Oligodendrocyte Progenitor Differentiation Research Articles

Voltage-Activated K+Channels and Membrane Depolarization Regulate Accumulation of the Cyclin-Dependent Kinase Inhibitors p27Kip1and p21CIP1in Glial Progenitor Cells

Neural cell development is regulated by membrane ion channel activity. We have previously demonstrated that cell membrane depolarization with veratridine or blockage of K+ channels with tetraethylammonium (TEA) inhibit oligodendrocyte progenitor (OP) proliferation and differentiation (); however the molecular events involved are largely unknown. Here we show that forskolin (FSK) and its derivative dideoxyforskolin (DFSK) block K+ channels in OPs and inhibit cell proliferation. The antiproliferative effects of TEA, FSK, DFSK, and veratridine were attributable to OP cell cycle arrest in G1 phase. In fact, (1) cyclin D accumulation in synchronized OP cells was not affected by K+ channel blockers or veratridine; (2) these agents prevented OP cell proliferation only if present during G1 phase; and (3) G1 blockers, such as rapamycin and deferoxamine, mimicked the anti-proliferative effects of K+ channel blockers. DFSK also prevented OP differentiation, whereas FSK had no effect. Blockage of K+ channels and membrane depolarization also caused accumulation of the cyclin-dependent kinase inhibitors p27(Kip1) and p21(CIP1) in OP cells. The antiproliferative effects of K+ channel blockers and veratridine were still present in OP cells isolated from INK4a-/- mice, lacking the cyclin-dependent kinase inhibitors p16(INK4a) and p19(ARF). Our results demonstrate that blockage of K+ channels and cell depolarization induce G1 arrest in the OP cell cycle through a mechanism that may involve p27(Kip1) and p21(CIP1) and further support the conclusion that OP cell cycle arrest and differentiation are two uncoupled events.

Neurotransmitter receptor activation triggers p27(Kip1 )and p21(CIP1) accumulation and G1 cell cycle arrest in oligodendrocyte progenitors.

We examined the pathways that link neurotransmitter receptor activation and cell cycle arrest in oligodendrocyte progenitors. We had previously demonstrated that glutamate receptor activation inhibits oligodendrocyte progenitor proliferation and lineage progression. Here, using purified oligodendrocyte progenitors and cerebellar slice cultures, we show that norepinephrine and the beta-adrenergic receptor agonist isoproterenol also inhibited the proliferation, but in contrast to glutamate, isoproterenol stimulated progenitor lineage progression, as determined by O4 and O1 antibody staining. This antiproliferative effect was specifically attributable to a beta-adrenoceptor-mediated increase in cyclic adenosine monophosphate, since analogs of this cyclic nucleotide mimicked the effects of isoproterenol on oligodendrocyte progenitor proliferation, while alpha-adrenoceptor agonists were ineffective. Despite the opposite effects on lineage progression, both isoproterenol and the glutamate receptor agonist kainate caused accumulation of the cyclin-dependent kinase inhibitors p27(Kip1)and p21(CIP1), and G1 arrest. Studies with oligodendrocyte progenitor cells from INK4a-/- mice indicated that the G1 cyclin kinase inhibitor p16(INK4a) as well as p19(ARF)were not required for agonist-stimulated proliferation arrest. Our results demonstrate that beta-adrenergic and glutamatergic receptor activation inhibit oligodendrocyte progenitor proliferation through a mechanism that may involve p27(Kip1) and p21(CIP1); but while neurotransmitter-induced accumulation of p27(Kip1) is associated with cell cycle arrest, it does not by itself promote oligodendrocyte progenitor differentiation.

Clocks regulating developmental processes

Developmental clocks are hypothetical embryonic time-measuring devices — some are run by oscillators, whereas otherdepend on rate-limiting processes. Their existence has been deduced from recent studies of the timing of the midblastula transition, the opening of the Hox cluster during organogenesis, and oligodendrocyte progenitor differentiation; however, the mechanisms underlying their function remain largely unknown.

Mitochondrial Ca2+ uptake and release influence metabotropic and ionotropic cytosolic Ca2+ responses in rat oligodendrocyte progenitors.

1. Many physiologically important activities of oligodendrocyte progenitor cells (O-2A cells), including proliferation, migration and differentiation, are regulated by cytosolic Ca2+ signals. However, little is known concerning the mechanisms of Ca2+ signalling in this cell type. We have studied the interactions between Ca2+ entry, Ca2+ release from endoplasmic reticulum and Ca2+ regulation by mitochondria in influencing cytosolic Ca2+ responses in O-2A cells. 2. Methacholine (MCh; 100 microM) activated Ca2+ waves that propagated from several initiation sites along O-2A processes. 3. During a Ca2+ wave evoked by MCh, mitochondrial membrane potential was often either depolarized (21 % of mitochondria) or hyperpolarized (20 % of mitochondria), as measured by changes in the fluorescence of 5,5',6,6'-tetrachloro-1,1',3, 3'-tetraethylbenzimidazole carbocyanine iodide (JC-1). 4. Stimulation with kainate (100 microM) evoked a slowly rising, sustained cytosolic Ca2+ elevation in O-2A cells. This also, in some cases, resulted in either a depolarization (15 % of mitochondria) or hyperpolarization (12 % of mitochondria) of mitochondrial membrane potential. 5. Simultaneous measurement of cytosolic (fluo-3 AM) and mitochondrial (rhod-2 AM) Ca2+ responses revealed that Ca2+ elevations in the cytosol evoked by either MCh or kainate were translated into long-lasting Ca2+ elevations in subpopulations of mitochondria. In some mitochondria, Ca2+ signals appeared to activate Ca2+ release into the cytosol. 6. Inhibition of the mitochondrial Na+-Ca2+ exchanger by CGP-37157 (25 microM) decreased kainate Ca2+ response amplitude and increased the rate of return of the response to basal Ca2+ levels. 7. Thus, both ionotropic and metabotropic stimulation evoke changes in mitochondrial membrane potential and Ca2+ levels in O-2A cells. Ca2+ uptake into some mitochondria is activated by Ca2+ entry into cells or release from stores. Mitochondrial Ca2+ release appears to play a key role in shaping kainate-evoked Ca2+ responses.

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. © 1997 Wiley-Liss, Inc.

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 PDGFalpha 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-.

Isolation and Purification of Primary Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system, and are avidly studied by investigators interested in neural cell lineages, myelination, multiple sclerosis, and oligodendroglioma. This unit presents a strategy for manipulating the division and differentiation of oligodendrocyte progenitors by isolating O-2A progenitors from optic nerve and then purifying and expanding them by culture in a defined medium supplemented with platelet-derived growth factor (PDGF) plus basic fibroblast growth factor (bFGF). Cells are then allowed to differentiate through removal of the growth factors. A protocol is also provided for monitoring the cell type composition of the cultures by immunohistochemistry. In addition to making it possible to generate large numbers of cells, this approach allows access to intermediate differentiation stages. Cells can thus be studied as they differentiate into oligodendrocytes, making the actual process of differentiation open to examination.

GGF/Neuregulin Is a Neuronal Signal That Promotes the Proliferation and Survival and Inhibits the Differentiation of Oligodendrocyte Progenitors

We show that GGF/neuregulin is a mitogen for pro-oligodendrocytes (O4+/O1− cells), oligodendrocytes (O4+/O1+ cells), and type-2 astrocytes. Heregulin β1, another neuregulin isoform, is also mitogenic. The proliferative effect of glial growth factor (GGF) does not require, but is greatly potentiated by, serum factors. GGF also promotes the survival of pro-oligodendrocytes under serum-free conditions. High levels of GGF reversibly inhibit the differentiation and lineage commitment of oligodendrocyte progenitors and, in differentiated cultures, result in loss of O1 and myelin basic protein expression. All three erbB receptors are expressed by progenitors and are activated by GGF; the relative abundance of these receptors changes during differentiation. Finally, cortical neurons release a soluble mitogen for pro-oligodendrocytes that is specifically blocked by antibodies to GGF. These results implicate the neuregulins in the neuronal regulation of oligodendrocyte progenitor proliferation, survival, and differentiation.

Oligodendrocyte progenitors are generated throughout the embryonic mouse brain, but differentiate in restricted foci.

Recent evidence from studies mapping the expression of putative oligodendrocyte progenitor specific mRNAs has suggested that oligodendrocyte progenitors arise during embryogenesis, in specific foci of the neuroectoderm. In order to test this hypothesis, we have assayed different regions of the embryonic central nervous system for their ability to generate oligodendrocytes following transplantation into neonatal cerebrum. To allow identification of donor-derived oligodendrocytes in wild-type host brain, we used the MbetaP transgenic mouse, which expresses lacZ in oligodendrocytes, as donor tissue. We found that tissue fragments derived from several levels of the anterior-posterior axis of the neural tube at E14.5 and E12.5, chosen to include (hindbrain, cervical and lumbar spinal cord), or exclude (dorsal telencephalon) putative foci of oligodendrocyte progenitors, all produced oligodendrocytes following transplantation. In addition, these same regions taken from E10.5, prior to the appearance of putative oligodendrocyte progenitor markers, also all yielded oligodendrocytes on transplantation. This indicates that precursor cells that can generate oligodendrocytes are widespread throughout the neuroectoderm as early as E10.5. We have also used the oligodendrocyte lineage-specific glycolipid antibodies O4, R-mAb and O1 to identify those regions of the developing brain that first support the differentiation of oligodendrocytes from their progenitor cells. We found that the first oligodendrocytes arise in prenatal brain at E14.5, in a restricted zone adjacent to the midline of the medulla. These cells are mitotically inactive, differentiated oligodendrocytes and, using light and electron microscopy, we show that they become functional, myelin-bearing oligodendrocytes. We have mapped the subsequent appearance of differentiated oligodendrocytes in the prenatal brain and show that they appear in a restricted, tract-specific manner. Our results suggest that oligodendrocytes are generated from neuroectodermal cells positioned throughout the rostrocaudal axis of the neural tube, rather than at restricted locations of the neuroectoderm. By contrast, the differentiation of such cells into oligodendrocytes does occur in a restricted manner, consistent with local regulation of oligodendrocyte progenitor differentiation.

Activation of mitogen-activated protein kinases in oligodendrocytes.

The proliferation and differentiation of oligodendrocyte progenitors are stringently controlled by an interacting network of growth and differentiation factors. Not much is known, however, about the intracellular signaling pathways activated in oligodendrocytes. In this study, we have examined the activation of mitogen-activated protein (MAP) kinase [also called extracellular signal-regulated protein kinases (ERKs)] in primary cultures of developing oligodendrocytes and in a primary oligodendrocyte cell line, CG4, in response to platelet-derived growth factor (PDGF) and basic fibroblast growth factor. MAP kinase activation was determined by an ingel protein kinase renaturation assay using myelin basic protein (MBP) as the substrate. The specificity of MAP kinase activation was further confirmed by an immune complex kinase assay using anti-MAP kinase antibodies. Stimulation of oligodendrocyte progenitors with the growth factors PDGF and basic fibroblast growth factor and a protein kinase C-activating tumor promoter, phorbol 12-myristate 13-acetate, resulted in a rapid activation of p42mapk (ERK2) and, to a lesser extent, p44mapk (ERK1). Immunoblot analysis with anti-phosphotyrosine antibodies revealed an increased Tyr phosphorylation of a 42-kDa phosphoprotein band cross-reacting with anti-MAP kinase antibodies. The phosphorylation of p42mapk in PDGF-treated oligodendrocyte progenitors was preceded by a robust autophosphorylation of the growth factor receptor. Immunoblot analysis with anti-pan-ERK antibodies indicated the presence of ERK-immunoreactive species other than p42mapk and p44mapk in oligodendrocytes. The presence of some of the same pan-ERK-immunoreactive species and certain renaturable MBP kinase activities was also demonstrable in myelin preparations from rat brain, suggesting that MAP kinases (and other MBP kinases) may function not only during oligodendrogenesis but also in myelinogenesis.

A sulfatide-reactive human monoclonal antibody obtained from a multiple sclerosis patient selectively binds to the surface of oligodendrocytes

In a previous paper, we described the production of a sulfatide-reactive IgM antibody-secreting B cell line that was obtained by Epstein-Barr virus transformation of peripheral B cells from a patient with multiple sclerosis (MS) (Uhlig and Dernick, 1989). In the present study, we demonstrate that this human monoclonal antibody (humAb) DS1F8 selectively binds to the surface of living oligodendrocytes in mixed brain cell cultures of newborn rats. Since a mouse mAb reactive with sulfatide was shown to inhibit oligodendrocyte progenitor differentiation, autoantibodies with binding specificities similar to DS1F8 could play a role in the demyelinating process in the CNS.

Inhibition of Protein and Lipid Sulfation in Oligodendrocytes Blocks Biological Responses to FGF-2 and Retards Cytoarchitectural Maturation, but Not Developmental Lineage Progression

We have examined the interfaces between protein and lipid sulfation, the response of progenitor cells to specific growth factors, and developmental lineage progression. The experimental system used is the oligodendrocyte (OL), the cell responsible for myelination of axons in the central nervous system. The ability to regulate the growth and differentiation of oligodendrocytes in primary cultures growing in defined medium offers a particularly attractive model for these interrelated areas of cell and developmental biology. Sulfation was inhibited in this system by growing cells in sodium chlorate, a competitive inhibitor of sulfation. Four principal conclusions are drawn from the data. First, heparan sulfate proteoglycan is a necessary cofactor for the environmental regulation by FGF-2 of nontransformed oligodendrocyte progenitors, including both the mitogenic response and the inhibition of terminal differentiation at a specific stage of the lineage. Second, the differentiation of oligodendrocyte progenitors from an early to a mature stage can occur in the absence of sulfation. Third, however, the formation of cellular processes and myelin-like sheets is retarded, leading to the prediction that sulfated molecules are critically involved in aspects of oligodendrocyte differentiation directly relating to myelination per se. Fourth, the developmental proligodendroblast antigen, POA, is a sulfated molecule. The results provide clear evidence for the importance of sulfation for the responses of OL progenitors to FGF-2 and for OL cytoarchitectural maturation, while demonstrating an ability of OL progenitors to undergo lineage progression in its absence.

Reversible inhibition of oligodendrocyte progenitor differentiation by a monoclonal antibody against surface galactolipids.

We have hypothesized that oligodendrocyte (OL) surface glycolipids, specifically galactocerebroside and sulfatide, play a role in the regulation of OL development by acting as sensors/transmitters of environment information. In support of this hypothesis we report here a reversible inhibition of OL progenitor cell differentiation by a monoclonal antibody [Ranscht mAb (R-mAb); Ranscht, B., Clapshaw, P. A. & Seifert, W. (1982) Proc. Natl. Acad. Sci. USA 79, 2709-2713] that reacts with these glycolipids. When isolated OL progenitors or mixed primary cultures are grown in the presence of the antibody, myelinogenic development is blocked in a dose-dependent manner at concentrations as low as 2 micrograms of IgG per ml. The inhibited cells express the OL progenitor markers O4 and vimentin but are negative for galactosylcerebroside, sulfatide, 2',3'-cyclic nucleotide 3'-phosphohydrolase, myelin basic protein, and myelin basic protein RNA expression. In contrast, the levels of total cellular protein and the expression of astrocytic glial fibrillary acidic protein in mixed cultures are not affected. Antibody-blocked cells have a distinctive morphology in which long, sparsely branched processes emanate from round cell bodies. Upon removing the perturbing antibody, the cells rapidly resume differentiation. Reverted mixed primary cultures, in which OL progenitors of several sequential developmental stages are present at the time of plating, differentiate more rapidly than control cultures, suggesting that the antibody-induced block results in a synchronization of developmental progression along the OL lineage by accumulating cells at the inhibition point. However, the normal temporal sequence of marker expression is maintained. Control studies with several other antibodies recognizing OL cell surface antigens, including HNK-1, neural cellular adhesion molecule (N-CAM), 1A9, anticholesterol, and O1, did not inhibit development. Since the inhibition occurs in highly enriched populations of OL progenitors, the inhibition does not involve cell-cell interactions between OLs and other cell types but concerns interactions of OLs with themselves, soluble factors, or OL extracellular matrix molecules and adhesion factors that provide essential environmental signals required for normal myelinogenic development.