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Myocilin Is Involved in Ngr1/Lingo-1-Mediated Oligodendrocyte Differentiation and Myelination of the Optic Nerve

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Myocilin Is Involved in Ngr1/Lingo-1-Mediated Oligodendrocyte Differentiation and Myelination of the Optic Nerve The Journal of Neuroscience, April 16, 2014 • 34(16):5539–5551 • 5539 Cellular/Molecular Myocilin Is Involved in NgR1/Lingo-1-Mediated Oligodendrocyte Differentiation and Myelination of the Optic Nerve Heung Sun Kwon,1 Naoki Nakaya,1 Mones Abu-Asab,2 Hong Sug Kim,3 and Stanislav I. Tomarev1 1Retinal Ganglion Cell Biology Section, Laboratory of Retinal Cell and Molecular Biology and 2Histology Core, National Eye Institute, National Institutes of Health (NIH), Bethesda, Maryland 20892, and 3Neuro-Oncology Branch, National Cancer Institute, NIH, Bethesda, Maryland 20892 Myocilin is a secreted glycoprotein that belongs to a family of olfactomedin domain-containing proteins. Although myocilin is detected in several ocular and nonocular tissues, the only reported human pathology related to mutations in the MYOCILIN gene is primary open-angle glaucoma. Functions of myocilin are poorly understood. Here we demonstrate that myocilin is a mediator of oligodendrocyte differentiation and is involved in the myelination of the optic nerve in mice. Myocilin is expressed and secreted by optic nerve astrocytes. Differentiation of optic nerve oligodendrocytes is delayed in Myocilin-null mice. Optic nerves of Myocilin-null mice contain reduced levels of several myelin-associated proteins including myelin basic protein, myelin proteolipid protein, and 2Ј3Ј-cyclic nucleotide 3Ј- phosphodiesterase compared with those of wild-type littermates. This leads to reduced myelin sheath thickness of optic nerve axons in Myocilin-null mice compared with wild-type littermates, and this difference is more pronounced at early postnatal stages compared with adult mice. Myocilin also affects differentiation of oligodendrocyte precursors in vitro. Its addition to primary cultures of differentiating oligodendrocyte precursors increases levels of tested markers of oligodendrocyte differentiation and stimulates elongation of oligoden- drocyte processes. Myocilin stimulation of oligodendrocyte differentiation occurs through the NgR1/Lingo-1 receptor complex. Myocilin physically interacts with Lingo-1 and may be considered as a Lingo-1 ligand. Myocilin-induced elongation of oligodendrocyte processes may be mediated by activation of FYN and suppression of RhoA GTPase. Key words: astrocyte; differentiation; Lingo-1; myelin; myocilin; oligodendrocyte Introduction deterioration of trabecular meshwork function and elevation of MYOCILIN (MYOC) was the first gene in which identified mu- intraocular pressure. The pathological role of mutated myocilin tations were found to cause glaucoma, the second leading cause in other ocular and nonocular tissues is less clear. Myoc-null mice of blindness in the world (Adam et al., 1997, Stone et al., 1997). do not show any detectable changes in intraocular pressure or MYOC encodes a secreted glycoprotein and is expressed in ocular defects in the optic nerve head (Kim et al., 2001). However, Myoc- and several nonocular tissues. In the eye, MYOC expression has null mice exhibit reduced cortical bone thickness (Kwon et al., been detected in the tissues responsible for aqueous humor pro- 2013b), a moderate reduction in the amount of dystrophin- duction (ciliary body) and outflow (trabecular meshwork), as associated syntrophin in the skeletal muscle (Joe et al., 2012), and well as in the iris, sclera, retinal pigmented epithelium, and optic defects in sciatic nerve myelination (Kwon et al., 2013a) as com- nerve (Adam et al., 1997, Ortego et al., 1997, Stone et al., 1997, pared with wild-type littermates. In the sciatic nerve, myocilin is Tomarev et al., 2003). Available data suggest that expression of expressed in Schwann cells (Ohlmann et al., 2003) and localized mutated myocilin in the trabecular meshwork leads to the acti- to the nodes of Ranvier where it interacts with gliomedin, neuro- vation of an unfolded protein response (Joe et al., 2003, Joe and fascin, and NrCAM, proteins essential for node formation and Tomarev, 2010, Zode et al., 2011) and increases sensitivity of cells function (Eshed et al., 2005, Kwon et al., 2013a). Sciatic nerves of to oxidative stress (Joe and Tomarev, 2010). This may lead to Myoc-null mice have thinner myelin sheaths and partial disorga- nization of Ranvier nodes when compared with wild-type litter- Received Nov. 7, 2013; revised Jan. 24, 2014; accepted March 11, 2014. mates (Kwon et al., 2013a). Author contributions: H.S. Kwon designed research; H.S. Kwon, N.N., and M.A.-A. performed research; H.S. Kim In the CNS, including the optic nerve, oligodendrocytes play a contributed unpublished reagents/analytic tools; H.S. Kwon analyzed data; H.S. Kwon and S.I.T. wrote the paper. critical role in the myelination of axons. Several regulators that This work was supported by the Intramural Research Programs of the National Eye Institute, National Institutes of Health. We thank Drs. Haohua Qian for help with flash visual evoked potentials recording and Thomas V. Johnson negatively or positively regulate myelination in the CNS have for critical reading of this manuscript. been identified (Emery, 2010). One of the negative regulators of The authors declare no competing financial interests. myelination is leucine-rich repeat and Ig domain-containing 1 Correspondence should be addressed to Stanislav I. Tomarev, Building 6, Room 212, 6 Center Drive, National Eye (Lingo-1), a transmembrane signaling protein expressed in both Institute, NIH, Bethesda, MD 20892. E-mail: [email protected]. DOI:10.1523/JNEUROSCI.4731-13.2014 oligodendrocytes and neurons, but not in astrocytes (Mi et al., Copyright © 2014 the authors 0270-6474/14/345539-13$15.00/0 2005). In neurons, Lingo-1 is a coreceptor of the Nogo receptor 5540 • J. Neurosci., April 16, 2014 • 34(16):5539–5551 Kwon et al. • Myocilin Participates in Myelination of the Optic Nerve complex that may mediate the inhibition of axon growth (Mi et PVDF membrane (Life Technologies). The membrane was blocked with al., 2004). We have recently shown that olfactomedin 1 (Olfm1), 5% skim milk in Tris-buffered saline. Primary and secondary antibodies a protein belonging to the same superfamily as myocilin, interacts and SuperSignal West Dura Chemiluminescent Substrate (Thermo with the Nogo receptor complex preferentially binding NgR1. Fisher Scientific) were used to detect proteins. All Western blotting ex- Olfm1 caused the inhibition of NgR1 signaling by interfering periments were repeated at least three times. Primary astrocyte and oligodendrocyte cultures. Mouse optic nerve as- with interaction between NgR1 and its coreceptors, p75NTR or trocytes were derived from the anterior portions of C57BL/6 mouse optic LINGO-1 (Nakaya et al., 2012). nerves. The posterior pole of the eye was dissected, and the optic nerve In the present communication, we investigated a role of myo- head was freed from sclera and other neighboring tissues. The optic nerve cilin in the optic nerve. Myoc-null mice show defects in optic head was sliced sagittally and the anterior portion of the optic nerve was nerve myelination. We demonstrate that myocilin is expressed in carefully dissected from the prelaminar and postlaminar regions under a astrocytes and plays a role in differentiation of oligodendrocytes dissection microscope. Two or three explants of the anterior region were acting through the Lingo-1/NgR1 complex. obtained from each eye. The explants were put into T-25 cm 2 plastic tissue culture flasks, which had been conditioned with DMEM/F-12 sup- Materials and Methods plemented with 10% FBS. The first passage cells were characterized by Animals, plasmids, and antibodies. Mice were maintained in accordance immunostaining with antibodies against GFAP and Olig2 to identify with guidelines set forth by the National Eye Institute Committee on the astrocytes and oligodendrocytes, respectively. Astrocytes were selected Use and Care of Animals. Myoc-null mice (B6/129 mixed genetic back- from primary cultures by growing cells for 1 week in modified astrocyte- ground) have been described previously (Kim et al., 2001). Mice of either defined, serum-free medium (ADM; Clonetics) containing forskolin to sex were used in experiments. Human FLAG- and alkaline phosphatase suppress fibroblast growth. More than 95% of cells in second-passage (AP)-tagged myocilin, myocilin-⌬C, and myocilin-⌬N constructs have cultures were positive for GFAP. They were grown to 60–80% conflu- been described (Kwon et al., 2009). Antibodies were obtained from fol- ency and starved in serum-free medium for 1 week before being used for lowing sources: HSC70 (Santa Cruz Biotechnology); MBP and Lingo-1 experiments. (Abcam); neurofilament H, MBP, RhoA, Olig2, A2B5, and O4 (Milli- Immature oligodendrocytes were isolated using the MACS procedure pore); Nogo, phosphor-Fyn, ErbB2, and ErbB3 (Cell Signaling Technol- following the manufacturer’s instructions (Miltenyi Biotec). Optic nerve ogy). Anti-mouse or rabbit IgG antibody conjugated to horseradish and brain tissues were collected from two to three P5–P7 mice for each peroxidase were from GE. Alexa 488- or Alexa 594-conjugated anti- isolation. For oligodendrocyte selection, cells were incubated with mouse, anti-rabbit, or anti-goat IgG was from Life Technologies. Anti- anti-O4 monoclonal antibodies magnetic beads in MACS BSA buffer bodies against mouse myocilin were described previously (Malyukova et (Miltenyi Biotec) for 15 min at 4°C. Cells were applied to type MS Mini al., 2006). The human Lingo-1 extracellular domain sequence corre- MACS columns in the presence of a strong magnetic field
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