S. E. Pfeiffer

The oligodendrocyte and its many cellular processes

The oligodendrocyte (OL) is increasingly providing a model system for probing central issues of cell biology. During development, OL progenitors undergo controlled migration, proliferation and differentiation, secrete and respond to a number of growth factors, and dramatically change their cellular architecture, culminating in the formation of the myelin sheath. This review examines some facets of the OL that make it an especially attractive tool for studying many basic questions in cell biology.

Proligodendroblast antigen (POA), a developmental antigen expressed by A007/O4-positive oligodendrocyte progenitors prior to the appearance of sulfatide and galactocerebroside.

Abstract: Evidence is presented for the immunological identification of a developmental antigen appearing at a critical point in the oligodendroglial lineage. Specifically, monoclonal antibody A007 recognizes cells in the oligodendrocyte lineage at two distinct stages. Analyses of purified lipid standards and lipid extracts from galactocerebroside‐positive (GalC+) oligodendrocytes by enzyme‐linked immunosorbent assay, lipid dot blot, and immuno‐TLC demonstrated that A007 recognizes sulfatide (SUL) and seminolipid. However, neither 35SO4 incorporation into SUL nor SUL accumulation could be detected in A007‐positive cells lacking galactocerebroside (i.e., A007+GalC− progenitor cells) present early in development. These data suggest that A007 also recognizes an antigen, named proligodendroblast antigen (POA), that appears during the late stage of oligodendrocyte progenitor development prior to the expression by oligodendrocytes of SUL and GalC. We have previously reported that monoclonal antibody O4 also recognizes not only SUL and seminolipid, but in addition an antigen that appears prior to the expression of SUL and galactocerebroside. In the present study all A007+ cells were also O4+ (and vice versa), and the developmental patterns of the two antibodies appeared to be identical. We conclude that (1) A007 is similar or identical to O4 with respect to its antigenic specificity, and (2) during oligodendrocyte lineage progression both antibodies react first with antigen POA on the surface of the oligodendrocyte progenitor cell prior to the expression of SUL [i.e., A007+O4+(POA+)SUL−GalC− proligodendroblasts], and only later with SUL as terminally differentiating oligodendrocytes emerge (i.e., A007+‐O4+SUL+GalC+ oligodendrocytes).

Regulation of FGF Receptors in the Oligodendrocyte Lineage

Fibroblast growth factors (FGFs) affect a broad spectrum of developmentally regulated cellular responses involved in the control of growth and differentiation. To identify specific FGF receptor forms involved in these responses, we have characterized FGF receptor transcript expression, and its modulation by FGF-2, as enriched populations of oligodendrocyte progenitors differentiate into mature oligodendrocytes. The data demonstrate that the levels of mRNA expression for FGF high-affinity receptors-1, -2, and -3 are differentially regulated during lineage progression: FGF receptor-1 expression increases with lineage progression, FGF receptor-2 is predominantly expressed by terminally differentiated oligodendrocytes, and FGF receptor-3 reaches a peak level of expression in late progenitors and then declines upon further differentiation; FGF receptor-4 expression was not detected in oligodendrocytes. Distinct patterns of alternatively spliced variants of FGF receptor-1 and -2 transcripts are expressed: the predominant FGF receptor-1 transcripts contain three Ig-like domains (FGF receptor-1 alpha), whereas the FGF receptor-2 transcripts contain two Ig-like domains (FGF receptor-2 beta 2) and this form is up-regulated as oligodendrocytes differentiate. In addition, the expression of these receptors is differentially regulated by the ligand, FGF-2: FGF receptor-1 mRNA expression is up-regulated in early progenitors, and FGF receptor-2 mRNA expression is down-regulated in mature oligodendrocytes. Finally, astrocytes express FGF receptor-1, -2, and -3 transcripts, but at different levels and with different exon utilization (FGF receptor-1 beta, FGF receptor-2 beta 1/beta 2) compared to oligodendrocytes. To our knowledge this is the first report that demonstrates that the mRNA expression of these three FGF receptor types is differentially regulated in primary cells as they differentiate along a lineage from progenitors to terminally differentiated cells. We propose that this pattern of expression provides a molecular basis for the developmentally varying response of cells to a common ligand. For example, according to this hypothesis, in response to FGF-2, FGF receptor-1 transduces signals that stimulate the prolonged proliferation and migration of early progenitors, FGF receptor-3 promotes the proliferation and arrest of differentiation of late progenitors, and FGF receptor-2 transduces signals for terminal differentiation, but not proliferation, in mature oligodendrocytes.

FGF‐2 converts mature oligodendrocytes to a novel phenotype

Fibroblast growth factor (FGF)‐2 differentially regulates oligodendrocyte progenitor proliferation and differentiation in culture, and modulates gene expression of its own receptors, in a developmental and receptor type‐specific manner (Bansal et al., 1996a,b). Three FGF receptors (types 1, 2, 3) are expressed in postmitotic, terminally differentiating oligodendrocytes. Exposure of such cells to FGF‐2 results in: (a) the down‐regulation of myelin‐specific gene expression (e.g., ceramide galactosyltransferase, 2′,3′‐cyclic nucleotide 3′‐phosphohydrolase, myelin basic protein, proteolipid protein), (b) dramatic increases in the length of cellular processes in a time‐ and dose‐dependent manner, (c) re‐entrance into the cell cycle without accompanying mitosis, and (d) the alteration of the expression of both low‐ and high‐affinity FGF receptors. Compared to oligodendrocyte progenitors, the differentiated oligodendrocytes treated with FGF‐2 incorporate BrdU at a slower rates, exhibit different patterns of both FGF high‐ and low‐affinity (syndecans) receptors, and are morphologically very different. In addition, they do not re‐express the progenitor markers A2B5, NG2 or PDGFα receptor. Therefore, although the FGF‐treated cells lose their differentiated OL/myelin markers, they do not revert to progenitors and clearly represent a different, apparently novel, phenotype both morphologically and biochemically, which we have termed NOLs. These data indicate that terminally differentiated oligodendrocytes retain the plasticity to reprogram their differentiation fate under the influence of environmental factors. The possible significance of this response to FGF relative to normal and pathological physiology is discussed. In particular, on the basis of these data we predict the appearance of cells in and around multiple sclerosis plaques with the phenotype O4+, NG2−, A2B5−, O1−, MBP−. J. Neurosci. Res. 50:215–228, 1997.

Monoclonal autoantibody SCH94.03, which promotes central nervous system remyelination, recognizes an antigen on the surface of oligodendrocytes

A monoclonal antibody SCH94.03, made in syngeneic mice by injection of spinal cord homogenate, promotes central nervous system remyelination when injected into SJL/J mice chronically infected with Theilers virus. To elucidate the mechanism of antibody‐mediated remyelination, SCH94.03 was investigated by immunocytochemistry, flow cytometry, immunoelectron microscopy, Western blotting, and immuno‐thin layer chromatography. All cell types investigated in vitro showed strong cytoplasmic staining with a pattern resembling a cytoskeletal protein. In contrast, among the primary cultured cells studied, only oligodendrocytes showed strong surface reactivity. Other cell types, including astrocytes, microglia, Schwann cells, myoblasts, and T and B lymphocytes, were negative. Mouse and rat oligodendrocytes which showed strong surface reactivity exhibited a well‐differentiated morphology, and ∼50% expressed myelin basic protein. Since oligodendrocyte progenitors were negative for surface staining, the expression of the antigens recognized by this monoclonal antibody appears to be developmentally regulated, i.e., transiently expressed on younger, terminally differentiating oligodendrocytes. Among the cell lines studied, only two rat oligodendrocyte lineage cell lines showed surface reactivity with SCH 94.03. Western blotting of secondary isolated oligodendrocytes lysates revealed reactivity with multiple protein bands of 27, 32, 50, 100, and 106 kDa, whereas there was no reactivity to lipid antigens by immuno‐thin layer chromatography. These results raise the possibility that SCH94.03 recognizes a novel oligodendrocyte‐specific surface antigen, and may act directly on oligodendrocytes to promote remyelination.

Differential expression of rab3 isoforms in oligodendrocytes and astrocytes.

The small GTP‐binding protein Rab3a is involved in regulated secretory pathways and is enriched in synaptic and neuroendocrine secretory vesicles. We have reported previously the developmental regulation of Rab3a in oligodendrocytes in culture and purified central nervous system myelin (Huber et al.: FEBS Lett 347: 273–278, 1994). Since multiple rab3 isoforms exist in the brain and may be associated with different secretory pathways, we have investigated the differential expression of the rab3 isoforms in oligodendrocytes, astrocytes, and Schwann cell line RT4‐D6P2T. The expression of specific rab3 isoforms (rab3a‐c) was detected by polymerase chain reaction (PCR) amplification and confirmed by sequence analyses. These data show that in addition to the previously reported expression in neurons, the two macroglial populations, astrocytes and oligodendrocytes, also express rab3 isoforms. Rab3b was preferentially amplified from purified, cultured astrocytes, while rab3a and rab3c were preferentially amplified from highly enriched populations of both cultured oligodendrocytes and those isolated directly from the brain by immunopanning. No novel rab3 isoform was detected in glia. These results indicate that glial cells in the brain express specific isoforms of the vesicular trafficking Rab3 protein family.

Multiple splice isoforms of proteolipid M6B in neurons and oligodendrocytes

Proteolipids are abundant integral membrane proteins, initially described as structural proteins of CNS myelin. More recently, two neuronal proteins related to proteolipid protein (PLP), termed M6A and M6B, were identified, suggesting a common function of proteolipids in oligodendrocytes and neurons. We have analyzed the X-linked M6B gene and discovered an unexpected complexity of protein isoforms. Two promoters and alternative exons yield at least eight M6B proteins and polypeptides, differentially expressed in neurons and oligodendrocytes. Six isoforms are tetraspan membrane proteins that differ by highly conserved amino- and carboxy-terminal domains, termed alpha, beta, psi, and omega. In MDCK cells, the beta-domain of M6B stabilizes tetraspan proteolipids at the cell surface, whereas non-beta isoforms are more abundant in intracellular compartments. Cotransfection experiments suggest a physical interaction of M6B and mutant PLP, when retained in the endoplasmic reticulum, that may also contribute to oligodendrocyte dysfunction in Pelizaeus-Merzbacher disease.

Developmental and FGF-2-Mediated Regulation of Syndecans (1–4) and Glypican in Oligodendrocytes

Differentiating cells undergo developmentally regulated changes in cell-cell and cell-matrix adhesion that control migration through microenvironments, proliferation, and differentiation. The diversity of the patterns of expression of heparan sulfate proteoglycans (HSPGs), coupled with their interactions with extracellular matrix, cell adhesion molecules, and growth factors, has emphasized their critical importance in the regulation of these events. Syndecans (1-4), glypican, and cerebroglycan are membrane-associated HSPGs that have been implicated in these events in various tissues and several tumor cell lines. We have examined the developmental expression and FGF-2-mediated regulation of these HSPGs during differentiation within a specific lineage of primary cells, oligodendrocytes (OL). Northern analyses of highly purified, developmentally synchronized populations of OL-lineage cells at three stages of differentiation (early and late progenitors and mature OLs) showed that the expression of individual forms of these syndecans and glypican are developmentally regulated. Specifically, the level of expression of syndecan-2 and -4 and glypican mRNAs increased as the cells differentiated from proliferative late progenitors to postmitotic mature cells. The expression of syndecan-1 and -3 had the inverse developmental pattern. Therefore, these two sets of molecules may have different roles in regulating the onset of terminal differentiation in OLs. The levels of mRNA expression were regulated by FGF-2: in late progenitors, FGF-2 induced a doubling of the mRNA levels of syndecan-2, -3, and -4, while those for syndecan-1 and glypican remained unaffected; in mature OLs, the levels of syndecan-1 mRNA were up-regulated, the levels of syndecan-2 and -4 and glypican were down-regulated. These results suggest that the individual syndecan molecules have distinct functions during the differentiation process and that multiple levels of regulation must exist, leading to a changing repertoire of these molecules during OL lineage progression and myelinogenesis.

Mice with Conditional Inactivation of Fibroblast Growth Factor Receptor-2 Signaling in Oligodendrocytes Have Normal Myelin But Display Dramatic Hyperactivity when Combined with Cnp1 Inactivation

Fibroblast growth factor receptors (Fgfr) comprise a widely expressed family of developmental regulators implicated in oligodendrocyte (OL) maturation of the CNS. Fgfr2 is expressed by OLs in myelinated fiber tracks. In vitro, Fgfr2 is highly upregulated during OL terminal differentiation, and its activation leads to enhanced growth of OL processes and the formation of myelin-like membranes. To investigate the in vivo function of Fgfr2 signaling by myelinating glial cells, we inactivated the floxed Fgfr2 gene in mice that coexpress Cre recombinase (cre) as a knock-in gene into the OL-specific 2′,3′-cyclic nucleotide phosphodiesterase (Cnp1) locus. Surprisingly, no obvious defects were detected in brain development of these conditional mutants, including the number of OLs, the onset and extent of myelination, the ultrastructure of myelin, and the expression level of myelin proteins. However, unexpectedly, a subset of these conditional Fgfr2 knock-out mice that are homozygous for cre and therefore are also Cnp1 null, displayed a dramatic hyperactive behavior starting at ∼2 weeks of age. This hyperactivity was abolished by treatment with dopamine receptor antagonists or catecholamine biosynthesis inhibitors, suggesting that the symptoms involve a dysregulation of the dopaminergic system. Although the molecular mechanisms are presently unknown, this novel mouse model of hyperactivity demonstrates the potential involvement of OLs in neuropsychiatric disorders, as well as the nonpredictable role of genetic interactions in the behavioral phenotype of mice.

SNARE complex proteins, including the cognate pair VAMP-2 and syntaxin-4, are expressed in cultured oligodendrocytes

Abstract : Myelin membrane synthesis in the CNS by oligodendrocytes (OLs) involves directed intracellular transport and targeting of copious amounts of specialized lipids and proteins over a relatively short time span. As in other plasma membrane‐directed fusion, this process is expected to use specific trafficking and vesicle fusion proteins characteristic of the SNARE model. We have investigated the developmental expression of SNARE proteins in highly enriched primary cultures of OLs at discrete stages of differentiation. VAMP‐2/synaptobrevin‐2, syntaxin‐2 and ‐4, nsec‐1/munc‐18‐1, Rab3a, synaptophysin, and synapsin were expressed. During differentiation, expression of the vesicular SNARE VAMP‐2, the small GTP‐binding protein Rab3a, and the target SNARE syntaxin‐4 were up‐regulated. VAMP‐2 and Rab3 proteins detected immunocytochemically in cultured OLs were localized within the developing process network ; in situ anti‐VAMP‐2 antibody stained the perikarya of rows of cells with the distribution and appearance of OLs. We discuss the potential involvement of SNARE complex proteins in a plasma membrane‐directed transport mechanism targeting nascent myelin vesicles to the forming myelin sheath.