Myelin Basic Protein Transcription Research Articles

Hyaluronan hydrates and compartmentalises the CNS/PNS extracellular matrix and provides niche environments conducive to the optimisation of neuronal activity.

The central nervous system/peripheral nervous system (CNS/PNS) extracellular matrix is a dynamic and highly interactive space-filling, cell-supportive, matrix-stabilising, hydrating entity that creates and maintains tissue compartments to facilitate regional ionic micro-environments and micro-gradients that promote optimal neural cellular activity. The CNS/PNS does not contain large supportive collagenous and elastic fibrillar networks but is dominated by a high glycosaminoglycan content, predominantly hyaluronan (HA) and collagen is restricted to the brain microvasculature, blood-brain barrier, neuromuscular junction and meninges dura, arachnoid and pia mater. Chondroitin sulphate-rich proteoglycans (lecticans) interactive with HA have stabilising roles in perineuronal nets and contribute to neural plasticity, memory and cognitive processes. Hyaluronan also interacts with sialoproteoglycan associated with cones and rods (SPACRCAN) to stabilise the interphotoreceptor matrix and has protective properties that ensure photoreceptor viability and function is maintained. HA also regulates myelination/re-myelination in neural networks. HA fragmentation has been observed in white matter injury, multiple sclerosis, and traumatic brain injury. HA fragments (2 × 105 Da) regulate oligodendrocyte precursor cell maturation, myelination/remyelination, and interact with TLR4 to initiate signalling cascades that mediate myelin basic protein transcription. HA and its fragments have regulatory roles over myelination which ensure high axonal neurotransduction rates are maintained in neural networks. Glioma is a particularly invasive brain tumour with extremely high mortality rates. HA, CD44 and RHAMM (receptor for HA-mediated motility) HA receptors are highly expressed in this tumour. Conventional anti-glioma drug treatments have been largely ineffective and surgical removal is normally not an option. CD44 and RHAMM glioma HA receptors can potentially be used to target gliomas with PEP-1, a cell-penetrating HA-binding peptide. PEP-1 can be conjugated to a therapeutic drug; such drug conjugates have successfully treated dense non-operative tumours in other tissues, therefore similar applications warrant exploration as potential anti-glioma treatments.

Icariin Ameliorates Alzheimer's Disease Pathology by Alleviating Myelin Injury in 3 × Tg-AD Mice.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by excessive deposition of β amyloid (Aβ), hyperphosphorylation of tau protein, and neuronal cell death. Recent studies have shown that myelin cell damage, which leads to cognitive dysfunction, occurs before AD-related pathological changes. Here, we examine the effect of icariin (ICA), a prenylated flavonol glycoside, in improving cognitive function in AD model mice. ICA has been reported to exhibit cardiovascular protective functions and antiaging effects. In this study, we used 3 × Tg-AD mice as an AD model. The Morris water maze and Y maze tests were performed to assess the learning and memory of the mice. Immunofluorescence analysis of Aβ1-42 deposition and myelin basic protein (MBP) expression in the mouse hippocampus was performed. Tau protein phosphorylation and MBP protein expression in the hippocampus were further analyzed by Western blotting. Myelin damage in the mouse optic nerve was evaluated by electron microscopy, and LFB staining was performed to assess myelin morphology in the mouse corpus callosum. MBP, Mpp5, and Egr2 transcript levels were quantified by qPCR. We observed that ICA treatment improved the learning and memory of 3 × Tg-AD mice and reduced Aβ deposition and tau protein phosphorylation in the hippocampus. Moreover, this treatment protocol increased myelin-related gene expression and reduced myelin damage.

A novel myelin basic protein transcript variant in the murine central nervous system.

Myelin basic protein is a multifunctional protein whose primary role is to adhere membranes of the myelin sheath. There are various isoforms that have been identified, 6 distinct isoforms in human and 13 distinct isoforms in mice. These distinct isoforms are the product of alternative splicing of a single gene. The present study sought out to identify the different isoforms found in the murine central nervous system. Neuronal tissue (brain) from five different C57BL6/J mice at 2 months of age was harvested and used for mRNA extraction. mRNA was reversed transcribed to cDNA and transcripts were detected through PCR amplification and DNA agarose gel separation. Primers for exon 1, exon 5b and exon 11 of the myelin basic protein gene were used to capture all the possible transcripts that are naturally found in the murine central nervous system. Unknown transcript was sequenced at Genewiz facilities (South Plainfield, NJ) and mass spectrometry protein sequence analysis demonstrated the presence of a novel myelin basic protein transcript variant. We identified a novel transcript variant of myelin basic protein. This novel transcript variant corresponds to a myelin basic protein of 32.5 kDa which has not been previously reported. This novel transcript variant presents relevant clinical significance to various demyelinating diseases due to its contribution to the understanding of the natural state of the murine central nervous system.

Conditional knockout of TOG results in CNS hypomyelination.

The tumor overexpressed gene (TOG) protein is present in RNA granules that transport myelin basic protein (MBP) mRNA in oligodendrocyte processes to the myelin compartment. Its role was investigated by conditionally knocking it out (KO) in myelinating glia in vivo. TOG KO mice have severe motor deficits that are already apparent at the time of weaning. This phenotype correlates with a paucity of myelin in several CNS regions, the most severe being in the spinal cord. In the TOG KO optic nerve <30% of axons are myelinated. The number of oligodendrocytes in the corpus callosum, cerebellum, and cervical spinal cord is normal. In the absence of TOG, the most patent biochemical change is a large reduction in MBP content, yet normal amounts of MBP transcripts are found in the brain of affected animals. MBP transcripts are largely confined to the cell body of the oligodendrocytes in the TOG KO in contrast to the situation in wild type mice where they are found in the processes of the oligodendrocytes and in the myelin compartment. These findings indicate that MBP gene expression involves a post-transcriptional TOG-dependent step. TOG may be necessary for MBP mRNA assembly into translation permissive granules, and/or for transport to preferred sites of translation. GLIA 2017;65:489-501.

Different isoforms of deiminated myelin basic protein have different adsorption capacities to the myelin lipids

Myelin basic protein (MBP) is one of the most abundant proteins of the mammalian myelin sheath and plays a structural role in maintaining the stability of myelin. This lipid-rich membrane is essential for proper nerve conduction and the maintenance of neuronal health. MBP is localized on the cytoplasmic side of the membrane. There are 4 isoforms of MBP in the myelin sheath [1]. The mouse MBP transcription unit has been shown to contain 11 exons [2]. The gene contains three different transcription start sites that regulate production of the different MBP-related products [3]. The most downstream transcription start site 3 controls the classic MBP isoforms, with molecular sizes ranging from 14 to 21 kDa. In addition, each protein isoform can exist in the form of several different charge isomers. Alteration of MBP cationicity may represent a regulatory mechanism for normal myelin assembly or a degradative mechanism in demyelinating disorders [4]. MBP shows extensive posttranslational modifications, including deimination of arginine residues. The fractions isolated by cation exchange chromatography on a CM52 column [5] have been named C1, C2, C3, ... , C8. The first big peak to elute is the least cationic isomer, i.e., MBP C8, which is further divided in two subfractions C8a and C8b [6]. It has been shown that fraction C8b contains only MBP, but the main component of fraction C8a has been identified as stathmin [6], which is a microtubule-destabilizing protein; it prevents healing in the demyelinating brain [7]. Deiminated MBP is structurally less ordered and more susceptible to proteolytic attack than the native form. The reduction in cationicity of deiminated MBP impedes membrane assembly and exposes an immunodominant epitope in the membrane-bound protein to proteases [5]. This exposure may then cause the deiminated, highly immunogenic epitope to be released, priming the innate cells of the CNS [4]. In a previous study, we have found that citrullination (i.e., deimination of arginine residues) of myelin basic protein leads to abnormal formation of the myelin membrane [8]. The citrullinated peptide 45-89 released from modified deiminated MBP activates microglial cells and subsequent generation of nitric oxide induces damage of oligodendrocytes, which may be a first step in the initiation of demyelinating disorders [9].

Myelin Basic Protein as a Novel Genetic Risk Factor in Rheumatoid Arthritis—A Genome-Wide Study Combined with Immunological Analyses

Rheumatoid arthritis (RA) is a major cause of adult chronic inflammatory arthritis and a typical complex trait. Although several genetic determinants have been identified, they account for only a part of the genetic susceptibility. We conducted a genome-wide association study of RA in Japanese using 225,079 SNPs genotyped in 990 cases and 1,236 controls from two independent collections (658 cases and 934 controls in collection1; 332 cases and 302 controls in collection2), followed by replication studies in two additional collections (874 cases and 855 controls in collection3; 1,264 cases and 948 controls in collection4). SNPs showing p<0.005 in the first two collections and p<10−4 by meta-analysis were further genotyped in the latter two collections. A novel risk variant, rs2000811, in intron2 of the myelin basic protein (MBP) at chromosome 18q23 showed strong association with RA (p = 2.7×10−8, OR 1.23, 95% CI: 1.14–1.32). The transcription of MBP was significantly elevated with the risk allele compared to the alternative allele (p<0.001). We also established by immunohistochemistry that MBP was expressed in the synovial lining layer of RA patients, the main target of inflammation in the disease. Circulating autoantibody against MBP derived from human brain was quantified by ELISA between patients with RA, other connective tissue diseases and healthy controls. As a result, the titer of anti-MBP antibody was markedly higher in plasma of RA patients compared to healthy controls (p<0.001) and patients with other connective tissue disorders (p<0.001). ELISA experiment using citrullinated recombinant MBP revealed that a large fraction of anti-MBP antibody in RA patients recognized citrullinated MBP. This is the first report of a genetic study in RA implicating MBP as a potential autoantigen and its involvement in pathogenesis of the disease.

Phosphorylation Regulates OLIG2 Cofactor Choice and the Motor Neuron-Oligodendrocyte Fate Switch

SummaryA fundamental feature of central nervous system development is that neurons are generated before glia. In the embryonic spinal cord, for example, a group of neuroepithelial stem cells (NSCs) generates motor neurons (MNs), before switching abruptly to oligodendrocyte precursors (OLPs). We asked how transcription factor OLIG2 participates in this MN-OLP fate switch. We found that Serine 147 in the helix-loop-helix domain of OLIG2 was phosphorylated during MN production and dephosphorylated at the onset of OLP genesis. Mutating Serine 147 to Alanine (S147A) abolished MN production without preventing OLP production in transgenic mice, chicks, or cultured P19 cells. We conclude that S147 phosphorylation, possibly by protein kinase A, is required for MN but not OLP genesis and propose that dephosphorylation triggers the MN-OLP switch. Wild-type OLIG2 forms stable homodimers, whereas mutant (unphosphorylated) OLIG2S147A prefers to form heterodimers with Neurogenin 2 or other bHLH partners, suggesting a molecular basis for the switch.

Sp1 phosphorylation is involved in myelin basic protein gene transcription

Myelin basic protein (MBP), which helps form compact myelin sheets, is a major protein expressed during oligodendrocyte (OL) differentiation. Myelin basic protein expression is regulated mainly at the transcriptional level. Previous studies showed that the transcription factor Sp1 can activate the MBP promoter. Data from the laboratory also indicate that Sp1 is expressed highly in both growing and differentiated cells. Because this is true, we wanted to understand how Sp1 activity is regulated such that it increases MBP gene transcription only in differentiating cells. Phosphorylation is one major way to regulate transcription factor activity. Our results show that there is more Sp1 binding to the MBP promoter in differentiating OLs. Sp1 is also more phosphorylated in differentiating OLs than in precursor cells. Using inhibitors of different pathways, we found that the protein kinase C (PKC) modulator phorbol 12-myristate 13-acetate (PMA) can increase Sp1 phosphorylation when the cells are treated for 1 hr and can decrease Sp1 phosphorylation with long treatment (12 hr). The increased phosphorylation of Sp1 induced by PMA in short treatments could be abolished by the extracellular signal-regulated kinases (ERK) pathway inhibitor PD98059. This indicates that PKC phosphorylates Sp1 through the ERK pathway. Mutation of Sp1 threonines 453 and 739, which are phosphorylated by ERK, decreased MBP transcriptional activity. Furthermore, we found that PKC regulates Sp1 phosphorylation only in differentiating OLs. In conclusion, our results indicate that, in OLs, Sp1 phosphorylation can be regulated by PKC-ERK pathways. This phosphorylation is important for MBP transcription and oligodendrocyte differentiation.

LINGO-1-mediated inhibition of oligodendrocyte differentiation does not require the leucine-rich repeats and is reversed by p75NTR antagonists

LINGO-1 is a potent negative regulator of oligodendrocyte differentiation and hence may play a pivotal restrictive role during remyelination in demyelinating diseases such as multiple sclerosis. However, little is known as to which stages of oligodendrocyte differentiation are inhibited by LINGO-1, which domains of the protein are involved and whether accessory proteins are required. Here, we show that LINGO-1 expression in the human oligodendroglial cell line MO3.13 inhibited process extension and this was reversed by an anti-LINGO-1 antibody or the antagonist LINGO-1-Fc. LINGO-1 expression was also found to inhibit myelin basic protein transcription in the rat oligodendroglial cell line CG4. Both of these inhibitory actions of LINGO-1 were abrogated by deletion of the entire ectodomain or cytoplasmic domains but, surprisingly, were unaffected by deletion of the leucine-rich repeats (LRRs). As in neurons, LINGO-1 physically associated with endogenous p75NTR in MO3.13 cells and, correspondingly, its inhibition of process extension was reversed by antagonists of p75NTR. Thus, LINGO-1 inhibits multiple aspects of oligodendrocyte differentiation independently of the LRRs via a process that requires p75NTR signalling.

A Novel Nuclear Signaling Pathway for Thromboxane A2 Receptors in Oligodendrocytes: Evidence for Signaling Compartmentalization during Differentiation

The present study investigated G protein expression, localization, and functional coupling to thromboxane A(2) receptors (TPRs) during oligodendrocyte (OLG) development. It was found that as OLGs mature, the expression levels of G(q) increase while those of G(13) decrease. In contrast, the expression levels of G(s), G(o), and G(i) do not change significantly. Localization studies revealed that G(q), G(13), and G(i) are present only in the extranuclear compartment, whereas G(s) and G(o) are found in both the extranuclear and the nuclear compartments. Purification of TPR-G protein complexes demonstrated that TPRs couple to both G(q) and G(13) in the extranuclear compartment but only to G(s) in the nuclear compartment. Furthermore, functional analysis revealed that stimulation of nuclear TPR in OLGs stimulates CREB phosphorylation and myelin basic protein transcription and increases survival. Collectively, these results demonstrate that (i) OLGs selectively modulate the expression of certain G proteins during development, (ii) G proteins are differentially localized in OLGs leading to subcellular compartmentalization, (iii) TPRs couple to G(q) and G(13) in the extranuclear compartment and to G(s) only in the nucleus, (iv) mature OLGs have a functional nuclear TPR-G(s) signaling pathway, and (v) nuclear TPR signaling can stimulate CREB phosphorylation and myelin gene transcription and increase cell survival. These findings represent a novel paradigm for selective modulation of G protein-coupled receptor-G protein signaling during cell development.

Calpain inhibitor prevented apoptosis and maintained transcription of proteolipid protein and myelin basic protein genes in rat spinal cord injury

Spinal cord injury (SCI) is associated with progressive neurodegeneration and dysfunction. Multiple cellular and molecular mechanisms are involved in this pathogenesis. In particular, the activation of proteases following trauma can cause apoptosis in the spinal cord. Calpain, a calcium-dependent cysteine protease, plays a major role in apoptosis following trauma. We identified apoptosis and decrease in transcription of the genes for proteolipid protein (PLP) and myelin basic protein (MBP) in five 1-cm long spinal cord segments (S1, distant rostral; S2, near rostral; S3, lesion; S4, near caudal; and S5, distant caudal) 24 h after induction of SCI (40 g·cm force) in rats by weight-drop method. Sham rats underwent laminectomy and did not receive injury. Internucleosomal DNA fragmentation occurred prominently in the lesion (S3), moderately in near segments (S2 and S4), and slightly in distant segments (S1 and S5) of injured rats, indicating the occurrence of apoptosis in the lesion and penumbra. Levels of transcription of PLP and MBP were reduced highly in the lesion and moderately in near segments, suggesting that apoptotic loss of cells impaired biosynthesis of two important structural components of myelin. Immediate administration of the calpain inhibitor E-64-d (1 mg/kg) to injured rats prevented apoptosis and restored transcription of these genes, indicating the therapeutic efficacy of calpain inhibitor for treatment of SCI.

Characterization of myelination in the developing zebrafish.

Myelination, the process by which glial cells ensheath and electrically insulate axons, has been investigated intensely. Nevertheless, knowledge of how myelination is regulated or how myelinating cells communicate with neurons is still incomplete. As a prelude to genetic analyses of these processes, we have identified zebrafish orthologues of genes encoding major myelin proteins and have characterized myelination in the larval zebrafish. Expression of genes corresponding to proteolipid protein (PLP/DM20), myelin protein zero (P0), and myelin basic protein (MBP) is detected at 2 days postfertilization (dpf), first in the ventral hindbrain, close to the midline. During the next 8 days, expression spreads rostrally to the midbrain and optic nerve, and caudally to the spinal cord. DM20 is expressed in the CNS only, while MBP transcripts are detected both in the CNS and in Schwann cells of the lateral line, cranial nerves, and spinal motor nerves. Unlike its closest homologue, trout IP1, zebrafish P0 transcripts were restricted to the CNS. Ultrastructurally, the expression of myelin genes correlated well with myelination, although myelination showed a temporal lag. Myelinated axons were first detected at 4 dpf in the ventral hindbrain, where they were loosely wrapped by processes of glia cells. By 7 dpf, bundles of heavily myelinated axons were observed in the same region. Axons in the lateral line and optic nerves were also surrounded by compact myelin. Conservation in gene expression patterns and the early appearance of myelinated axons, support using the zebrafish to dissect the process of myelination by a genetic approach.

Expression of Golli mRNA during development in primary immune lymphoid organs of the rat

The gene-of-the-oligodendrocyte lineage (Golli)–MBP transcription unit contains three Golli-specific exons together with eight exons of the “classical” myelin basic protein (MBP) gene, yielding alternatively spliced proteins which share amino acid sequence with MBP. Unlike MBP, a late antigen expressed only in the nervous system, Golli gene products are expressed pre- and post-natally at many sites. In this study, we determined the sequence of Golli in rat by RT-PCR and 5′ RACE and showed that Golli sequences are expressed in primary lymphoid organs as early as e16.5, which could explain the anergic rat T cell response we previously observed in Golli-induced meningitis.

Distinct molecular mechanisms lead to diminished myelin basic protein and 2',3'-cyclic nucleotide 3'-phosphodiesterase in qk(v) dysmyelination.

The genetic lesion of quakingviable (qk(v)) causes diminished expression of the QKI RNA-binding protein in myelin producing cells. Consequently, several structural myelin proteins are severely reduced. Among these affected proteins, the reduction of the myelin basic protein (MBP) results from post-transcriptional abnormalities of the MBP mRNA, presumably due to the lack of interactions with QKI. However, whether this is the common mechanism for reduced expression of other myelin proteins in qk(v) dysmyelination remains unclear. Here we report that distinct molecular mechanisms underlie the reduction of MBP and the 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) in qk(v) dysmyelination. MBP transcripts bind QKI strongly and are markedly reduced in the qk(v)/qk(v) oligodendrocytes in which QKI is almost completely lost. In contrast, CNP transcripts bind QKI weakly and are only slightly affected by the lack of QKI. None the less, CNP proteins are severely reduced in the qk(v)/qk(v) brain. Since CNP transcripts are predominantly associated with translating polyribosomes, diminished CNP expression in qk(v) dysmyelination is unlikely to be due to translational failures, but more likely results from accelerated protein degradation.

Distinct molecular mechanisms lead to diminished myelin basic protein and 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase in qkv dysmyelination

The genetic lesion of quakingviable (qkv) causes diminished expression of the QKI RNA‐binding protein in myelin producing cells. Consequently, several structural myelin proteins are severely reduced. Among these affected proteins, the reduction of the myelin basic protein (MBP) results from post‐transcriptional abnormalities of the MBP mRNA, presumably due to the lack of interactions with QKI. However, whether this is the common mechanism for reduced expression of other myelin proteins in qkv dysmyelination remains unclear. Here we report that distinct molecular mechanisms underlie the reduction of MBP and the 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase (CNP) in qkv dysmyelination. MBP transcripts bind QKI strongly and are markedly reduced in the qkv/qkv oligodendrocytes in which QKI is almost completely lost. In contrast, CNP transcripts bind QKI weakly and are only slightly affected by the lack of QKI. None the less, CNP proteins are severely reduced in the qkv/qkv brain. Since CNP transcripts are predominantly associated with translating polyribosomes, diminished CNP expression in qkv dysmyelination is unlikely to be due to translational failures, but more likely results from accelerated protein degradation.

Association of JC virus large T antigen with myelin basic protein transcription factor (MEF-1/Puralpha) in hypomyelinated brains of mice transgenically expressing T antigen.

Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease caused by cytolytic destruction of oligodendrocytes, the myelin-producing cells of the central nervous system, by the human neurotropic JC virus (JCV). The early protein of JCV, T antigen, which is produced at the early stage of infection, is important for orchestrating the events leading to viral lytic infection and cytolytic destruction of oligodendrocytes. Results from transgenic mouse studies, however, have revealed that, in the absence of lytic infection, this protein can induce brain hypomyelination and suppression of myelin gene expression. Since expression of the gene encoding myelin basic protein, the major component of myelin, can be regulated by a DNA-binding transcription factor, MEF-1/Puralpha, (Puralpha), we have examined the level of this protein in transgenic mouse brains. Results from immunoprecipitation and Western blots showed that while there was no drastic decrease in the level of MEF-1/Puralpha in transgenic mouse brains, JCV T antigen was found in a complex with MEF-1/Puralpha. Immunohistological studies revealed abnormal oligodendrocytes in white matter, where MEF-1/Puralpha and T antigen were detected. Furthermore, immunogold electron microscopic studies revealed that Puralpha and T antigen are colocalized in the nucleus of the oligodendrocytes and in hippocampal neurons. Interestingly, results from cell culture studies revealed that incubation of oligodendrocytes with JCV led to a drastic decrease in the level of MEF-1/Puralpha protein. These observations provide insight into the molecular pathogenesis of PML and support a model for a dual effect of JCV on inducing hypomyelination by (i) affecting myelin gene expression via interaction of JCV T antigen and the myelin gene transcription factor, MEF-1/Puralpha, and (ii) causing a decline in the level of the host regulatory proteins, including MEF-1/Puralpha, which are involved in myelin gene expression.

MEBA Derepresses the Proximal Myelin Basic Protein Promoter in Oligodendrocytes

The central nervous system expression of myelin basic protein (MBP) is restricted to oligodendrocytes and is developmentally regulated; these regulatory features are transcriptionally mediated. We have previously shown that the proximal 149 nucleotides of the MBP promoter were both necessary and sufficient to activate the transcription of MBP in cultured oligodendrocytes, but not in other cell types. Sequences within the distal portion of this promoter, which contains a nuclear factor 1 (NF1) binding site, repressed activation of the MBP promoter in Cos-7 cells, but not in oligodendrocytes. We now describe a sequence upstream of and partially overlapping the NF1 site that activates the MBP promoter in oligodendrocytes, but not in Cos-7 cells. A protein complex binds to this site, designated MEBA (myelinating glia-enriched DNA binding activity), and is enriched in nuclear extracts prepared from the brain, oligodendrocytes, and Schwann cells. The amount of MEBA parallels MBP expression and myelinogenesis in the developing brain and parallels new MBP expression as purified oligodendrocytes differentiate. Mutational analyses of binding and function distinguish MEBA, an activator, from NF1, a repressor of MBP transcription, and suggest that MEBA consists of at least two proteins. Because the binding sites of MEBA and NF1 overlap, we suggest that MEBA may either compete with or modify NF1 binding, thereby activating the MBP promoter in oligodendrocytes.

Two proteins bind to a novel motif in the promoter of the myelin basic protein gene from mouse.

The box 1 and 2 motif of the myelin basic protein (MBP) promoter is a potential regulatory sequence of the MBP transcription unit. A DNA fragment that contained the sequence of the box 1 and 2 motif from mouse was synthesized, and its protein binding properties were examined by gel-shift assays. The box 1 and 2 probe and nuclear extracts from mouse brain generated a pattern of six major DNA-protein complexes (a, b, c, d, e, and f). The box 1 and 2 probe and nuclear extracts from oligodendrocyte-like glioma cells 1C10 generated a pattern of DNA-protein complexes that exhibited only complexes a, b, e, and f. Complex b generated by extracts from 1C10 cells, however, was very intense compared to any of the other complexes. It was determined that dephosphorylation of the proteins in nuclear extracts from 1C10 cells with acid phosphatase significantly altered their DNA binding properties. Two proteins of minimum M, approximately 32 and approximately 38 kDa (MBP32 and MBP38) that bind to the box 1 and 2 motif were identified in these nuclear extracts by using a UV crosslinking method. MBP32 and MBP38 are found in cell types and tissues known to express the golli transcription unit of the golli-MBP gene complex and may be involved in the modulation of the MBP unit in those cells.

Exon 2 containing myelin basic protein (MBP) transcripts are expressed in lesions of experimental allergic encephalomyelitis (EAE)

Exon 2 containing myelin basic protein (MBP) transcripts are expressed during developmental myelination in mice and humans, and during remyelination subsequent to virally induced demyelination in adult mice. Since remyelination characterizes CNS lesions during experimental allergic encephalomyelitis (EAE) and multiple sclerosis (MS), we investigated whether exon 2 containing isoforms of MBP are expressed in EAE lesions during relapsing disease. Exon 2 containing MBP transcripts were detected by in situ hybridization in 17 of 52 EAE mice and in 16 of 30 mice at the peak of the first or second episode of paralysis. Thus exon 2 containing MBP transcripts are expressed in lesions of the CNS during active phases of chronic relapsing autoimmune disease. Implications of these findings with respect to future therapies aimed toward enhancing remyelination in EAE and, possibly MS, are discussed.

Origin of Oligodendrocytes within the Human Spinal Cord

To determine the time and site of origin of the oligodendrocyte lineage in the developing human spinal cord, we have examined tissues from 45 to 83 d postconception (dpc) using sets of probes and antibodies recognizing oligodendrocyte-specific glycolipids, transcripts, and proteins. We found that two clusters of oligodendrocyte precursors appear on or before 45 dpc on each side of the cord ventral ependyma above the floor plate. These precursors express glycolipids recognized by the O4 and Rmab antibodies, platelet-derived growth factor alpha-receptor, myelin basic protein (MBP), and 2', 3'-cyclic nucleotide 3' phosphodiesterase as well as MBP and proteolipid transcripts. Expression of the morphogen sonic hedgehog was detected in the floor plate at 45 dpc and decreased at 58 dpc. During this period, oligodendrocyte precursors emerged in the ventral and lateral region of the forming white matter, a process occurring first in cervical and later in lumbar cord. The majority of O4(+) cells express the proliferating cell nuclear antigen (PCNA), and their pattern of dispersion suggests that these cells progressively populate the lateral and dorsal cord regions. Oligodendrocytes expressing galactocerebroside appeared at 53 dpc and did not express PCNA. Oligodendrocyte precursors were detected in dorsal cord regions at 74 dpc and at 83 dpc when myelination started in the ventral roots. Thus, oligodendrocyte precursors expressing myelin transcripts and proteins emerge in the ventral region of the embryonic cord several weeks before myelination.