Stem cell therapy in multiple sclerosis: promise and controversy.

Stem cells offer the potential for regeneration of lost tissue in neurological disease, including multiple sclerosis (MS). Their development in vitro and their use in vivo in animal models of degenerative neurological disease and recent first efforts in human clinical trials were the topics of a recent international meeting sponsored by the Multiple Sclerosis International Federation and the National Multiple Sclerosis Society on "Stem Cells & MS: Prospects and Strategies" Participants reviewed the current state of knowledge about the potential use of stem and progenitor cells in MS and other degenerative neurological disorders and outlined a series of urgent fundamental and applied clinical research priorities that should allow the potential of regeneration of damaged tissue in MS to be assessed and pursued.

Normal CNS myelination in transgenic mice overexpressing MHC class I H-2L(d) in oligodendrocytes.

In demyelinating diseases, such as multiple sclerosis, an upregulation of MHC class I expression is thought to contribute to oligodendrocyte/myelin damage. In order to investigate potential physiological consequences of upregulated MHC class I expression in oligodendrocytes, we generated transgenic mice that overexpress H-2L(d) under the control of the proteolipid protein (PLP) promoter (PLP-L(d) mice). We focused our studies on the MHC class I molecule H-2L(d), because of its unique intracellular transport characteristics. In the CNS of PLP-L(d) mice, H-2L(d) was expressed by oligodendrocytes. Furthermore, H-2L(d) protein was transported to and expressed on the surface of oligodendrocytes. Most importantly, this upregulation of MHC class I expression in the CNS of PLP-L(d) mice did not by itself result in a de- or dysmyelinating phenotype. These transgenic mice are likely to provide a unique and novel tool for the analysis of potential roles of MHC class I-mediated mechanisms in demyelinating pathologies.

Akt-mediated survival of oligodendrocytes induced by neuregulins.

Neuregulins have been implicated in a number of events in cells in the oligodendrocyte lineage, including enhanced survival, mitosis, migration, and differentiation. At least two signaling pathways have been shown to be involved in neuregulin signaling: the phosphatidylinositol (PI)-3 kinase and the mitogen-activated protein kinase pathways. In the present studies, we examined the signaling pathway involved in the survival function of heregulin, focusing on heregulin-induced changes in Akt activity in cultured glial cells, and the consequences of Akt activation in cells in the oligodendrocyte lineage. Heregulin binds erbB receptors, and in our studies, primary cultures of both oligodendrocyte progenitor cells and differentiating oligodendrocytes expressed erbB2, erbB3, and erbB4 receptors. In C6 glioma cells and primary cultures of oligodendrocytes, heregulin induced time- and dose-dependent Akt phosphorylation at Ser(473) in a wortmannin-sensitive manner. To investigate further the signaling pathway for heregulin in glial cells, BAD was overexpressed in C6 glioma cells. In these cells, heregulin induced phosphorylation of BAD at Ser(136). Apoptosis of oligodendrocyte progenitor cells induced by growth factor deprivation was effectively blocked by heregulin in a wortmannin-sensitive manner. Overexpression of dominant negative Akt but not of wild-type Akt by adenoviral gene transfer in primary cultures of both oligodendrocytes and their progenitors induced significant apoptosis through activation of the caspase cascade. The present data suggest that the survival function of heregulin is mediated through the PI-3 kinase/Akt pathway in cells in the oligodendrocyte lineage and that the Akt pathway may be quite important for survival of cells in this lineage.

Proteolipid protein mRNA stability is regulated by axonal contact in the rodent peripheral nervous system.

Proteolipid protein (PLP) and its alternatively spliced isoform, DM20, are the main intrinsic membrane proteins of compact myelin in the CNS. PLP and DM20 are also expressed by Schwann cells, the myelin-forming cells in the PNS, and are necessary for normal PNS function in humans. We have investigated the expression of PLP in the PNS by examining transgenic mice expressing a LacZ transgene under the control of the PLP promoter. In these animals, myelinating Schwann cells expressed beta-galactosidase more prominently than nonmyelinating Schwann cells. PLP/DM20 mRNA levels, but not those of LacZ mRNA, increased during sciatic nerve development and decreased after axotomy, with resultant Wallerian degeneration. PLP/DM20 transcription rates, in nuclear run off experiments, however, did not increase in developing rat sciatic nerve despite robust increases in PLP/DM20 mRNA levels during the same period. In RNAse protection studies, PLP mRNA levels fell to undetectable levels following nerve transection whereas levels of DM20 were essentially unchanged despite both being transcribed from the same promoter. Finally, cotransfection studies demonstrated that PLP-GFP, but not DM20-GFP mRNA is down-regulated in Schwann cells cultured in the absence of forskolin. Taken together these data demonstrate that steady state levels of PLP mRNA are regulated at a posttranscriptional level in Schwann cells, and that this regulation is mediated by Schwann cell-axonal contact. Since the difference between these two mRNAs is a 105-bp sequence in PLP and not in DM20, this sequence is likely to play a role in the regulation of PLP mRNA.

Purification and analysis of in vivo-differentiated oligodendrocytes expressing the green fluorescent protein.

A complete understanding of the molecular mechanisms involved in the formation and repair of the central nervous system myelin sheath requires an unambiguous identification and isolation of in vivo-differentiated myelin-forming cells. In order to develop a novel tool for the analysis of in vivo-differentiated oligodendrocytes, we generated transgenic mice expressing a red-shifted variant of the green fluorescent protein under the control of the proteolipid protein promoter. We demonstrate here that green fluorescent protein-derived fluorescence in the central nervous system of 9-day- to 7-week-old mice is restricted to mature oligodendrocytes, as determined by its spatiotemporal appearance and by both immunocytochemical and electrophysiological criteria. Green fluorescent protein-positive oligodendrocytes could easily be visualized in live and fixed tissue. Furthermore, we show that this convenient and reliable identification now allows detailed physiological analyses of differentiated oligodendrocytes in situ. In addition, we developed a novel tissue culture system for in vivo-differentiated oligodendrocytes. Initial data using this system indicate that, for oligodendrocytes isolated after differentiation in vivo, as yet unidentified factors secreted by astrocytes are necessary for survival and/or reappearance of a mature phenotype in culture.

Peripheral neuropathy caused by proteolipid protein gene mutations.

Pelizaeus-Merzbacher disease (PMD) is a dysmyelinating disorder of the central nervous system typically caused by duplications or missense mutations of the proteolipid protein (PLP) gene. Most investigators have found that peripheral nerve function and structure is normal in PMD patients. We have found that null mutations of the PLP gene cause demyelinating peripheral neuropathy, whereas duplications and a proline 14 to leucine mutation do not affect nerve function. A family with a nonsense mutation at position 144, which affects only PLP but not the alternatively spliced gene product DM20, has a very mild syndrome, including normal peripheral nerve function. Our findings suggest that DM20 alone is sufficient to maintain normal nerve function and that there may be domains of PLP/DM20 that have a relatively more active role in the peripheral nervous system compared with that in the central nervous system.

Disruption of the murine nuclear factor I-A gene (Nfia) results in perinatal lethality, hydrocephalus, and agenesis of the corpus callosum.

The phylogenetically conserved nuclear factor I (NFI) family of transcription/replication proteins is essential both for adenoviral DNA replication and for the transcription of many cellular genes. We showed previously that the four murine NFI genes (Nfia, Nfib, Nfic, and Nfix) are expressed in unique but overlapping patterns during mouse development and in adult tissues. Here we show that disruption of the Nfia gene causes perinatal lethality, with >95% of homozygous Nfia(-/-) animals dying within 2 weeks after birth. Newborn Nfia(-/-) animals lack a corpus callosum and show ventricular dilation indicating early hydrocephalus. Rare surviving homozygous Nfia(-/-) mice lack a corpus callosum, show severe communicating hydrocephalus, a full-axial tremor indicative of neurological defects, male-sterility, low female fertility, but near normal life spans. These findings indicate that while the Nfia gene appears nonessential for cell viability and DNA replication in embryonic stem cells and fibroblasts, loss of Nfia function causes severe developmental defects. This finding of an NFI gene required for a developmental process suggests that the four NFI genes may have distinct roles in vertebrate development.

Digitized image analysis reveals diffuse abnormalities in normal-appearing white matter during acute experimental autoimmune encephalomyelitis.

Demyelination of the central nervous system is a hallmark of multiple sclerosis and its widely used animal model, experimental autoimmune encephalomyelitis (EAE). Recent studies using magnetic resonance imaging and spectroscopy on multiple sclerosis patients have revealed abnormalities of central nervous system normal-appearing white matter suggesting that micro-demyelination and/or extensive membrane turnover accompanies and perhaps precedes the appearance of manifest inflammatory lesions. In the present study, we induced EAE in SWXJ mice and analyzed digitized images of immunocytochemically stained spinal cord for detection of myelin proteolipid protein (PLP). We found that digitized image analysis is a highly sensitive, objective methodology for measuring the extent of myelin loss during EAE. Our data show that two-thirds of the measured reduction of myelin PLP occurring in EAE spinal cord could be attributed to a loss of myelin in normal-appearing white matter. The marked decrease in detection of PLP was accompanied by a corresponding decrease in PLP mRNA in the central nervous system. Our results indicate that during acute EAE, diffuse myelin abnormalities extend far beyond visibly detectable inflammatory foci and are characterized by a global decrease in the expression of myelin genes and their encoded proteins.

Multiple restricted origin of oligodendrocytes.

The plp gene encodes the proteolipid protein and its alternatively spliced product DM-20, major proteins of CNS myelin. In the mouse, plp/dm-20 transcripts are expressed beginning at embryonic day 9.5 (E9.5) by restricted foci of germinative neuroepithelial cells. To determine the identity of the neural precursors expressing plp/dm- 20, a zeomycin resistance gene fused to the lacZ reporter was expressed in transgenic mice under the control of the plp regulatory sequences. In the three different lines generated, the pattern of beta-galactosidase expression was similar and superimposable on the expression pattern of endogenous plp/dm-20. Both in vivo and in vitro, the transgene was expressed by O4(+) pre-oligodendrocytes, and later by RIP+ differentiated oligodendrocytes, but not by neuronal cells, astrocytes, or radial glial cells. After zeomycin selection, a dramatic enrichment in O4(+) pre-oligodendrocytes was observed in cultures derived from E12.5 transgenic embryos. This enrichment indicates the oligodendroglial specification of neural precursors that continuously express plp/dm-20. Early plp/dm-20-expressing precursors, however, appear to be a separate population from previously described PDGFRalpha oligodendrocyte precursors, as shown by the striking differences in their (1) patterns of distribution and (2) responsiveness to PDGF. These data suggest that oligodendrocytes have a plural origin and that early plp/dm-20 defines one of the neural lineages generating oligodendrocytes.

Phosphodiesterase I, a novel adhesion molecule and/or cytokine involved in oligodendrocyte function.

One of the more complex developmental processes occurring postnatally in the CNS is the formation of the myelin sheath by oligodendrocytes. To examine the molecular events that take place during myelination, we isolated oligodendrocyte-derived cDNA clones, one of which (p421.HB) represents a putative alternatively spliced isoform of rat brain-specific phosphodiesterase I (PD-Ialpha) and a species homolog of the human cytokine autotaxin. Analysis of the structural composition of the p421.HB/PD-Ialpha protein suggests a transmembrane-bound ectoenzyme, which, in addition to the phosphodiesterase-active site contains presumed cell recognition and Ca2+-binding domains. Consequently, it may be involved in extracellular signaling events. Expression of p421.HB/PD-Ialpha is enriched in brain and spinal cord, where its mRNA can be detected in oligodendrocytes and in cells of the choroid plexus. Expression in the brain increases during development with an intermediate peak of expression around the time of active myelination and maximal expression in the adult. We have identified four presumably alternatively spliced isoforms, two of which appear to be CNS-specific. Decreased levels of p421.HB/PD-Ialpha mRNA in the dysmyelinating mouse mutant jimpy, but not shiverer, suggest a role for p421.HB/PD-Ialpha during active myelination and/or late stages of oligodendrocyte differentiation. Furthermore, p421.HB/PD-Ialpha mRNA levels were reduced in the CNS at onset of clinical symptoms in experimental autoimmune encephalomyelitis. These data together implicate the importance of p421.HB/PD-Ialpha in oligodendrocyte function, possibly through cell-cell and/or cell-extracellular matrix recognition.

Differentiation and death of premyelinating oligodendrocytes in developing rodent brain.

Previous studies have indicated that newly formed oligodendrocytes are dynamic cells whose production, survival, and differentiation depend upon axonal influences. This study has characterized the appearance and fate of newly formed oligodendrocytes in developing rat brain. Oligodendrocytes appear in predictable locations and radially extend DM-20-positive processes that cover 80-microm domains in the cortex and 40-microm domains in the corpus callosum. These premyelinating oligodendrocytes have one of two fates: they myelinate axons or degenerate. Between 7 and 21 d after birth, approximately 20% of premyelinating oligodendrocytes identified in the cerebral cortex were degenerating. Oligodendrocytes that ensheathed axons expressed and selectively targeted proteolipid protein to compact myelin and did not degenerate. These observations support the hypothesis that axonal influences affect oligodendrocyte survival, differentiation, and expression of proteolipid protein gene products.

Regulation of murine myelin proteolipid protein gene expression.

To identify putative sequences that direct cell type-specific expression and/or enhance proteolipid protein (PLP) gene expression, glial or nonglial cells were transfected with various PLP-luciferase constructs that collectively span the entire mouse PLP-specific DNA present in a transgene known to direct cell type specificity in transgenic mice. These constructs were transfected into murine oligodendrocyte cell lines that transcribe the PLP gene and, hence, should contain the requisite trans-acting factors necessary for PLP gene expression. Mouse NIH/3T3 fibroblasts were used as a nonglial model. We have finely mapped the PLP promoter region for transcriptional regulatory elements and demonstrate both positive and negative elements, none of which appear to extinguish expression in nonglial cells. The 5'-flanking PLP DNA tested did not enhance the basal herpes simplex-1 virus thymidine kinase (TK) promoter, nor did PLP sequences present in the distal half of intron 1. The 5' portion of intron 1 did enhance TK promoter activity, suggesting that this region of the gene may contain enhancer elements that modulate PLP gene expression; however, the enhancement did not appear to be cell type-specific. Intriguingly, a 541 bp region of the intron that significantly enhanced TK promoter activity contains multiple JC virus repeated elements and other elements known to be important in restricting the virus to oligodendrocytes. These results suggest that intron 1 sequences may modulate expression of the PLP gene.

Expression of molecular chaperones and vesicle transport proteins in differentiating oligodendrocytes.

The major stages of oligodendrocyte differentiation are defined by the presence or absence of certain myelin-specific proteins. Events leading to the successful processing of these proteins, such as the folding, assembly, and trafficking of these proteins through the biosynthetic pathway, are largely undefined. In the present study, we have examined both cultured primary oligodendrocytes and immortalized oligodendrocyte cell lines for the presence of molecular chaperones and/or vesicle transport proteins. We find that a select set of these proteins are expressed relatively early in oligodendrocyte differentiation, whereas a characteristically different set of proteins are expressed at later stages of oligodendrocyte differentiation. In other systems, these proteins participate in the folding and assembly of protein complexes, in the prevention of protein aggregation, as well as the trafficking of proteins via vesicles to specific subcellular destinations including the plasma membrane. Some of the chaperones and/or vesicle transport proteins investigated in this study may play a pivotal role in the certain aspect of myelin biogenesis.

GapIII, a new brain-enriched member of the GTPase-activating protein family.

Ras GTPase-activating proteins (GAPs) are negative regulators of ras, which controls proliferation and differentiation in many cells. Ras GAPs have been found in a variety of species from yeast to mammals. We describe here a newly identified mammalian GAP, GapIII, which was obtained by differential screening of a rat oligodendrocyte cDNA library. GapIII putatively encodes a 834 amino acid protein with a predicted molecular weight of 96 kDa, which contains a consensus GAP-related domain (GRD). The protein encoded by this cDNA has high homology with Gap1m, which was recently identified as a putative mammalian homolog of Drosophila Gap1. These proteins contain three structural domains, an N-terminal calcium-dependent phospholipid binding domain, GRD, and a C-terminal PH/Btk domain. Because of the sequence homology and the structural similarities of this protein with Gap1m, we hypothesize that GapIII and Gap1m may be members of a mammalian GAP gene family, separate from p120GAP, neurofibromin (NF1), and IQGAP. To confirm the GapIII protein activity, constructs containing different GapIII-GRD domains were transformed into iral mutant yeast to determine their relative ability to replace IRA1 functionally. Constructs that contained essentially the full-length protein (all three domains), the GRD alone, or the GRD plus PH/Btk domain suppressed heat shock sensitivity of ira1, whereas constructs that contained the GRD with part of the PH/Btk domain had only a weak ability to suppress heat shock sensitivity. These results suggest that the GapIII GRD itself is sufficient to down-regulate ras proteins in yeast. Expression of GapIII mRNA (4.2 kb) was examined by Northern analysis and in situ hybridization. This mRNA was expressed at highest levels in the brain, where its expression increased with development. Lower levels of the mRNA were expressed in the spleen and lung. Among neural cells, GapIII mRNA was expressed in neurons and oligodendrocytes, but not in astrocytes. Interestingly, the expression pattern in brain is reminiscent of type 1 NF1 expression reported by Gutmann et al. (Cell Growth Differ in press, 1995). We propose that in addition to p120GAP and neurofibromin, the GapIII/Gap1m family may be important for modulating ras activity in neurons and oligodendrocytes during normal brain development and in particular in the adult brain.

Expression of cell surface markers and myelin proteins in cultured oligodendrocytes from neonatal brain of rat and mouse: a comparative study.

Dissociated brain cell cultures are a useful model for investigating development and differentiation of oligodendrocytes in vitro. The current studies compare the developmental patterns of expression for oligodendrocyte lineage/myelin markers in both primary and secondary oligodendrocyte cultures derived from mouse and rat neonates. The rat and mouse dissociated brain cell cultures express the same myelin-specific antigens, but mouse oligodendrocytes produce a larger and more elaborate sheet-like membrane than rat oligodendrocytes, and some of the myelin markers (O4, GC, and MBP) show more intense membrane staining in mouse cultures. GD3 appears to be a good oligodendrocyte marker for rat cells, but it is nonspecific in mouse cells. There are fewer oligodendrocytes in mouse cultures, and they appear to require a longer differentiation time than rat oligodendrocytes. These same results are also observed in secondary oligodendrocyte cultures, although in general late myelin markers such as MBP and PLP are expressed at a much lower level in mouse cells than rat cells.

Developmental expression of protein kinase C isozymes in oligodendrocytes and their differential modulation by 4 beta-phorbol-12,13-dibutyrate.

Myelin gene expression in normal oligodendrocytes (OLG) depends on developmentally regulated protein kinase C (PKC) enzyme activity (Asotra and Macklin: J Neurosci Res 34:571-588, 1993). We studied the developmental expression of the Ca(++)-dependent PKC-alpha, -beta 1, -beta II and -gamma isozymes, and the Ca(++)-independent PKC-delta, -epsilon, -zeta and -eta isozymes in enriched rat brain OLG cultures. In A2B5+ O-2A progenitors, only PKC-delta, PKC-epsilon and PKC-zeta were detected immunocytochemically. In 04+ proligondendrocytes, PKC-beta I, -delta and -zeta were expressed moderately and low levels of PKC-alpha and -epsilon were detected. GD3+ OLG, GC+ OLG and MBP+ OLG showed increased levels of PKC-alpha, -beta I, -delta and -zeta isozymes. PKC-beta II, -gamma and -eta were poorly expressed in OLG. On immunoblots, PKC-alpha was present early and increased continually up to 18 days but PKC-beta I increased until 12 days in cultured OLG. High levels of PKC-delta, PKC-epsilon and PKC-zeta, the most abundant PKC isozymes in OLG, were maintained up to 12 days and were then slightly reduced. Interestingly, relatively high levels of PKC-alpha, PKC-beta I, PKC-beta II, PKC-gamma and PKC-epsilon isozymes were detected in purified myelin membrane although greater levels of PKC-delta were found in OLG than in purified myelin. Thus, most of the PKC isozymes found in cultured OLG were also present in myelin, although at different levels. Treatment with 50 nM 4 beta-phorbol-12,13-dibutyrate (PDB) caused a delayed downregulation of PKC-delta levels after 8 hr without modulating the expression of other PKC isozymes in 1-day OLG; in the 3-day-old and 6-day-old OLG, PDB downmodulated PKC-beta I, -delta and epsilon isozymes with only a minor effect on PKC-alpha and no reduction in PKC-zeta. Induction or downmodulation of individual PKC isozymes by phorbol esters appears to depend on the differentiation state of OLG. These data suggest that PKC-beta I, -delta and -epsilon isozymes have an important function in different cellular events of OLG differentiation. We conclude that the PKC-dependent modulation of myelin gene expression in OLG results predominantly from the Ca(++)-dependent PKC-beta I isozyme activity and the CA(++)-independent PKC-delta and PKC-epsilon activitives in a cell differentiation state-dependent manner.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification of novel mRNAs expressed in oligodendrocytes.

To identify new proteins, which are expressed in oligodendrocytes and which may have a functional role in myelination, a rat oligodendrocyte cDNA library was screened using differential and subtractive screening techniques. Ten clones that have elevated levels of expression in brain were isolated. Two of these clones were characterized further and one clone, pC26.H2, was found to be closely related to mouse stearoyl-CoA desaturase 2 (SCD2), which catalyzes the synthesis of unsaturated fatty acid. From Northern blot and in situ hybridization studies, SCD2 mRNA was expressed primarily in brain with lower levels found in lung and spleen. In brain sections, SCD2 mRNA was found primarily in oligodendrocytes, although mRNA was detected at a low level in neurons, in particular in Purkinje cells in the cerebellum. Northern blot analysis of the other clone, p973.HB, indicated that it was expressed more selectively in brain. In mixed glial cultures oligodendrocytes were the only cells that expressed this mRNA, whereas in brain, neurons expressed this mRNA at a higher level than in oligodendrocytes. This clone is being actively pursued because of its unique expression exclusively in oligodendrocytes and neurons.

A myelin proteolipid protein-LacZ fusion protein is developmentally regulated and targeted to the myelin membrane in transgenic mice.

Transgenic mice were generated with a fusion gene carrying a portion of the murine myelin proteolipid protein (PLP) gene, including the first intron, fused to the E. coli LacZ gene. Three transgenic lines were derived and all lines expressed the transgene in central nervous system white matter as measured by a histochemical assay for the detection of beta-galactosidase activity. PLP-LacZ transgene expression was regulated in both a spatial and temporal manner, consistent with endogenous PLP expression. Moreover, the transgene was expressed specifically in oligodendrocytes from primary mixed glial cultures prepared from transgenic mouse brains and appeared to be developmentally regulated in vitro as well. Transgene expression occurred in embryos, presumably in pre- or nonmyelinating cells, rather extensively throughout the peripheral nervous system and within very discrete regions of the central nervous system. Surprisingly, beta-galactosidase activity was localized predominantly in the myelin in these transgenic animals, suggesting that the NH2-terminal 13 amino acids of PLP, which were present in the PLP-LacZ gene product, were sufficient to target the protein to the myelin membrane. Thus, the first half of the PLP gene contains sequences sufficient to direct both spatial and temporal gene regulation and to encode amino acids important in targeting the protein to the myelin membrane.

Enhanced degradation of messenger RNA encoding myelin proteins by terminal complement complexes in oligodendrocytes.

Sublytic terminal C complexes (TCC) are capable of stimulating cells and affect the target cell activity. Activation of TCC that generates leukotriene B4 in oligodendrocytes, the myelin-forming cells of the central nervous system, is also a required process in antibody-mediated demyelination of rodent cerebellar explants. In the present study, the effect of TCC on myelin protein gene expression was studied in primary rat oligodendrocytes in culture. Sublytic activation of serum C reduced accumulation of mRNA encoding proteolipid protein (PLP) and myelin basic protein (MBP) within 1 h, but not beta-actin mRNA. C activation, on the other hand, induced sustained expression of c-jun mRNA. Experiments using C7-deficient human serum to determine the role of TCC showed that selective MBP and PLP mRNA down-regulation was achieved only when C7 was reconstituted to form TCC. The C7 requirement was also observed in the presence of alpha-amanitin. Post-transcriptional regulation was explored by determining mRNA decay, which demonstrated that the MBP and PLP mRNA were selectively destabilized when C7 was reconstituted. Limited exploration of the signals responsible for the TCC effect revealed that down-regulation of mRNA by TCC was significantly influenced by Ca2+ on PLP, whereas MBP did not show the same Ca2+ sensitivity as PLP. The TCC-mediated MBP mRNA decay was completely abrogated by HA1004, an inhibitor for the cAMP- and cGMP-dependent protein kinases, but not by H7, a protein kinase C inhibitor.

Protein kinase C activity modulates myelin gene expression in enriched oligodendrocytes.

Protein kinase C (PKC) and its potential role in myelin gene expression were investigated in primary cultured rat oligodendrocytes. The major myelin genes were expressed in a developmentally coordinated manner in cultured oligodendrocytes. PKC activity in these cells was similarly regulated with differential expression of PKC isozyme mRNAs. PKC-gamma mRNA was expressed transiently and was most abundant in 9-day cells in vitro. PKC-alpha and PKC-beta mRNAs were present at low levels throughout development in these cells, and their expression increased in 18-25 day cells. Immunocytochemical colocalization of PKC with oligodendrocyte-specific markers--O4, galactosyl cerebroside, MBP, and PLP--in enriched oligodendrocyte cultures suggested that the PKC enzyme activities assayed in these cultures were predominantly contributed by oligodendrocytes. PKC inhibition resulting from long-term exposure to 4 beta-phorbol-12,13-dibutyrate (4 beta-PDB) reduced steady-state levels of MBP, PLP, MAG, CNP, and PKC-alpha mRNAs, as detected by slot blots or in situ hybridization, and downregulated the oligodendrocyte-specific markers O4, galactosyl cerebroside, and the major constituent proteins MBP and PLP, as detected by immunocytochemistry. PKC-mediated downmodulation of myelin gene expression was most profound in normally differentiating oligodendrocytes at or before the onset of myelin protein synthesis. Six-day oligodendrocytes were most susceptible to such modulation. To elucidate the mechanism of reduction in various myelin gene messages upon modulation of PKC, we analyzed mRNA levels in oligodendrocytes, which were pretreated with either the transcriptional inhibitor actinomycin D or the protein synthesis blocker cycloheximide before exposure to 4 beta-PDB. Our results demonstrate that the PKC inhibition-mediated loss in myelin mRNA levels did not require the transcription of any genes, but appeared to be at least partially dependent on continuous protein synthesis.