesion microenvironment through the reprogramming of myeloid cells. Future investigations into the intrinsic and extrinsic properties elucidated in this dissertation will inform treatment strategies aimed at promoting remyelination in individuals affected by MS.