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The Role of mTOR in Cytoskeletal Organization During Oligodendrocyte Differentiation and Developmental Myelination"
Cell Biology, Neuroscience and Physiology Program
B.S. 2009, Delaware State University, Dover, DE
M.S. 2011, The City University of New York, NY, NY
Thesis Advisor: Terri Wood, PhD
Department of Pharmacology, Physiology & Neuroscience
Monday, November 27, 2017
10:00 A.M., Cancer Center G1196
Oligodendrocytes are macroglial cells of the central nervous system (CNS) whose plasma membrane extends processes to form myelin sheaths that surround axons, to propagate rapid nerve signal transmission through saltatory conduction. Oligodendrocyte development occurs in multiple stages from initial precursor cells (OPC) to mature myelinating cells. During development, OPCs morphologically transition during differentiation to extend a network of processes through a sequence of events involving cytoskeletal organization. Our laboratory has published data demonstrating that oligodendrocyte differentiation is regulated by the mammalian target of rapamycin (mTOR), a downstream target of the of PI3K/Akt pathway (Tyler et al, 2009; Wahl et al, 2014). Pharmacological inhibition of mTOR by rapamycin results in a significant reduction in process extension. Moreover, our in vivo studies demonstrate that conditional knockout of mTOR (mTOR cKO) in oligodendrocytes results in hypomyelination in the spinal cord reflected by a reduction in the number of myelinated axons as well as in thickness of myelin membrane around axons. Thus, both in vitro and in vivo studies support the hypothesis that normal mTOR signaling is crucial for proper morphological development of oligodendrocytes.
Extension of cellular processes during morphological differentiation of oligodendrocytes involves protrusion at the leading edge initiated by actin filaments positioned immediately beneath the cell membrane. A recent publication by Zuchero and colleagues revealed that F-actin stability and branching is required for axon ensheathment at the onset of myelination, a process involving polymerization of actin (Zuchero et al, 2015). The goal of the studies here was to test the hypothesis that mTOR signaling regulates the oligodendrocyte cytoskeletal changes necessary for proper morphological differentiation and myelination. Dynamics of actin filaments are regulated by profilin, an actin polymerizing factor, and cofilin, an actin depolymerizing factor. Western immunoblot analysis of primary cell cultures revealed a significant reduction in total-profilin2 and phospho-cofilin in mTOR-inhibited cells undergoing differentiation. This reduction was apparent in the first two days of differentiation. In vivo, we also found a reduction in total-profilin2 positive oligodendrocytes in the mTOR cKO, at the peak of differentiation. This suggests delays in axon ensheathment after differentiation and supports our previous data showing a reduction in total number of myelinated axons. In addition, our in vitro staining revealed cytoplasmic accumulation of myelin basic protein (MBP) in cells lacking mTOR signaling, and we observed a decrease in myelin membrane spread in these cells. This suggests a role for mTOR in regulating MBP mRNA transport to the oligodendrocyte processes where it is normally translated during myelination. In support of this conclusion, we observed a decrease in expression of the kinesin Kif1b that is known to facilitate MBP mRNA transport in oligodendrocytes.
These data demonstrate a role for mTOR in cytoskeleton reorganization and modulation of cytoskeleton associated protein expression specifically early during morphological differentiation and initiation of myelination. We also reveal a critical role for mTOR in proper expression and localization of MBP that is necessary for myelin membrane wrapping. Results from these studies have the potential to contribute to the development of therapeutic strategies to promote remyelination in Multiple Sclerosis and other demyelinating diseases.