Oligodendrocyte maturation is inhibited by bone morphogenetic protein

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Abstract

Mature oligodendrocytes myelinate axons in the CNS. The development of the myelin sheath is dependent on the proper maturation of oligodendrocytes from precursors cells, a spatially restricted process that is regulated by inductive and repressive cues. Several members of the bone morphogenetic protein family (BMP2 and 4) have been implicated as repressors of oligodendrocyte development in vitro by shifting oligodendrocyte precursors into the astrocyte lineage. We now report on a second role of BMPs in oligodendrocyte development, regulation of myelin protein expression in immature oligodendrocytes. Purified immature rodent oligodendrocytes treated with BMP4 maintained galactocerebroside (GalC) expression, whereas the expression of three key myelin proteins, proteolipid protein (PLP), myelin basic protein (MBP), and 2′-3′-cyclic nucleotide 3′-phosphodiesterase (CNP), was severely decreased. Paradoxically, BMP-treated oligodendrocytes show increased process extension and complexity, normally a feature of oligodendrocyte maturation. We also investigated whether BMP4 could inhibit myelin protein expression in an E 12.5 mouse explant culture of cervical spinal cord and hindbrain that maintains the in vivo cellular relationships and architecture. Beads soaked in BMP protein implanted into these explants inhibited the expression of myelin proteins, proteolipid protein, and myelin-associated glycoprotein (MAG), in the local area surrounding the bead. Since these explants also contained precursors cells, expression of galactocerebroside and O4, an oligodendrocyte marker, were also decreased by BMP treatment but to a much lesser degree than the myelin markers. Together, these data indicate that BMPs have multiple roles in oligodendrocyte development. At earlier stages, they affect cell lineage decisions and at later stages, they inhibit cell specialization.

Introduction

Myelination in the CNS assures the rapid and efficient conduction of electrical impulses. Myelin is synthesized by oligodendrocytes arising from precursors that have progressed through several phenotypic stages. Immature oligodendrocytes express galactocerebroside (GalC) and begin to increase process extension and complexity. Mature oligodendrocytes express myelin proteins, such as proteolipid protein (PLP), myelin basic protein (MBP), 2′-3′cyclic nucleotide 3′-phosphodiesterase (CNP), and myelin-associated glycoprotein (MAG), resulting in synthesis of a myelin membrane. The timing of myelination is extremely precise for each region of the CNS, suggesting that it is regulated by local signals, possibly both inductive and repressive. However, the actual controls of this differentiation process are poorly understood.

Oligodendrocytes are distributed throughout the CNS; however, precursors are generated in a restricted area of the ventral neuroepithelium. These precursors form a column of only one to two cell layers in width on either side of the central canal in the ventral epithelium of the neural tube (Pringle and Richardson, 1993). A gradient of the secreted protein sonic hedgehog (Shh) appears to be necessary and sufficient for the specification of these cells Orentas et al., 1999, Poncet et al., 1996. Although oligodendrocyte precursors do not appear to be generated in dorsal neuroepithelium, the application of Shh to dorsal epithelium can direct oligodendrocyte precursor specification Poncet et al., 1996, Pringle et al., 1996. Thus, it is likely that factors present in the dorsal neuroepithelium repress the generation of oligodendrocytes precursors. Direct evidence for a dorsal inhibitory factor first emerged from coculture experiments showing that the presence of dorsal cervical spinal cord and hindbrain inhibited oligodendrocyte generation from ventral explants (Wada et al., 2000) and, more recently, from experiments in which ablation of dorsal tissue permitted the generation of ectopic oligodendrocytes (Mekki-Dauriac et al., 2002).

Bone morphogenetic proteins, members of the TGFβ superfamily of secreted signaling molecules, are the first class of candidate molecules for this inhibitory action (BMPs). BMPs have established roles in dorsal cell specification and they inhibit ventralizing signals (for review, see Lee and Jessell, 1999, Liem et al., 1995). In vitro, BMPs inhibit oligodendrocyte preprogenitors or precursors from becoming immature oligodendrocytes and promote astrogliogenesis Grinspan et al., 2000, Mabie et al., 1997. Follastatin, chordin, and noggin are BMP-antagonists expressed in the notocord and floor plate (Tanabe and Jessell, 1996) possibly creating a region of ventral neuroepithelium that is permissive for oligodendrogliogenesis by blocking the action of BMPs. The role of BMPs in vivo has only recently begun to be explored. Overexpression studies in chick and xenopus showed inhibition of generation of oligodendrocyte precursors following exogenous addition of BMP Mekki-Dauriac et al., 2002, Miller et al., 2004, and a BMP-overexpressing mouse line had somewhat reduced numbers of oligodendrocytes (Gomes et al., 2003). In addition, oligodendrocyte lineage cells were generated in ectopic locations following inhibition of BMP by ablation of dorsal tissue, use of inhibitors such as noggin, or function blocking antibodies Mekki-Dauriac et al., 2002, Miller et al., 2004.

These previous studies implicate BMPs in cell fate specification. We now show that BMP4 plays another role in oligodendrocyte development by inhibiting the expression of multiple myelin proteins by maturing oligodendrocytes. BMP-treated oligodendrocytes in culture show a severe decrease in the expression of three myelin proteins, PLP, MBP, and CNP, although they continued to express GalC and, paradoxically, show increased process extension and complexity. Explant cultures prepared from E12.5 mouse cervical spinal cord and hindbrain, which maintain cellular relationships, also exhibited an inhibition of PLP and MAG expression in response to ventrally localized BMPs. Since these explants contained many oligodendrocyte precursors as well as immature oligodendrocytes, BMP bead treatment caused an expected decrease in the number of cells expressing O4 and Gal C but to a much smaller extent than the decrease in myelin protein expression. These data directly implicate BMPs as myelin protein inhibitors in immature oligodendrocytes.

Section snippets

Oligodendrocytes at three stages of differentiation express BMP receptors R1A and R1B

Oligodendrocyte differentiation in tissue culture can be roughly divided into three stages: precursors, immature oligodendrocytes, and mature oligodendrocytes. We generated purified cultures of cells in these stages, prepared RNA, and performed RT-PCR analysis to determine if cells in the three stages expressed the same BMP receptors. BMP signals through two types of receptors: types I and II. The type I receptors, A and B, are thought to confer specificity (Massague, 1998). We demonstrated the

Discussion

Investigations into the molecular basis of oligodendrocyte development have identified promoters and inhibitors of the lineage. The major inhibitors appear to be members of the BMP family of secreted proteins. However, to date, where BMPs affect the lineage has not been clearly defined. In our present studies, BMP4 specifically blocks the expression of several myelin structural proteins, preventing myelination. Our previous studies and those of other laboratories, both in vivo and in vitro,

Cell culture generation of primary cultures and immunoselection procedures

Cultures of oligodendrocyte precursors were established from the forebrains of 1-day-old Sprague–Dawley rats and seeded on 100 mm Petri dishes in serum-containing medium as previously described (Grinspan and Franceschini, 1995). After 24 h, the cultures were fed N1 medium consisting of 5 μg/ml transferrin, 16.1 μg/ml putrescene, 6.3 ng/ml progesterone, 3.3 ng/ml selenium, 10 ng/ml biotin, 2 mM glutamine, and 5 μg/ml insulin in DMEM. This medium was supplemented with 30% medium conditioned over

Acknowledgements

This work was supported by RO1 NS43422 (JBG). The authors thank MacLean Pancoast for technical assistance.

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