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LINGO-1 Regulates Oligodendrocyte Differentiation Through the Cytoplasmic Gelsolin Signaling Pathway

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LINGO-1 Regulates Oligodendrocyte Differentiation Through the Cytoplasmic Gelsolin Signaling Pathway The Journal of Neuroscience, March 22, 2017 • 37(12):3127–3137 • 3127 Cellular/Molecular LINGO-1 Regulates Oligodendrocyte Differentiation through the Cytoplasmic Gelsolin Signaling Pathway Zhaohui Shao, Xinhua Lee, Guanrong Huang, Guoqing Sheng, Christopher E. Henderson, XDaniel Louvard, Jiho Sohn, Blake Pepinsky, and XSha Mi Biogen, Inc., Cambridge, Massachusetts 02142 Differentiation and maturation of oligodendrocyte progenitor cells (OPCs) involve the assembly and disassembly of actin microfila- ments. However, how actin dynamics are regulated during this process remains poorly understood. Leucine-rich repeat and Ig-like domain-containing Nogo receptor interacting protein 1 (LINGO-1) is a negative regulator of OPC differentiation. We discovered that anti-LINGO-1 antibody-promoted OPC differentiation was accompanied by upregulation of cytoplasmic gelsolin (cGSN), an abundant actin-severing protein involved in the depolymerization of actin filaments. Treating rat OPCs with cGSN siRNA reduced OPC different- iation, whereas overexpression of cGSN promoted OPC differentiation in vitro and remyelination in vivo. Furthermore, coexpression of cGSN and LINGO-1 blocked the inhibitory effect of LINGO-1. Our study demonstrates that cGSN works downstream of LINGO-1 signaling pathway, which enhances actin dynamics and is essential for OPC morphogenesis and differentiation. This finding may lead to novel therapeutic approaches for the treatment of demyelinating diseases such as multiple sclerosis (MS). Key words: cytoplasmic gelsolin; LINGO-1; multiple sclerosis; oligodendrocyte; remyelination; therapeutic Significance Statement Myelin loss and subsequent axon degeneration contributes to a variety of neurological diseases, such as multiple sclerosis (MS). Understandingtheregulationofmyelinationbyoligodendrocytesisthereforecriticalfordevelopingtherapiesforthetreatmentof MS. We previously demonstrated that leucine-rich repeat and Ig-like domain-containing Nogo receptor interacting protein 1 (LINGO-1) is a negative regulator of oligodendrocyte differentiation and that anti-LINGO-1 promotes remyelination in preclinical animal models for MS and in a phase II acute optic neuritis clinical trial (RENEW). The mechanism by which LINGO-1 regulates oligodendrocyte differentiation is unknown. Here, we demonstrate that LINGO-1 regulates oligodendrocyte differentiation and maturation through the cytoplasmic gelsolin signaling pathway, providing new drug targets for the treatment of demyelination diseases. Introduction (MS). Many factors have been implicated in regulating oligoden- Oligodendrocyte differentiation and myelination are tightly reg- drocyte progenitor cell (OPC) differentiation and maturation ulated processes that are essential to normal CNS development (Bercury and Macklin, 2015). OPC differentiation requires dy- and function. Understanding how the processes are regulated namic assembly and disassembly of actin microfilaments to should provide insights for developing new therapies for the allow the plasma membrane extension necessary for lamellipo- treatment of demyelinating diseases such as multiple sclerosis dia/filopodia growth, membrane protrusion, and focal adhesions (Bercury and Macklin, 2015). Two recent studies suggest that Received Dec. 4, 2016; revised Jan. 19, 2017; accepted Feb. 6, 2017. actin dynamics are regulated in distinct manners for different Author contributions: D.L. and S.M. designed research; Z.S., X.L., G.H., G.S., and J.S. performed research; G.H., phases of myelination: actin assembly is required for oligoden- C.E.H., B.P., and S.M. analyzed data; Z.S. and S.M. wrote the paper. drocyte process extension, whereas actin disassembly leads to We thank Colin Smith and Chris-Ann McKenzie at University of Edinburgh for providing the fresh-frozen brain myelin wrapping (Nawaz et al., 2015; Zuchero et al., 2015). samples from human MS and non-MS patients for Western blot analysis, and Yun Bian for helping to harvest tissues from the animals. Actin exists in cells in filamentous (F-actin) and globular/ The authors hold stock in Biogen, Inc. monomeric (G-actin) states. The ratio of F-actin to G-actin, D. Louvard’s present address: Unite´ Mixte de Recherche 144 Centre National de la Recherche Scientifique/Insti- which is tightly controlled by actin-binding proteins such as tute Curie, Paris, France. gelsolin (dos Remedios et al., 2003), regulates cell morphology Correspondence should be addressed to Sha Mi, Ph.D., Department of Neurology Research, Biogen, Inc., 115 Broadway, Cambridge, MA 02142. E-mail: [email protected]. and can change rapidly under physiological conditions. Gelsolin DOI:10.1523/JNEUROSCI.3722-16.2017 is an abundant actin-binding protein and plays a critical role Copyright © 2017 the authors 0270-6474/17/373127-11$15.00/0 in the regulation of actin dynamics (Li et al., 2012). Gelsolin exists 3128 • J. Neurosci., March 22, 2017 • 37(12):3127–3137 Shao et al. • LINGO-1 Regulates Oligodendrocyte Differentiation Table 1. Human MS brain tissues and sex- and age-matched control samples Group 1 (MS) Group 2 (control) # Sex Age Lesion # Sex Age Neuropath classifications 1 F 42 CL 21 F 42 No significant abnormalities 2 F 53 AL 22 F 53 No significant abnormalities 3 F 53 CL 23 F 47 No significant abnormalities 4 F 44 CL 24 F 44 No significant abnormalities 5 M 46 CAL 25 M 46 Control, vascular disease, other 6 F 53 AL/CAL 26 F 51 No significant abnormalities 7 M 45 CL 27 M 45 No significant abnormalities 8 F 57 CL 28 F 57 Depression, recurrent; dependence, alcohol; control, vascular disease, other 9 M 43 CAL 29 M 43 No significant abnormalities 10 F 48 CAL 30 F 48 No significant abnormalities 11 M 45 AL 31 M 44 No significant abnormalities 12 F 71 CAL 32 F 71 Control, vascular disease, other 13 F 71 AL/CAL 33 F 71 No significant abnormalities 14 F 53 CAL 34 F 49 No significant abnormalities 15 F 42 AL 35 F 41 No significant abnormalities 16 F 50 AL/CAL 36 F 50 Control, vascular disease, other 17 F 44 AL 37 F 44 No significant abnormalities 18 F 50 CAL 38 F 51 No significant abnormalities 19 F 48 AL/CAL 39 F 49 Control, substance abuse, alcohol; dependence, alcohol; no significant abnormalities 20 F 57 AL 40 F 57 No significant abnormalities AL, Active lesion; CAL, chronic active lesion; CL, chronic lesion. in the extracellular (secreted, pGSN) and intracellular (cytoplas- tissue. Equal amounts of protein were loaded onto 4–15% SDS-PAGE gel mic, cGSN) forms, which are expressed from an alternatively and subjected to Western blot analysis probed with antibodies against spliced gene under different promoters. pGSN contains an addi- LINGO-1 (3C11, 2 ␮g/ml; Biogen), GSN (12953, RRID: AB-2632961, tional N-terminal 24 aa compared with cGSN (Kwiatkowski et 1:1000; Cell Signaling Technology), or pGSN (NBP2-27566, RRID: AB- al., 1986). Both forms of gelsolin are potent actin filament- 2632960, 2 ␮g/ml; Novus Biologicals). Western blot membranes were severing proteins, converting F-actin to G-actin. pGSN plays an then stripped and reprobed with anti-GAPDH (NB600-502, RRID: AB- important role in the clearance of actin aggregates in the blood 350715, 1:5000 or NB100-56875, RRID: AB-838305, 1:500; Novus Bio- after tissue-injury-induced cell death (Lee and Galbraith, 1992; logicals) as loading controls. Band intensities of the Western blot TIFF images were quantified by the Image Studio software (LI-COR). Spinardi and Witke, 2007; Bucki et al., 2008; Li-ChunHsieh et al., Primary OPC culture. Enriched populations of rat OPCs were purified 2015). cGSN is ubiquitously expressed inside cells including as described previously (Mi et al., 2005) and maintained in a defined myelin-forming oligodendrocytes. However, its function in OPC DMEM (DMEM with 4 mM glutamine, 1 mM sodium pyruvate, 0.1% w/v differentiation and myelination is unknown. bovine serum albumin, 50 ␮g/ml apo-transferrin, 5 ␮g/ml insulin, 30 nM LINGO-1, a membrane protein selectively expressed in the sodium selenite, 10 nMD-biotin, and 10 nM hydrocortisone) plus 10 CNS, has been identified as a negative regulator of oligodendro- ng/ml FGF and PDGF (Peprotech). For differentiation, the A2B5ϩ OPCs cyte differentiation and myelination (Mi et al., 2005; Mi et al., were cultured on PDL-coated plates (Fisher) in the defined DMEM 2013). Blocking LINGO-1 function by antagonists including supplemented with 15 nM T3 (Sigma-Aldrich) and 10 ng/ml ciliary anti-LINGO-1 antibodies promotes oligodendrocyte differentia- neurotrophic factor (Peprotech) for 3 d with 2 ␮g/ml anti-LINGO-1 tion and myelination in vitro and in vivo (Mi et al., 2007; Mi et al., antibody (BIIB033; Biogen) or an aglycosyl IgG1 isotype control anti- 2009). Opicinumab (BIIB033), an anti-LINGO-1 antibody, im- body (5C8; Biogen). All experiments related to OPC differentiation proved visual evoked potentials in a phase II acute optic neuritis were repeated two to three times with two biological replicates for each trial, suggesting a positive effect on remyelination (Cadavid et al., experiment. 2015). The downstream players that participate in the LINGO-1 For transfection of plasmid or siRNA into OPCs, the electroporation signaling pathway remain unclear. Here, we report that cGSN method was used with an Amaxa kit (Lonza). Transfected OPCs were then plays a key role in oligodendrocyte differentiation, acting down- plated onto PDL-coated plates in growth medium and then switched to stream of the LINGO-1 signaling pathway. differentiation medium for3dasdescribed above. All siRNAs were from Life Technology, including custom-ordered cGSN and pGSN-specific siRNA. Materials and Methods Rat GSN siRNA (ID# s150340), sense sequence (5Ј–3Ј) GGACAGUUUC AACAACGGUtt. Rat cGSN siRNA (ID# s529615), sense sequence (5Ј–3Ј) Quantitative RT-PCR. RNA was isolated from cells using the Absolutely CCACAGCAGGUACCACCAAtt. Rat pGSN siRNA (ID# s529575), sense RNA Miniprep Kit (Agilent Technologies). Quantitative RT-PCR was Ј Ј used to quantify GSN and myelin basic protein (MBP) mRNA levels with sequence (5 –3 ) CAAAGUCGGGUGUCUGAGAtt. ␤-actin used as an internal control. All primer sets were from Life Tech- For assessing protein expression by Western blot, cells were lysed in RIPA nologies, including custom-made cGSN and pGSN probes.
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