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Mechanistic Target of Rapamycin Regulates the Oligodendrocyte Cytoskeleton During Myelination

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Mechanistic Target of Rapamycin Regulates the Oligodendrocyte Cytoskeleton During Myelination Research ReportDevelopment/Plasticity/Repair Mechanistic Target of Rapamycin Regulates the Oligodendrocyte Cytoskeleton during Myelination https://doi.org/10.1523/JNEUROSCI.1434-18.2020 Cite as: J. Neurosci 2020; 10.1523/JNEUROSCI.1434-18.2020 Received: 31 May 2018 Revised: 23 February 2020 Accepted: 26 February 2020 This Early Release article has been peer-reviewed and accepted, but has not been through the composition and copyediting processes. The final version may differ slightly in style or formatting and will contain links to any extended data. Alerts: Sign up at www.jneurosci.org/alerts to receive customized email alerts when the fully formatted version of this article is published. Copyright © 2020 Musah et al. 1 Mechanistic Target of Rapamycin Regulates the Oligodendrocyte Cytoskeleton during 2 Myelination 3 1Aminat S. Musah, 2Tanya L. Brown, 1Marisa A. Jeffries, 1Quan Shang, 2Hirokazu Hashimoto, 4 1Angelina V. Evangelou, 3Alison Kowalski, 3Mona Batish, 2Wendy B. Macklin and 1Teresa L. 5 Wood 6 1Department of Pharmacology, Physiology & Neuroscience, New Jersey Medical School, 7 Rutgers University, Newark, NJ U.S.A. 07101, 2Department of Cell and Developmental Biology, 8 University of Colorado School of Medicine, Aurora, CO, U.S.A. 80045, 3Department of Medical 9 and Molecular Sciences, University of Delaware, Newark, DE 19716 10 11 Abbreviated Title: mTOR Regulates the Oligodendrocyte Cytoskeleton 12 Corresponding Author: Teresa L. Wood, PhD, Department Pharmacology, Physiology & 13 Neuroscience, New Jersey Medical School Cancer Center H1200, Rutgers University, 205 S. 14 Orange Ave, Newark, NJ 07101-1709 15 [email protected] 16 Number of pages: 39 17 Number of Figures: 10 18 Number of Tables: 0 19 Number of Words: 20 Abstract: 242 21 Introduction: 687 22 Discussion: 1075 23 Conflict of Interest: The authors declare no competing financial interests. 24 Acknowledgements: This work was supported by National Institute of Neurological Disorders & 25 Stroke NS082203 to WM and TLW, National Multiple Sclerosis Society RG5371-A-4 to TLW 26 and NIH Director’s Early Independence Award DP5 OD012160 to Mona Batish. The authors 27 wish to thank Luke Fritzky for technical assistance and Luipa Khandker for critical reading of 28 the manuscript. 29 2 30 Abstract 31 During differentiation, oligodendrocyte precursor cells (OPCs) extend a network of 32 processes that make contact with axons and initiate myelination. Recent studies revealed that 33 actin polymerization is required for initiation of myelination whereas actin depolymerization 34 promotes myelin wrapping. Here, we used primary OPCs in culture isolated from neonatal rat 35 cortices of both sexes and young male and female mice with oligodendrocyte-specific deletion of 36 mechanistic target of rapamycin (mTOR) to demonstrate that mTOR regulates expression of 37 specific cytoskeletal targets and actin reorganization in oligodendrocytes during developmental 38 myelination. Loss or inhibition of mTOR reduced expression of profilin2 and ARPC3, actin 39 polymerizing factors, and elevated levels of active cofilin, which mediates actin 40 depolymerization. The deficits in actin polymerization were revealed in reduced phalloidin and 41 deficits in oligodendrocyte cellular branching complexity at the peak of morphological 42 differentiation and a delay in initiation of myelination. We further show a critical role for mTOR 43 in expression and localization of myelin basic protein (Mbp) mRNA and MBP protein to the 44 cellular processes where it is necessary at the myelin membrane for axon wrapping. Mbp mRNA 45 transport deficits were confirmed by single molecule RNA FISH. Moreover, expression of the 46 kinesin family member 1B, an Mbp mRNA transport protein, was reduced in CC1+ cells in the 47 mTOR cKO and in mTOR inhibited oligodendrocytes undergoing differentiation in vitro. These 48 data support the conclusion that mTOR regulates both initiation of myelination and axon 49 wrapping by targeting cytoskeletal reorganization and MBP localization to oligodendrocyte 50 processes. 51 52 Significance Statement 53 Myelination is essential for normal central nervous system (CNS) development and adult axon 54 preservation and function. The mechanistic target of rapamycin (mTOR) signaling pathway has 55 been implicated in promoting CNS myelination; however, there is a gap in our understanding of 56 the mechanisms by which mTOR promotes developmental myelination through regulating 57 specific downstream targets. Here, we present evidence that mTOR promotes the initiation of 58 myelination through regulating specific cytoskeletal targets and cellular process expansion by 59 oligodendrocyte progenitor cells as well as expression and cellular localization of myelin basic 60 protein. 61 4 62 Introduction 63 Progression through specific stages of oligodendrocyte differentiation has been well 64 characterized and involves extensive morphological changes in preparation for axon contact and 65 initiation of myelination (for reviews, see (Michalski and Kothary, 2015; Snaidero and Simons, 66 2017; Brown and Macklin, 2019)). Oligodendrocyte precursor cells (OPCs) transition into 67 immature oligodendrocytes characterized by increased number and length of cellular processes 68 to contact axons and initiate myelination. Final maturation of oligodendrocytes requires the cell 69 membrane to flatten out to wrap axons in the process of myelination (Nawaz et al., 2015; 70 Zuchero et al., 2015). The inability of OPCs to differentiate contributes to impaired 71 developmental myelination as well as to reduced myelin repair after demyelination in diseases 72 such as multiple sclerosis (for reviews, see (Franklin and Ffrench-Constant, 2017; Snaidero and 73 Simons, 2017; Abu-Rub and Miller, 2018)). 74 The evolving morphology of oligodendrocytes during cellular differentiation and 75 myelination requires a dynamic cytoskeleton (for reviews, see (Michalski and Kothary, 2015; 76 Snaidero and Simons, 2017; Brown and Macklin, 2019; Thomason et al., 2019)). The actin 77 cytoskeleton exists in monomer and filament form; the balance between polymerization and 78 depolymerization is tightly regulated and controls cytoskeletal reorganizations. Numerous 79 laboratories have contributed to our current understanding of cytoskeletal dynamics in 80 developing oligodendroglia. Prior reports support two phases of developmental myelination 81 involving opposing cytoskeletal processes in differentiating oligodendrocytes; phase I requires 82 actin polymerization for growth cone formation mediated process extension (Fox et al., 2006; 83 Zuchero et al., 2015), whereas phase II requires actin depolymerization for proper axon wrapping 84 and myelination (Nawaz et al., 2015; Zuchero et al., 2015). Actin binding proteins that regulate 5 85 actin cytoskeletal reorganization include the actin polymerizing protein profilin, the ARP2/3 86 branched actin nucleator complex, and the depolymerizing protein family actin depolymerizing 87 factor (ADF)/cofilin1. The ARP 2/3 complex in oligodendroglia is known to regulate axon 88 ensheathment (Zuchero et al., 2015). In contrast, ADF/cofilin1 regulates filamentous actin (F- 89 actin) turnover for proper myelin wrapping; however, it is hypothesized that F-actin at the 90 membrane leading edge provides the driving force needed for myelin growth (Nawaz et al., 91 2015). Moreover, the involvement of F-actin at the inner tongue of myelinating cells helps create 92 the balance between myelin compaction and cytoplasmic channels (Snaidero and Simons, 2017). 93 The presence of myelin basic protein (MBP) in the oligodendrocyte processes also has been 94 proposed to be important in the wrapping phase of myelination to enable MBP to displace cofilin 95 at the membrane (Zuchero et al., 2015). The membrane displacement of cofilin then may 96 facilitate actin depolymerization necessary for wrapping (Zuchero et al., 2015). 97 The function of specific intracellular signaling pathways in the developmental 98 progression of oligodendroglia has been the focus of numerous publications (Sperber et al., 99 2001; Zou et al., 2011; Ishii et al., 2012; Bercury et al., 2014; Dai et al., 2014; Wahl et al., 2014; 100 Furusho et al., 2017). However, it is unknown how these pathways orchestrate the morphological 101 changes observed during differentiation and myelination. Moreover, the studies on actin 102 dynamics in developing oligodendrocytes and myelination did not identify upstream regulators 103 (Nawaz et al., 2015; Zuchero et al., 2015). Studies in a variety of other cell types including 104 neutrophils, fibroblasts and tumor cells demonstrate a role for the mechanistic target of 105 rapamycin (mTOR) pathway in regulating the cytoskeleton (Jacinto et al., 2004; Sarbassov et al., 106 2004; Liu et al., 2008; He et al., 2013), however, these studies have provided little information 107 on specific cytoskeletal targets downstream of mTOR. Our previous studies revealed that mTOR 6 108 regulates oligodendrocyte differentiation, initiation of myelination and myelin thickness in the 109 spinal cord (Tyler et al., 2009; Bercury et al., 2014; Wahl et al., 2014). Collectively, these 110 findings led us to hypothesize that mTOR regulates cytoskeletal dynamics in oligodendrocytes 111 necessary for initiation of myelination and for proper myelin wrapping. 112 The goal of the studies presented here was to determine the function of mTOR signaling 113 in regulating cytoskeletal reorganization during oligodendrocyte development. 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