Akt-Mediated Survival of Oligodendrocytes Induced by Neuregulins

The Journal of Neuroscience, October 15, 2000, 20(20):7622–7630 Akt-Mediated Survival of Oligodendrocytes Induced by Neuregulins Ana I. Flores,1 Barbara S. Mallon,1 Takashi Matsui,2 Wataru Ogawa,3 Anthony Rosenzweig,2 Takashi Okamoto,1 and Wendy B. Macklin1 1Department of Neurosciences, The Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, 2Cardiovascular Research Center, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts 02139, and 3Second Department of Internal Medicine, Kobe University School of Medicine, Chuo-ku, Kobe 650–0017, Japan Neuregulins have been implicated in a number of events in cells heregulin in glial cells, BAD was overexpressed in C6 glioma in the oligodendrocyte lineage, including enhanced survival, mi- cells. In these cells, heregulin induced phosphorylation of BAD at tosis, migration, and differentiation. At least two signaling path- Ser 136. Apoptosis of oligodendrocyte progenitor cells induced by ways have been shown to be involved in neuregulin signaling: the growth factor deprivation was effectively blocked by heregulin in phosphatidylinositol (PI)-3 kinase and the mitogen-activated pro- a wortmannin-sensitive manner. Overexpression of dominant tein kinase pathways. In the present studies, we examined the negative Akt but not of wild-type Akt by adenoviral gene transfer signaling pathway involved in the survival function of heregulin, in primary cultures of both oligodendrocytes and their progeni- focusing on heregulin-induced changes in Akt activity in cultured tors induced significant apoptosis through activation of the glial cells, and the consequences of Akt activation in cells in the caspase cascade. The present data suggest that the survival oligodendrocyte lineage. Heregulin binds erbB receptors, and in function of heregulin is mediated through the PI-3 kinase/Akt our studies, primary cultures of both oligodendrocyte progenitor pathway in cells in the oligodendrocyte lineage and that the Akt cells and differentiating oligodendrocytes expressed erbB2, pathway may be quite important for survival of cells in this erbB3, and erbB4 receptors. In C6 glioma cells and primary lineage. cultures of oligodendrocytes, heregulin induced time- and dose- dependent Akt phosphorylation at Ser 473 in a wortmannin- Key words: oligodendrocyte; survival; Akt; neuregulin; multiple sensitive manner. To investigate further the signaling pathway for sclerosis; apoptosis Oligodendrocyte loss and demyelination in the CNS have been tion of the heart and nervous system (Meyer and Birchmeier, 1995; proposed to result from apoptotic cell death during normal devel- Kramer et al., 1996; Erickson et al., 1997; Riethmacher et al., 1997; opment (Barres et al., 1992), after growth factor withdrawal in vitro Vartanian et al., 1999; Golding et al., 2000; Lin et al., 2000; (Yasuda et al., 1995), and in pathological conditions such as mul- Wolpowitz et al., 2000). tiple sclerosis (MS), Theiler’s murine encephalomyelitis virus The NRG-1/neuregulin family includes heregulin, acetylcholine (TMEV) infection, and experimental allergic encephalitis (EAE; receptor inducing activity, neu differentiation factor, glial growth Raine and Scheinberg, 1988; Ozawa et al., 1994; Tsunoda and factor (GGF), and sensorimotor-derived factor (Burden and Fujinami, 1996; Hisahara et al., 2000). Growth factors and recep- Yarden, 1997; Meyer et al., 1997). The heregulin (HRG) members tors can protect cells from apoptosis by blocking cell death signals contain two isoforms differing in their EGF-like domain (isoforms: (Raff et al., 1993). The neuregulin/erbB receptor system seems an ␣, ␤). The ␣- and ␤-variants bind differentially to receptors and ideal candidate to provide survival signals for oligodendrocytes in elicit different biological responses (Marikovsky et al., 1995; Baek vivo. In the CNS, neurons, oligodendrocytes, and astrocytes both and Kim, 1998; Crovello et al., 1998). NRG-1 proteins mediate produce neuregulins and express neuregulin receptors (Chen et al., their action through erbB receptor tyrosine kinases, including 1994; Meyer and Birchmeier, 1994; Raabe et al., 1997a; Francis et erbB2, erbB3 and erbB4. al., 1999). Furthermore, neuregulins present on the surface of Although they are clearly involved in cell survival, neuregulins axons play a role in the maintenance of oligodendrocytes and their have other effects in the oligodendrocyte lineage, including prolif- progenitors (Canoll et al., 1996; Vartanian et al., 1997). eration or blockage of differentiation (Lemke, 1996; Burden and The neuregulins (NRGs) are a class of epidermal growth factor- Yarden, 1997). Also, they have been demonstrated to signal like molecules that arise by alternative splicing from a single gene through both the phosphatidylinositol (PI)-3 kinase and the MAP (NRG-1). Recently, three other neuregulin-like genes have been kinase pathways (Canoll et al., 1999). The precise signaling path- described: NRG-2 (Carraway et al., 1997; Chang et al., 1997), ways through which neuregulins induce survival in oligodendrocyte NRG-3 (Zhang et al., 1997), and NRG-4 (Harari et al., 1999). lineage cells remain to be established. Thus, understanding which Targeted disruptions of the NRG-1 gene as well as the neuregulin pathway is primarily involved in their survival function for oligo- receptors demonstrate that neuregulins are essential for the forma- dendrocytes and their progenitors is an important question. Be- cause activation of PI-3 kinase/Akt pathway plays a major role in Received May 30, 2000; revised July 27, 2000; accepted July 31, 2000. survival signals for multiple growth factors (Dudek et al., 1997; This study is supported by National Institutes of Health (NIH) Grant NS25304 Franke et al., 1995, 1997; Kennedy et al., 1997), we have focused (W.B.M.), Multiple Sclerosis Pilot Grant PP0649 (T.O.), NIH Grant R29-MH56036 this study on whether neuregulin induces cell survival in different (T.O.), Prentiss Foundation (T.O.), and a Postdoctoral Fellowship from the National stages of the oligodendrocyte lineage through the PI-3 kinase/Akt Multiple Sclerosis Society (A.I.F.). Correspondence should be addressed to Wendy B. Macklin, Department of Neu- pathway. rosciences NC30, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195. E-mail: [email protected]. MATERIALS AND METHODS Dr. Okamoto’s present address: Laboratory for Neurodegeneration and Signaling, Reagents. Recombinant heregulin (HRG ␤1) was obtained from NeoMar- Brain Science Institute, The Institute of Physical and Chemical Research (RIKEN), kers (Fremont, CA). ErbB3 and erbB4 receptor polyclonal antibodies were Wako-shi.Saitama 351-0198, Japan. obtained from Santa Cruz Biotechnology (Santa Cruz, CA), and erbB2 Copyright © 2000 Society for Neuroscience 0270-6474/00/207622-09$15.00/0 polyclonal antibody was obtained from NeoMarkers. Anti-total Akt, anti- Flores et al. • Heregulin Is a Potential Survival Factor in Oligodendrocytes J. Neurosci., October 15, 2000, 20(20):7622–7630 7623 phospho Akt (Ser 473), anti-phospho BAD (Ser 112), anti-phospho BAD least 15 random fields. The results were expressed as percentage of the (Ser 136) and anti-total BAD were obtained from New England Biolabs total O4ϩ cells. (Beverly, MA). Monoclonal anti-A2B5 antibody was obtained from Roche Caspase activity assay. Caspase activation was measured by CaspACE Molecular Biochemicals (Indianapolis, IN). Polyclonal anti-NG2 antibody FITC-VAD-FMK in situ marker (Promega, Madison, WI) according to was provided by W. B. Stallcup (The Burnham Institute, La Jolla Cancer the manufacturer’s instructions. Briefly, live oligodendrocyte progenitor Research Center, La Jolla, CA). Wortmannin, PD98059, Rapamycin, and cells infected with adenovirus as described above were treated with the SB203580 were obtained from Calbiochem. L-NAME and D-NAME were FITC conjugate of the permeable irreversible caspase inhibitor (VAD- purchased from Sigma (St. Louis, MO). C6 glioma cells were obtained FMK) for 20 min to allow binding to activated caspase. Cells were either from American Type Culture Collection (Manassas, VA). live-stained with a monoclonal anti-A2B5 antibody (1:100) before fixation Cell culture, transfections, and stimulation protocols. The C6 glioma cells or fixed and stained with NG2 antibody (1:200). TR-labeled secondary were grown in DMEM containing 10% fetal bovine serum (Life Technol- antibody was used at 1:200. The total number of cells was determined by ogies, Gaithersburg, MD), plus penicillin and streptomycin at 37°C in 5% DAPI staining. The number of positive cells was determined by counting ␤ ϫ 6 CO2 environment. For stimulation with HRG 1, 2 10 cellsina100 at least 1000 cells per well. The results were expressed as percentage of the mm plate were cultured in serum-free DMEM for 20–24 hr and stimulated total cells Ϯ SEM. as indicated in the figure legends with HRG ␤1, insulin-like growth Immunocytochemistry. C6 glioma cells were plated at 80,000 cells/well in factor-1 (IGF-1), or 0.1 mM pervanadate prepared as indicated in And- four-chamber slide (Becton Dickinson, Franklin Lakes, NJ). After fixation jelkovic et al. (1997). Cells were exposed to inhibitors for 30 min before in 4% paraformaldehyde, cells were permeabilized in 0.3% Triton X-100 HRG ␤1 addition. in PBS for 30 min at room temperature and stained with anti-phospho Akt For transient transfections, cells seeded at 1 ϫ 10 6/100 mm dishes were antibody (1:100) overnight at 4°C. FITC-labeled secondary antibodies ␮ were

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