Contrasting Effects of Mitogenic Growth Factors on Myelination in Neuron–Oligodendrocyte Co-cultures ZHEN WANG, 1 HOLLY COLOGNATO, 1 AND CHARLES FFRENCH-CONSTANT 1,2 * 1 Department of Pathology, University of Cambridge, Cambridge, United Kingdom 2 Cambridge Centre for Brain Repair, University of Cambridge, Cambridge, United Kingdom KEY WORDS neuregulin; platelet-derived growth factor; fibroblast growth factor; nerve growth factor; ErbB4; myelin ABSTRACT Mitogenic growth factors play an important role in the ini- tial stages of oligodendrocyte development, but their roles in the process of myelination itself remain less well defined. In order to study directly the effects of different growth fac- tors on myelination, we used a purified in vitro co-culture system with dorsal root ganglion neurons and oligodendro- cytes. Extensive myelination had occurred in these cultures 14 days after oligodendrocyte precursors (OPCs) were added, with the relationship between neurite density and the percentage of oligodendrocytes forming myelin sheaths providing a robust and straightforward means of quantify- ing myelination. Addition of soluble neuregulin (Nrg1), a mitogen for oligodendroglial cells that also provides an axo- nal signal implicated in oligodendrocyte survival, increased myelination. Conversely, the OPC mitogens FGF-2 and PDGF inhibited myelination. The inhibitory effect of these mitogens was reversible, as inhibition of PDGF allowed myelination to proceed. Taken together, these data indicate that different mitogenic growth factors can regulate myeli- nation by oligodendrocytes in addition to their well- described effects on earlier stages of oligodendroglial devel- opment. Moreover, the results highlight important differ- ences between the growth factors. V V C 2007 Wiley-Liss, Inc. INTRODUCTION Myelination in the CNS involves sequential develop- mental processes in which oligodendrocyte precursor cells (OPCs) migrate, proliferate, and differentiate into newly formed oligodendrocytes, after which those oligo- dendrocytes selected by target-dependent survival mech- anisms wrap myelin membrane around the axons to form the sheath. Each oligodendrocyte can myelinate many axons, with the number of wraps proportional to the axon diameter and regulated tightly by reciprocal signaling between oligodendrocyte and axons. The regulation of oligodendrocyte development has been studied extensively, leading to the identification of many signaling molecules that control OPC migration, proliferation, survival, and differentiation (Miller, 2002). Many of these molecules have been identified using in vitro methods to study oligodendrocytes, facilitated by the development of a method to grow oligodendrocytes by mechanical dissociation of OPCs from mixed glial cul- tures (McCarthy and de Vellis, 1980), thus allowing their differentiation in culture. These cultured oligoden- drocytes produce myelin-specific lipids and proteins with a timescale similar to that seen in vivo, and also gener- ate myelin-like membranes in the absence of neurons (Bansal and Pfeiffer, 1985; Knapp et al., 1987; Sarlieve et al., 1980; Szuchet et al., 1986). Using these methods, a number of growth factors have been shown to regulate oligodendrocyte development (reviewed by McMorris and McKinnon, 1996). For example, platelet-derived growth factor (PDGF) promotes OPC migration (Armstrong et al., 1990; Milner et al., 1997; Noble et al., 1988), sur- vival (Barres et al., 1993), and proliferation (Noble et al., 1988; Raff et al., 1988; Richardson et al., 1988). The key role of this growth factor was confirmed by changes in OPC numbers in transgenic mice that over- or underexpress PDGF-AA (Calver et al., 1998; Fruttiger et al., 1999). Fibroblast growth factor-2 (FGF-2) en- hances OPC migration (Eccleston and Silberberg, 1985; Milner et al., 1997), proliferation, and prevents their differentiation (Bogler et al., 1990; Eccleston and Silberberg, 1985; Gard and Pfeiffer, 1993; McKinnon et al., 1990). Insulin-like growth factor-I (IGF-I) also promotes OPC proliferation and survival (McMorris and Dubois-Dalcq, 1988) with an effect in vivo confirmed by experiments showing increased myelination in trans- genic mice (Carson et al., 1993). Transforming growth factor-b (TGF-b1) promotes oligodendrocyte differentia- tion (McKinnon et al., 1993), as does FGF-2 in the pre- sence of astrocytes (Mayer et al., 1993). Neuregulin-1 (Nrg1) promotes OPC proliferation (Canoll et al., 1996) and target-dependent survival (Colognato et al., 2002; Flores et al., 2000). Much less well understood, however, are the roles of growth factors in regulating the unique feature of oligo- dendrocyte biology, the formation of the sheath. Clearly, these roles cannot be studied in cultures of purified OPCs as there are no target axons on which the sheath Grant sponsors: Wellcome Trust; Multiple Sclerosis Society of Great Britain and Northern Ireland; National Multiple Sclerosis Society. Holly Colognato is currently at Department of Pharmacology, State University of New York, Stony Brook, New York NY11794. *Correspondence to: Charles ffrench-Constant, Department of Pathology, Univer- sity of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK. E-mail: cfc@mole.bio.cam.ac.uk Received 21 October 2006; Revised 30 November 2006; Accepted 1 December 2006 DOI 10.1002/glia.20480 Published online 18 January 2007 in Wiley InterScience (www.interscience. wiley.com). GLIA 55:537–545 (2007) V V C 2007 Wiley-Liss, Inc.