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N-Wasp Regulates Oligodendrocyte Myelination

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N-Wasp Regulates Oligodendrocyte Myelination Research Report: Regular Manuscript N-Wasp regulates oligodendrocyte myelination https://doi.org/10.1523/JNEUROSCI.0912-20.2020 Cite as: J. Neurosci 2020; 10.1523/JNEUROSCI.0912-20.2020 Received: 19 April 2020 Accepted: 20 May 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 the authors 1 N-Wasp regulates oligodendrocyte myelination 2 3 Christina Katanov1, Nurit Novak1, Anya Vainshtein1, Ofra Golani2, Jeffery L Dupree3, and Elior Peles1 4 5 1Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 76100, Israel. 6 2Department of Life Sciences Core Facilities, Weizmann Institute of Science, Rehovot 76100, Israel. 7 3Department of Anatomy and Neurobiology, Virginia Commonwealth University, Richmond, VA, 23284, USA. 8 9 Abbreviated title: Role of N-Wasp in CNS myelination 10 11 Address correspondence to: 12 Dr. Elior Peles 13 Department of Molecular Cell Biology 14 Weizmann Institute of Science 15 Rehovot, 76100, Israel 16 Email: [email protected] 17 18 Number of Pages 22; Figures 5; Tables 0 19 Number of Words: Abstract 200; Introduction 575; Discussion 1500 20 The authors declare no competing financial interests. 21 22 23 ACKNOWLEDGEMENTS 24 This work was supported by the NIH (R01NS097428), the Dr. Miriam and Sheldon G. Adelson Medical 25 Research Foundation, and a research grant from the National American Brain Foundation, Lilly Fulop 26 Fund for Multiple Sclerosis Research, Estate of David Georges Eskinazi, Dahlia and Philip Lawee, Gary 27 Clayman, and Ellie Adiel, and VA Merit Review Award (1l01BX002565-05). E.P. is the Incumbent of the 28 Hanna Hertz Professorial Chair for Multiple Sclerosis and Neuroscience. 29 30 ABSTRACT 31 Oligodendrocytes myelination depends on actin cytoskeleton rearrangement. Neural Wiskott-Aldrich 32 syndrome protein (N-Wasp) is an actin nucleation factor that promotes polymerization of branched actin 33 filaments. N-Wasp activity is essential for myelin membrane wrapping by Schwann cells, but its role in 34 oligodendrocytes and CNS myelination remains unknown. Here we report that oligodendrocytes-specific 35 deletion of N-Wasp in mice of both sexes resulted in hypomyelination i.e., reduced number of myelinated 36 axons and thinner myelin profiles, as well as substantial focal hypermyelination reflected by the formation of 37 remarkably long myelin outfolds. These myelin outfolds surrounded unmyelinated axons, neuronal cell 38 bodies, and other myelin profiles. The latter configuration resulted in pseudo-multimyelin profiles that were 39 often associated with axonal detachment and degeneration throughout the CNS, including in the optic nerve, 40 corpus callosum and the spinal cord. Furthermore, developmental analysis revealed that myelin 41 abnormalities were already observed during the onset of myelination, suggesting that they are formed by 42 aberrant and misguided elongation of the oligodendrocyte inner lip membrane. Our results demonstrate that 43 N-Wasp is required for the formation of normal myelin in the CNS. They also reveal that N-Wasp plays a 44 distinct role in oligodendrocytes compared to Schwann cells, highlighting a difference in the regulation of 45 actin dynamics during CNS and PNS myelination. 2 46 SIGNIFICANCE STATEMENT 47 Myelin is critical for the normal function of the nervous system by facilitating fast conduction of action 48 potentials. During the process of myelination in the CNS, oligodendrocytes undergo extensive 49 morphological changes that involves cellular process extension and retraction, axonal ensheathment, and 50 myelin membrane wrapping. Here we present evidence that the N-Wasp, a protein regulating actin filament 51 assembly through Arp2/3 complex-dependent actin nucleation, plays a critical role in CNS myelination, and 52 its absence leads to several myelin abnormalities. Our data provides an important step into the 53 understanding of the molecular mechanisms underlying CNS myelination. 3 54 INTRODUCTION 55 Myelin is a specialized membrane produced by oligodendrocytes and Schwann cells that spiral around 56 axons, thereby enabling fast conduction of action potentials, and providing axonal support (Nave and 57 Werner, 2014; Cohen et al., 2020). During myelination in the central nervous system (CNS), 58 oligodendrocytes undergo extensive morphological changes (Bauer et al., 2009; Brown and Macklin, 2019; 59 Seixas et al., 2019), beginning with the formation of exploratory processes that either make stable axonal 60 contact or retract (Czopka et al., 2013; Almeida and Lyons, 2014). This initial contact is then followed by 61 membrane ensheathment and wrapping, longitudinal extension of the forming myelin unit, and compaction 62 of the myelin membrane layers (Osso and Chan, 2017; Stadelmann et al., 2019). Oligodendrocytes contain 63 two major cytoskeletal systems, microtubules and actin filaments, which regulate process formation and 64 myelination (Bauer et al., 2009; Brown and Macklin, 2019; Seixas et al., 2019). The actin cytoskeleton has 65 a higher turnover and reorganization rate than microtubules, enabling fast reshaping of myelinating 66 oligodendrocytes (Song et al., 2001). Microtubules provide support to the morphology of the cell and allow 67 transport of myelin-specific proteins and mRNA (Lunn et al., 1997; Seiberlich et al., 2015). Moreover, 68 microtubules and actin filaments interact with each other leading to oligodendrocyte process outgrowth 69 (Song et al., 2001). Previous studies highlight the role of actin dynamics during myelin formation and 70 suggest a two-step model. First, oligodendrocyte process outgrowth and the initial ensheathment of the 71 axon are driven by Arp2/3 complex-dependent actin polymerization (Zuchero et al., 2015). Subsequently, 72 lateral spreading and growth of the myelin membrane during wrapping and compaction requires F-actin 73 disassembly by ADF/cofilin-1 (Nawaz et al., 2015; Zuchero et al., 2015). 74 Neural Wiskott-Aldrich Syndrome protein (N-Wasp) is a nucleation-promoting factor that drives 75 the generation of branched actin filaments (Alekhina et al., 2017). It regulates cortical actin filament 76 reorganization in response to extracellular stimuli by linking between small GTPases (i.e. Rac and Cdc42) 77 and actin polymerization through the regulation of the Arp2/3 complex (Takenawa and Miki, 2001). N-Wasp, 78 and Wasp family verprolin homologous protein-1 (WAVE1) are required for myelination in the peripheral 79 (PNS) and central nervous system (CNS), respectively, and regulate oligodendrocyte differentiation (Kim 80 et al., 2006; Bacon et al., 2007; Jin et al., 2011; Novak et al., 2011). N-Wasp is expressed by both 81 oligodendrocytes and myelinating Schwann cells (Tsuchiya et al., 2006; Novak et al., 2011; Zhang et al., 82 2014; Marques et al., 2016). It plays a crucial role in Schwann cell membrane wrapping and in longitudinal 4 83 extension of the myelin unit in the PNS, most likely via regulation of the actin cytoskeleton (Jin et al., 2011; 84 Novak et al., 2011). Schwann cells from N-Wasp-mutant mice formed less radial lamellipodia and shorter 85 axial processes that lacked defined F-actin-rich cones (Jin et al., 2011). In the absence of N-Wasp, 86 Schwann cells properly sort and ensheath the axons, but were not capable of proceeding with myelin 87 wrapping (Jin et al., 2011; Novak et al., 2011). In the CNS, N-Wasp was detected with components of the 88 Arp2/3 complex in newly formed oligodendrocytes and in purified myelin fractions (Bacon et al., 2007). 89 Moreover, pharmacological inhibition of N-Wasp prevented process extension and caused filopodium 90 retraction in cultured oligodendrocyte precursor cells (OPCs). Overall, these data suggests that cellular 91 process extension by OPCs is regulated by N-Wasp and Arp2/3-driven actin polymerization. Here we report 92 that genetic deletion of N-Wasp in oligodendrocytes results in hypomyelination, diverse myelin 93 abnormalities including outfoldings that enwrap neuronal cell bodies, as well as myelin and axonal 94 degeneration. 5 95 MATERIALS AND METHODS 96 Mice 97 Generation of N-Waspflx/flx (Cotta-de-Almeida et al., 2007) and Cnp-Cre (Lappe-Siefke et al., 2003) mice 98 were previously described and were always kept as heterozygous. Cnp-Cre/N-Waspflx/flx mice (i.e., 99 homozygous for N-Wasp) were obtained by a conventional breeding scheme. Genotypes were determined 100 by performing PCR on genomic DNA extracted from mice tails. Both male and female animals were used 101 in the study, with no detectable difference in myelin morphology. Rotarod motor test was performed using 102 an accelerating rotarod as previously described (Novak et al., 2011). All experiments were performed in 103 compliance with the relevant laws and institutional guidelines and were approved by the Weizmann 104 Institute's Animal Care and Use Committee. 105 106 Reverse transcription PCR 107 Total RNA was isolated from optic nerves as described (Novak et al., 2011). PCR was performed using N- 108 Wasp (5’-GTGCAGTTGTATGCAGCAGATCG-3’ and 5’-GGTGTGGGAGATGTTGTTG-3’)- or the 109 previously described actin specific primers (Novak et al., 2011). 110 111 Histochemistry and Immunofluorescence 112 Mice were anaesthetized and perfused with 2.5% PFA. Brains were isolated and post-fixed on ice for 30 113 min, and Luxol Fast Blue (LFB) staining was performed as described (Elazar et al., 2019b). For 114 immunofluorescence sections were washed with PBS, blocked and permeabilized in PBS containing 5% 115 normal goat serum, 0.5% Triton X-100 for 1h at RT. Samples were incubated overnight at 4°C with primary 116 antibodies to MBP (1:300; MAB386, chemicon) diluted in blocking solution with 0.1% Triton X-100, washed 117 with PBS, incubated for 45 min at RT with secondary antibodies coupled to Dylight 488 (from Jackson 118 ImmunoResearch) and then washed with PBS and mounted with fluoromount-G.
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