Effects of erythropoietin on glial cell development; oligodendrocyte maturation and astrocyte proliferation

Abstract

We investigated the effects of erythropoietin (Epo) in glial cell development, especially the maturation of late stage immature oligodendrocytes and the proliferation of astrocytes. Epo mRNA level in oligodendrocytes was much more prominent than those in neurons or astrocytes, which were the same as those in the young adult kidney, while Epo receptor (Epo-R) mRNA level were almost the same among neural cells, kidney and liver tissues. On immunohistochemical examination, Epo-R expression was also detected in O4-positive immature oligodendrocytes and glial fibrillary acidic protein positive astrocytes. These results suggested that types of both glial cells are responsive to Epo. The numbers of mature oligodendrocytes, which are characterized by myelin basic protein and process development, were increased by treatment with recombinant human Epo (rhEpo) (0.001–0.1 U/ml). The maturation of oligodendrocytes was also enhanced by coculture with astrocytes in vitro. However, when mixed cultured cells (oligodendrocytes+astrocytes) were treated with anti-Epo antibody and/or soluble Epo-R, the differentiation of oligodendrocytes was partially inhibited. Interestingly, high dose rhEpo (1, 3, 10 U/ml) markedly enhanced the proliferation of astrocytes. These results suggested that Epo not only promotes the differentiation and/or maturation in oligodendrocytes, but also enhances the proliferation of astrocytes. It is generally accepted that astrocytes produce Epo, and therefore Epo might act on astrocytes in an autocrine manner. The astrocytes stimulated with Epo may further accelerate the maturation of oligodendrocytes. These comprehensive effects of Epo might also affect the ability of oligodendrocyte lineage cells to promote myelin repair in the normal and damaged adult central nervous system.

Introduction

During early development, oligodendrocyte progenitor cells are generated in the restricted region of the ventral ventricular zone (Warf et al., 1991, Noll and Miller, 1993, Pringle and Richardson, 1993, Ono et al., 1995), but during late gestational and early postnatal development, glial progenitor cells exist in the subventricular zone (SVZ) (Paterson et al., 1973, Sturrock, 1976, Levison and Goldman, 1993) and migrate into the intermediate zone and cortical plate where they differentiate into immature oligodendrocytes, which subsequently further mature and myelinate axons (LeVine and Goldman, 1988a, LeVine and Goldman, 1988b, Hardy and Reynolds, 1991, Ellison et al., 1996). Oligodendrocyte progenitor cells, however, exist in the adult rodent SVZ (Levison et al., 1999) and subcortical white matter (Genser and Goldman, 1996, Genser and Goldman, 1997, Zhang et al., 1999), and even in the adult human white matter (Armstrong et al., 1992, Roy et al., 1999). Interestingly, such cells have also been reported in patients with multiple sclerosis (MS) (Wolswijk, 1998) and in rodents with chronic experimental allergic encephalomyelitis (Nishiyama et al., 1997). Though the presence of oligodendrocyte progenitor cells in the adult brain has not been fully accepted yet, these observations suggest that the remyelination process occurs in both physiological and pathological conditions, in the adult brain. In human demyelinating diseases such as MS, oligodendrocyte progenitors may divide and/or migrate in response to signals present in demyelinated lesions and thus facilitate remyelination (Armstrong et al., 1992, Roy et al., 1999). Regardless of the progenitor's presence in the lesions in demyelinating diseases, the remyelination process does not fully occur in such cases. The deficiency of repair in such demyelinating disorders may be improved by stimuli to enhance oligodendrocyte maturation. One such strategy is to promote survival of oligodendrocytes, or maturation of oligodendrocytes formed from progenitors, to extend their processes to wrap axons (Oh and Yong, 1996). This approach to enhance the extent of process formation has been documented around the edges of lesions in the MS (Prineas et al., 1989, Prineas et al., 1993, Raine and Wu, 1993, Raine et al., 1981).

The maturation of oligodendrocytes is regulated by growth factors (Bögler et al., 1990, Gard and Pfeiffer, 1993, McKinnon et al., 1993, Wolswijk and Noble, 1992, Barres et al., 1992, Barres et al., 1993, McMorris and Dubois-Dalcq, 1988), cytokines (Mayer et al., 1994) and the extracellular matrix (Oh and Yong, 1996). Cell–cell interactions also may play important roles in the maturation of oligodendrocytes. Axons control oligodendrocyte survival (Barres and Raff, 1999). Contact with central nervous system (CNS) myelin inhibits oligodendrocyte progenitor maturation (Robinson and Miller, 1999). We (Sakurai et al., 1998) and others (Bhat and Pfeiffer, 1986, Gard et al., 1995, Oh and Yong, 1996) reported previously that oligodendrocyte maturation in vitro is promoted by astrocytes, and astrocytes are involved in early events of myelinogenesis in vivo. Furthermore, abnormalities in the astroglia of the dysmyelinating Jimpy mouse may inhibit oligodendroglial cell development and myelination (Skoff, 1976). A soluble factor present in the extracts of astrocyte-enriched cultures enhances the expression of myelination-associated events (Bhat and Pfeiffer, 1986). Platelet-derived growth factor and leukemia inhibitory factor are mediators of astrocyte function as paracrine regulators of oligodendroblast and oligodendrocyte survival (Gard et al., 1995), and basic fibroblast growth factor (bFGF) is one of the candidates for the maturation factor released from astrocytes (Oh and Yong, 1996).

Erythropoietin (Epo) is a cytokine that acts in erythroid progenitor proliferation and differentiation. Recently, it has been reported that Epo is produced in astrocytes (Masuda et al., 1994), and protects hippocampal and cortical neurons against glutamate neurotoxicity in vitro (Morishita et al., 1997) or cerebral ischemia in vivo (Sakanaka et al., 1998, Brines et al., 2000). Both immunohistochemical localization and mRNA expression of Epo and its receptor change during development (Liu et al., 1994, Chin et al., 2000, Juul et al., 1998, Juul et al., 1999), suggesting that Epo may play important roles in development of the brain. The actions of Epo on glia cells, however, have not been investigated in detail. In this study, therefore, we examined whether Epo is one of the astrocyte-derived factors that cause oligodendrocyte maturation in vitro, using primary cultured late stage immature oligodendrocytes from the rat cerebral hemisphere.