Thrombocytopenic c-mpl(-/-) mice can produce a normal level of platelets after administration of 5-fluorouracil: the effect of age on the response.

Administration of 5-fluorouracil (5-FU) to mice results in a marked increase in the level of circulating platelets in 10 days. Mice lacking Mpl, the receptor for thrombopoietin (TPO), are thrombocytopenic. To gain insight into the mechanism by which 5-FU produces such a substantial stimulation of platelet production, this study investigated whether 5-FU (150 mg/kg) produced thrombocytosis in c-mpl(-/-) mice, thus establishing whether TPO was required for this response. A 5- to 6-fold increase in platelet levels in c-mpl(-/-) mice (to approximately 1000 x 10(9)/L) was observed on days 20 and 25 after 5-FU injection. Thus, at the peak of the response, c-mpl(-/-) mice had platelet levels comparable to those in normal mice. Administration of 5-FU also produced thrombocytosis in previously splenectomized c-mpl(-/-) mice. Comparison of the platelet response to 5-FU in young (6-12 weeks) and old (33-46 weeks) c-mpl(-/-) mice found that older mice produced a much more marked response than younger mice, with a mean maximum platelet level of approximately 1700 x 10(9)/L. To determine whether this increase in circulating platelets was preceded by an increase in hematopoietic progenitors, serial cultures of bone marrow and spleen were evaluated. A considerable increase in all colony types studied was observed on days 15 and 20 in spleens of c-mpl(-/-) mice, but no similar elevations were detected in bone marrow. These results indicate that c-mpl(-/-) mice can achieve a normal level of platelets after 5-FU injection, by means of a TPO-independent mechanism, and that they respond to 5-FU myelosuppression by producing large numbers of megakaryocytic, myeloid, and erythroid progenitors. (Blood. 2001;98:1019-1027)

Eliprodil stimulates CNS myelination: new prospects for multiple sclerosis?

OBJECTIVE: To examine the potential of eliprodil-a neuroprotective agent with a high affinity for sigma-receptors-to promote myelination in neuron-oligodendrocytes cocultures. BACKGROUND: Remyelination is one of the major therapeutic issues in MS. Because neuronal integrity is required for CNS myelination, the authors postulated that neuroprotective molecules might favor myelination. METHODS: Two experimental culture conditions were compared: standard medium Bottenstein and Sato ([B-S] medium) and a medium depleted of both thyroid hormones and progesterone (depleted [D] medium). Myelination was quantified by counting the number of myelinated internodes, identified immunocytochemically with an antimyelin basic protein (anti-MBP) antibody. RESULTS: The authors first confirmed that in D medium myelination was reduced by a factor of 3.5 compared with cultures maintained in B-S medium. Under both culture conditions, addition of 10(-6) M eliprodil did not modify significantly the total number of either microtubule associated protein-2-positive neurons or MBP-positive oligodendrocytes. However, eliprodil induced a twofold (p < 0.01) increase in myelination when added to B-S medium, and a 4.7-fold (p < 0.0001) increase when added to D medium. CONCLUSION: Although the molecular mechanism mediating the effect of the sigma-receptor agonist on myelination remains to be elucidated, these results strongly suggest that neuroprotective molecules may be of therapeutic interest in demyelinating diseases such as MS.

Thyroid hormone receptor isoforms are sequentially expressed in oligodendrocyte lineage cells during rat cerebral development.

In the mammalian brain, thyroid hormones regulate myelination. Their actions are mediated by interactions with nuclear receptors that function as ligand-regulated transcription factors. Two genes, alpha and beta, encode different isoforms, of which only the beta and alpha1 isoforms are authentic nuclear triiodothyronine (T3)-receptors (NT3R). In agreement with the important role of T3 on myelination and oligodendrocyte generation, the presence of NT3Rs has been reported in oligodendrocytes and their precursors. We and others have shown that both progenitors and oligodendrocytes in vitro express the alpha1 and alpha2 isoforms, but the expression of the beta1 isoform is confined to differentiated oligodendrocytes, suggesting that they have different functions. To establish if this is the case during development in vivo, we have studied NT3R isoform expression in glial cells isolated by density gradient centrifugation from rat brains of various ages. We report the presence of the alpha1 NT3R and its variant alpha2, but not that of the beta1 isoform, in newborn rat glial progenitors. The pattern of expression of beta1, both at the level of mRNA and protein, parallels the increase in the number of oligodendrocytes. We found a significant change in the kinetic parameters of [125I]-T3 binding to NT3Rs in these cells during the first month of life, consisting of an increase in the binding capacity that peaks with myelination, and a significative decrease in Kd that coincides with the switch from the alpha to the beta1 isoform. Thus, the expression of NT3R isoforms in the rat oligodendrocyte lineage changes radically from the alpha to the beta1 isoform during the period when oligodendrocytes differentiate from progenitors.

Transcription of myelin basic protein promoted by regulatory elements in the proximal 5' sequence requires myelinogenesis.

Myelination in the central nervous system requires synthesis by oligodendrocytes of enormous amounts of lipids and proteins for incorporation in the developing myelin membranes. To approach the regulatory events coordinating the transcriptional activation of the genes that encode myelin proteins, we examined control of the myelin basic protein (MBP) locus. MBP plays a major role in myelin compaction. During development, MBP is already expressed in mature non-myelinating oligodendrocytes. Here we show that, in transgenic animals in which the E. coli lacZ reporter gene is under the control of increasingly large portions (256, 1900 and 3200 bp) of the MBP promoter, 5' of the initiation of transcription site, reporter gene expression was initiated after myelin formation had started. This delayed expression of the transgene compared to MBP, strongly suggests that premyelinating expression is dependent on regulatory elements located outside of the 3200 bp sequence studied, while expression occurring at the time of myelin formation is dependent on the proximal promoter sequence.

Myelin/oligodendrocyte glycoprotein (MOG) expression is associated with myelin deposition.

We investigated the onset of expression of the myelin/oligodendrocyte glycoprotein (MOG) mRNA and protein in the developing mouse central nervous system. In situ hybridization on brain sections at different stages of embryonic and postnatal development showed that MOG transcripts were first detected at birth in the medulla oblongata. During the first week after birth, cells expressing MOG mRNA were located in the ventral longitudinal funiculus. During the second postnatal week, the pattern of MOG mRNA expression extended rostrally to the mid-forebrain regions and reached completion by the beginning of the third week. MOG transcription was delayed by several days with respect to myelin basic protein (MBP), and it appeared that while the MBP probe labeled both non-myelinating and myelinating oligodendrocytes, only the latter were MOG-positive. In vitro, immunocytochemical analysis of MOG protein expression, performed on myelinating cultures derived from mouse brain embryos at 15 days of gestation, confirmed the strict restriction of MOG expression to myelinating oligodendrocytes. In particular, oligodendrocytes lining up their processes along axons, but not yet having started to deposit a myelin sheath, were still MOG negative. However, in the same cultures, pseudo-myelinating oligodendrocytes (i.e., cells not associated with neurites, but forming whorls of myelin-like figures) were MOG positive. Similarly, rat CG4 cells, an oligodendrocyte-like cell line, expressed MOG only after they had extended sheet-like processes, which suggested that the activation of MOG transcription depends more on an intrinsic oligodendroglial maturation program of myelination than on a neuronal signal.

Induction of myelination in the central nervous system by electrical activity.

The oligodendrocyte is the myelin-forming cell in the central nervous system. Despite the close interaction between axons and oligodendrocytes, there is little evidence that neurons influence myelinogenesis. On the contrary, newly differentiated oligodendrocytes, which mature in culture in the total absence of neurons, synthesize the myelin-specific constituents of oligodendrocytes differentiated in vivo and even form myelin-like figures. Neuronal electrical activity may be required, however, for the appropriate formation of the myelin sheath. To investigate the role of electrical activity on myelin formation, we have used highly specific neurotoxins, which can either block (tetrodotoxin) or increase (alpha-scorpion toxin) the firing of neurons. We show that myelination can be inhibited by blocking the action potential of neighboring axons or enhanced by increasing their electrical activity, clearly linking neuronal electrical activity to myelinogenesis.

HIV-1 envelope glycoprotein gp120 does not bind to galactosylceramide-expressing rat oligodendrocytes.

It may be postulated that the encephalopathy induced by the human immunodeficiency virus HIV-1, in particular, the characteristic "myelin pallor," may result from binding of the envelope glycoprotein gp120 to galactosylceramide and/or its metabolite sulfatide in the plasma membrane of oligodendrocytes, the myelin forming cells in the central nervous system. (1) gp120 has been reported to have a high affinity for these molecules in vitro. (2) The binding of antibodies to these molecules increases intracellular free calcium levels, which may be cytotoxic. (3) The binding of gp120 to the CD4 receptor in the immune system has the same effect. We have investigated the binding of gp120 to rat oligodendrocytes in vitro by indirect immunofluorescence and have monitored changes in intracellular free calcium with the calcium-sensitive dye INDO-1, in individual oligodendrocytes exposed to the glycoprotein. Antibodies against galatosylceramide and sulfatide bound to the cell membrane, but gp120 did not. The antibodies also increased intracellular free calcium levels in the oligodendrocytes, whereas gp120 did not. It, therefore, seems highly improbable that the demyelination observed during HIV encephalopathy is a direct cytotoxic effect of gp120 on oligodendrocytes.

[Interaction between neurons and oligodendrocytes during myelination]. / Interactions neurones-oligodendrocytes au cours de la myélinisation.

Oligodendrocytes are the myelin-forming cells of the central nervous system. Despite close relationship between oligodendrocyte and neuron, little is known about the exact role and nature of oligodendrocyte/axons interactions. We used an in vitro myelinating system to study axonal signals involved in myelination and showed: i) that only axons are myelinated, suggesting the existence of an axonal recognition signal and ii) that electrical activity seems necessary for myelination to proceed, as nerve influx blockade by tetrodotoxin strongly inhibits myelination. Moreover, myelinating oligodendrocyte appears to modulate axonal neurofilament phosphorylation. Knowledge of these reciprocal interactions may modify our physiopathological and therapeutical conceptions in human demyelinating diseases like multiple sclerosis.

Even in culture, oligodendrocytes myelinate solely axons.

Cerebral hemispheres from mouse embryos at 15 days of gestation were dissociated and maintained in culture for several weeks in a medium which permitted homochronic and homotypic oligodendrocytes and neurons to interact in the presence of other central nervous system cells. After 13-14 days in culture a few oligodendrocytes changed from highly branched, "sun-like," nonmyelinating cells to sparcely branched myelinating cells. The number of fibers myelinated per oligodendrocyte ranged from 1 to 10, similar to that described previously in vivo in the corpus callosum. When an oligodendrocyte began to myelinate, it immediately myelinated a maximum number of fibers, suggesting that the number of axons to be myelinated by the oligodendrocyte was predetermined. When only one fiber was in the vicinity of a myelinating oligodendrocyte, whorls of myelin-like figures were seen at the tip of oligodendrocyte processes that had not reached an axon. Myelinated fibers were unambiguously identified as axons both by immunostaining and by electron microscopy. Myelin was not observed around astrocyte processes or around dendrites. The exclusive myelination of axons suggests the existence of a specific axonal recognition signal which attracts oligodendrocyte processes.

Clonal segregation of oligodendrocytes and astrocytes during in vitro differentiation of glial progenitor cells.

To study the clonal lineage of the glial progenitor population, isolated from newborn rat brain (Lubetzki et al. J Neurochem 56:671, 1991), we combined somatic transgenesis using a retroviral vector encoding a modified bacterial beta-galactosidase with nuclear localization, and triple immunofluorescence labeling with A2B5, anti-galactosylceramide, and anti-glial acidic fibrillary protein antibodies. This allowed clonal analysis of the postnatal glial lineage with precise phenotypic identification of each cell within the lacZ-positive clones. When infected cells were cultivated under constant conditions, in the presence of either 1% or 10% fetal calf serum (FCS)-containing medium, all the 250 lacZ-positive clusters examined were homogeneous, i.e., either oligodendroglial or astroglial. Mixed astrocyte-oligodendroglial clones were observed when cells cultivated in the presence of 1% FCS were switched to a 10% FCS-containing medium, confirming the bipotentiality of glial progenitor cells (Temple and Raff Nature 313:223, 1985). However, even under the switch culture conditions, segregation into homogeneous clones of either oligodendrocytes or astrocytes still predominated, and the percentage of mixed clones dropped from 25 to 8 or to 3, when the switch took place at 8, 16, or 22 days in vitro, respectively. Two additional observations lead us to suggest that microenvironmental factors are responsible for the clonal segregation of glial progenitor cells: 1) the uneven distribution of oligodendrocyte and astrocyte clusters, the latter being seen mostly on the edge of the coverslips; and 2) the presence, in the vicinity of an homogeneous lacZ-positive clone, of some lacZ-negative cells expressing the same phenotype.