Golli Myelin Basic Proteins Modulate Voltage-Operated Ca(++) Influx and Development in Cortical and Hippocampal Neurons.

The golli proteins, products of the myelin basic protein gene, are widely expressed in oligodendrocyte progenitor cells and neurons during the postnatal development of the brain. While golli appears to be important for oligodendrocyte migration and differentiation, its function in neuronal development is completely unknown. We have found that golli proteins function as new and novel modulators of voltage-operated Ca(++) channels (VOCCs) in neurons. In vitro, golli knock-out (KO) neurons exhibit decreased Ca(++) influx after plasma membrane depolarization and a substantial maturational delay. Increased expression of golli proteins enhances L-type Ca(++) entry and processes outgrowth in cortical neurons, and pharmacological activation of L-type Ca(++) channels stimulates maturation and prevents cell death in golli-KO neurons. In situ, Ca(++) influx mediated by L-type VOCCs was significantly decreased in cortical and hippocampal neurons of the golli-KO brain. These Ca(++) alterations affect cortical and hippocampal development and the proliferation and survival of neural progenitor cells during the postnatal development of the golli-KO brain. The CA1/3 sections and the dentate gyrus of the hippocampus were reduced in the golli-KO mice as well as the density of dendrites in the somatosensory cortex. Furthermore, the golli-KO mice display abnormal behavior including deficits in episodic memory and reduced anxiety. Because of the expression of the golli proteins within neurons in learning and memory centers of the brain, this work has profound implication in neurodegenerative diseases and neurological disorders.

Voltage-operated Ca(2+) and Na(+) channels in the oligodendrocyte lineage.

It is becoming increasingly clear that expression of Ca(2+) and Na(+) channels in the OL lineage is highly regulated and may be functionally related to different stages of development and myelination. Characterization of the mechanisms of voltage-dependent Ca(2+) and Na(+) entry are important because changes in intracellular Ca(2+) and Na(+) are central to practically all cellular activities. In nonexcitable cells, voltage-dependent Ca(2+) influx plays a key role in several important processes, including proliferation, apoptosis, and cell migration. It has been demonstrated that Ca(2+) signaling is essential in the development and functioning of OLs. For example, Ca(2+) uptake is required for the initiation of myelination, and perturbation of Ca(2+) homeostasis, e.g., overwhelming influxes of Ca(2+), leads to demyelination. Although OL progenitor cell Na(+) channels are present at a much lower density, their physiological properties appear to be indistinguishable from those recorded in neurons. Interestingly, recent data indicate that, as with neurons, some white matter OPCs possess the ability to generate Na(+)-dependent action potentials. This Mini-Review focuses on the mechanisms of Ca(2+) and Na(+) signaling in cells within the OL lineage mediated by voltage-operated ion channels, with a particular focus on the relevance of these voltage-dependent currents to oligodendroglial development, myelination, and demyelination. Overall, it is clear that cells in the OL lineage exhibit remarkable plasticity with regard to the expression of voltage-gated Ca(2+) and Na(+) channels and that perturbation of Ca(2+) and Na(+) homeostasis likely plays an important role in the pathogenesis underlying demyelinating diseases.

Expression and regulation of golli products of myelin basic protein gene during in vitro development of oligodendrocytes.

The myelin basic protein (MBP) gene produces two families of proteins, the classic MBPs, important for myelination of the CNS, and the golli proteins, whose biological role in oligodendrocytes (OLs) is still unknown. The goals of this work were to study the in vitro pattern of expression of the golli products during OL differentiation and to compare it with that of the classic MBP products of the gene. Mouse primary glial cultures were analyzed at the mRNA and protein levels with an array of techniques. We found that OLs express golli mRNA primarily during intermediate stages of differentiation, which was confirmed by immunocytochemical analysis. Golli expression was low in proliferating OL progenitors as well as in terminally mature OLs. Golli proteins were found associated with the OL cell soma and nuclei and, to a lesser extent, with the cellular processes. We also found that golli proteins are not targeted to myelin in vitro and in vivo, in contrast to the classic MBPs. Finally, we found that golli expression is regulated during OL development and can be manipulated by growth factors such as basic fibroblast growth factor, neurotrophin-3, and retinoic acid.

Classic and soma-restricted proteolipids are targeted to different subcellular compartments in oligodendrocytes.

The myelin proteolipid (PLP) gene is very active in oligodendrocytes (OLs) and generates at least four proteins: the classic PLP and DM20 proteolipids, which are associated with compact myelin and the srPLP and srDM20, which are associated with the cell soma. These proteins are extremely hydrophobic and appear to follow the biosynthetic route used by secretory proteins. In this study, we have analyzed the subcellular distribution of the newly described sr-proteolipids and compared it to that of the classic proteolipids. Immunocytochemical analysis indicates that the sr-proteolipids and classic proteolipids are found in association with the endoplasmic reticulum (ER) and Golgi apparatus of mature OLs in vitro. Whereas the classic proteolipids become associated with the myelin-like sheets elaborated by OLs, the sr-proteolipids are not targeted to the myelin leaflets. The sr-proteolipids were associated with endosomes and with recycling vesicles as determined by double immunocytochemistry with markers such as syntaxin 6 and clathrin. In vivo, immunohistochemical analysis showed a distribution of the sr-proteolipids that was similar to that obtained in vitro, with a total absence of incorporation of sr-proteolipids into compact myelin. This differential subcellular localization is further evidence for a biological role for these products of the PLP/DM20 gene, which is different from that of the classic proteolipids.

Differential sensitivity in the survival of oligodendrocyte cell lines to overexpression of myelin proteolipid protein gene products.

The proteolipid (PLP) gene encodes at least four proteins, including the classic PLP and DM20, which are important components of the myelin sheath, and the recently identified soma-restricted (sr) isoforms, srPLP and srDM20. The classic PLP and DM20 gene products have been implicated in oligodendrocyte survival by overexpression studies in vitro and in vivo. The classic and sr proteolipids are targeted to different cellular compartments in the oligodendrocyte, suggesting different cellular functions. Accordingly, we examined the effects of in vitro overexpression of the sr-PLP/DM20 isoforms on the survival of stably transfected, conditionally immortalized, oligodendroglial cell lines and compared this to overexpression of the classic and the jimpy-mutated proteolipids. The results indicate that overexpression of either normal or jimpy classic PLP/DM20 resulted in a dramatic reduction in the survival of the oligodendrocyte cell lines at the nonpermissive temperature, but not the COS-7 cell line, a cell line expressing the same oncogene constitutively. Survival of the oligodendrocyte cell lines was significantly less affected when either the sr-PLP/DM20 or the dopamine D-2 receptor, another cell membrane protein, was overexpressed in the cell lines. These results suggest that overexpression of the "classic" PLP or DM20 can compromise the survival of oligodendrocytes whether or not they are mutated. Furthermore, they suggest that the internal mechanisms for normal targeting of the PLP/DM20 isoforms of either the "classic" or the "sr" types influence the oligodendrocyte's ability to survive when these proteolipids are overexpressed.

Tbr1 regulates differentiation of the preplate and layer 6.

During corticogenesis, early-born neurons of the preplate and layer 6 are important for guiding subsequent neuronal migrations and axonal projections. Tbr1 is a putative transcription factor that is highly expressed in glutamatergic early-born cortical neurons. In Tbr1-deficient mice, these early-born neurons had molecular and functional defects. Cajal-Retzius cells expressed decreased levels of Reelin, resulting in a reeler-like cortical migration disorder. Impaired subplate differentiation was associated with ectopic projection of thalamocortical fibers into the basal telencephalon. Layer 6 defects contributed to errors in the thalamocortical, corticothalamic, and callosal projections. These results show that Tbr1 is a common genetic determinant for the differentiation of early-born glutamatergic neocortical neurons and provide insights into the functions of these neurons as regulators of cortical development.

The pathobiology of myelin mutants reveal novel biological functions of the MBP and PLP genes.

Substantial biological data indicate that the myelin basic protein (MBP) and myelin proteolipid protein (PLP/DM20) genes produce products with functions beyond that of serving as myelin structural proteins. Much of this evidence comes from studies on naturally-occurring and man-made mutations of these genes in mice and other species. This review focuses upon recent evidence showing the existence of other products of these genes that may account for some of these other functions, and recent studies providing evidence for alternative biological functions of PLP/DM20. The MBP and PLP/DM20 genes each encode the classic MBP and PLP isoforms, as well as a second family of proteins that are not involved in myelin structure. The biological roles of these other products of the genes are becoming clarified. The non-classic MBP gene products appear to be components of transcriptional complexes in the nucleus, and they also may be involved in signaling pathways in T-cells and in neural cells. The non-classic PLP/DM20 gene products appear to be components of intracellular transport vesicles in oligodendrocytes. There is evidence for other functions of the classic PLP/DM20 proteins, including a role in neural cell death mechanisms, autocrine and paracrine regulation of oligodendrocytes and neurons, intracellular transport and oligodendrocyte migration.

Identification of genes in the oligodendrocyte lineage through the analysis of conditionally immortalized cell lines.

The mouse oligodendrocyte cell lines, N19 and N20.1, were used as sources of potential stage-specific RNA in order to construct a subtraction library enriched in cDNAs expressed early in the oligodendrocyte (OL) lineage. From this library, 23 clones were examined and three were examined in most detail. The mRNAs of the three library clones were preferentially expressed in the N19 (progenitor) compared to the N20.1 (immature) OL line. One of these corresponded to the intermediate filament protein cytokeratin K19, which has not been reported to be expressed in OLs previously. Another was identified as the mouse homolog of T-cadherin, previously reported not to be present in OLs. Antisera raised against a T-cadherin peptide indicated the protein colocalized with the OL lineage markers A(2)B(5), A007, and 01 in mouse primary glial cultures. However, small round cells resembling OL precursors labeled intensely with T-cadherin, but were negative for the other markers, suggesting that this gene might be expressed earlier in the lineage. In early postnatal brain, in addition to the expected neuronal tracts, the T-cadherin antibody labeled small bipolar cells, approximately 8-10 microm in diameter, in white matter tracts. These cells had the morphology of OLs or their precursors and were identified within the cerebellar white matter and the corpus callosum, regions rich in OLs. The third clone, 3g5, was homologous to the P8 clone isolated from rat pancreas. It encoded an 80-amino-acid polypeptide with a protein kinase C domain suggesting a possible role in signal transduction. Antisera to this peptide also colocalized 3g5 with cells expressing A(2)B(5), A007, and 01 in culture and in cells within white matter tracts which had the same morphology as those labeled by T-cadherin in these regions. In addition to these, beta(10) thymosin and mevalonate kinase clones were also isolated from the screen.

Thymocytes express the golli products of the myelin basic protein gene and levels of expression are stage dependent.

The golli products of the myelin basic protein gene have been shown to be expressed in mouse thymus and brain. The full repertoire of thymic cell types expressing golli products has not yet been determined, although immunoreactivity has been found in some macrophages. We have analyzed the cellular expression of golli mRNAs and proteins in the thymus. The results showed that MTS5(+) cortical/MTS10(+) medullary epithelial cells and NLDC145(+) dendritic cells did not express golli, while some macrophages did exhibit strong immunoreactivity. GOLLI: mRNAs were not detected in macrophages by in situ hybridization. Thymocytes expressed significant levels of golli mRNAs and proteins by in situ hybridization and immunohistochemistry. Interestingly, golli immunoreactivity varied with thymocyte stage of differentiation. For example, CD4(-)CD8(-) (double-negative) thymocytes expressed relatively high levels of golli. Upon further differentiation into CD4(-)CD8(-) (double-positive) thymocytes, golli protein expression declined dramatically. When thymocytes developed into CD8(-) or CD4(+) (single-positive) thymocytes, golli protein expression increased again, but it never achieved the levels found in double-negative thymocytes. Thus, the altered levels of expression of golli proteins in developing thymocytes correlated with the transitions from double-negative to double-positive and double-positive to single-positive stages. The lack of significant golli expression in thymic stromal cells may offer an alternative explanation for the mechanism of inefficient negative selection of those autoreactive thymocytes with specificity for myelin basic proteins.

Immunogenicity of self antigens is unrelated to MHC-binding affinity: T-cell determinant structure of Golli-MBP in the BALB/c mouse.

The 'classical' myelin basic protein (MBP) exons belong to a much larger unit, termed the 'Golli-MBP' gene. Here we have examined the T-cell determinant structure of the Golli protein region in the BALB/c mouse. Golli p10-24, which was shown to have the strongest affinity for I-A(d), could not induce T-cell activation. Paradoxically, the poorer binding, overlapping p5-19 was effective at inducing T-cell proliferation. Thus, immunogenicity is not necessarily related to the MHC-binding affinity of self-peptides. In addition, MBP: p151-168-specific T cell clones responded only poorly to J37, a Golli-MBP protein, while MBP: 59-76-specific clones responded well to J37.

Platelet-derived growth factor and basic fibroblast growth factor regulate cell proliferation and the expression of notch-1 receptor in a new oligodendrocyte cell line.

We generated a new cell line, N38, by conditionally immortalizing mouse oligodendrocytes (OLs) at early stages of maturation. The morphology and marker expression pattern suggest N38 cells are similar to immature OLs. N38 cells were sensitive to changes in serum concentrations, and forcing the cells to differentiate in low serum at 39 degrees C significantly decreased the survival of the cells. Importantly, addition of PDGFaa, bFGF or astrocyte-conditioned medium had protective effects on the cells, by increasing cell proliferation but not cell differentiation. This effect was receptor-mediated. Exposure of N38 cells to differentiating signals such as retinoic acid did not cause further differentiation of the cells. The N38 cell line expresses the vertebrate homolog of the Drosophila notch-1 receptor, a molecule that appears to regulate OL differentiation. Notch-1 receptor was homogeneously distributed in the somas of N38 cells. Incubation of N38 cells with either PDGFaa or bFGF, however, induced a polarized distribution of the receptor in the majority of the cells as well as an upregulation of receptor protein levels. The upregulation of molecules, such the notch-1 receptor, in pathways that control differentiation might be an important mechanism for keeping OL precursors in an undifferentiated state during their exit of the germinal layer and migration in the developing central nervous system. This OL cell line might constitute a suitable model for studies of regulatory mechanisms at this stage of OL differentiation.

Identification of a novel silencer that regulates the myelin basic protein gene in neural cells.

The myelin basic protein gene produces two families of proteins, the golli proteins and the 'classic' myelin basic proteins from three transcription start sites (tsp). The golli proteins are expressed from the first tsp, and little is known about genetic elements that control its activity. We have examined elements that may regulate the expression of the golli products produced from this promoter in neural cell lines with constructs containing upstream portions of the first tsp by transient transfection assays. Three putative regulatory elements were identified, among them a 345bp novel silencer region, termed the golli silencer region (GSR), which was characterized in detail. This silencer was responsible for a significant (approx. 60%) inhibition of luciferase expression in PC12 cells. It was orientation-dependent and a double dose of this GSR completely abolished expression of the luciferase reporter activity. Transfections with deleted constructs identified three critical sites that bind at least two repressor proteins. We postulate that the silencer activity is the result of synergistic interactions between these repressor proteins and might involve the formation of a high-ordered protein-DNA structure.

New insights on the biology of myelin basic protein gene: the neural-immune connection.

In the past 6 years, our conception of the major myelin protein genes has begun to change significantly because of recent findings documenting the existence of new exons encoding other products of these genes. A decade ago the myelin basic protein (MBP) and proteolipid protein (PLP) genes were thought to be expressed solely in myelin-forming cells, and their products were thought to be structural components of myelin. Since then, abundant evidence has been gathered identifying the presence of products of these genes in nonmyelinating cell types including both the immune and the nervous systems. Furthermore, within the nervous system, products of these genes have been identified in neurons and embryonic cells, clearly indicating that these myelin protein genes have additional functions in a number of cell types that are unrelated to myelination. In this brief communication, we review the recent literature that has resulted in this revision of our understanding of the MBP gene structure, products and expression.

Zebra finch aromatase gene expression is regulated in the brain through an alternate promoter.

The learned singing behavior in songbirds is sex-steroid-dependent and sexually dimorphic. Estrogen plays a major role in masculinizing the song system in these songbirds. The songbird brain synthesizes large amounts of estrogen, which, in the case of zebra finches, have been found to enter the systemic circulation. Aromatase cytochrome P450 is the key enzyme catalyzing the conversion of androgens to estrogens. We have cloned a novel alternatively spliced form of aromatase cDNA expressed predominantly in the zebra finch brain. We have also isolated and characterized the gene coding for zebra finch aromatase which spans 20kb in length. The alternate forms of aromatase mRNA (ARO) differ in their 5'-untranslated regions encoded by either exon 1a or 1b. The putative promoter sequences controlling the regulation of the alternate forms of ARO in zebra finches contain consensus binding sites for various transcription factors. While both the promoters have binding sites for SRY-like transcription factor, a binding site for SF-1 is present only in the promoter 1b active in the ovary. Intriguingly, a 55bp segment within the promoter 1a sequence appears to be highly conserved among zebra finch, mouse and human aromatase promoters active in the brain.

Identification of a new exon in the myelin proteolipid protein gene encoding novel protein isoforms that are restricted to the somata of oligodendrocytes and neurons.

The myelin proteolipid protein (PLP) gene (i.e., the PLP/DM20 gene) has been of some interest because of its role in certain human demyelinating diseases, such as Pelizaeus-Merzbacher disease. A substantial amount of evidence, including neuronal pathology in knock-out and transgenic animals, suggests the gene also has functions unrelated to myelin structure, but the products of the gene responsible for these putative functions have not yet been identified. Here we report the identification of a new exon of the PLP/DM20 gene and at least two new products of the gene that contain this exon. The new exon, located between exons 1 and 2, is spliced into PLP and DM20 mRNAs creating a new translation initiation site that generates PLP and DM20 proteins with a 12 amino acid leader sequence. This leader sequence appears to target these proteins to a different cellular compartment within the cell bodies of oligodendrocytes and away from the myelin membranes. Furthermore, these new products are also expressed in a number of neuronal populations within the postnatal mouse brain, including the cerebellum, hippocampus, and olfactory system. We term these products somal-restricted PLP and DM20 proteins to distinguish them from the classic PLP and DM20 proteolipids. They represent putative candidates for some of the nonmyelin-related functions of the PLP/DM20 gene.

Developmental regulation of the distribution of aromatase- and estrogen-receptor- mRNA-expressing cells in the zebra finch brain.

Strong evidence exists for the masculinizing effects of estrogen on the neural network that controls song learning and behavior in zebra finches. However, the mechanisms by which estrogen acts to influence the development of this circuitry are not well understood. In this study, we used in situ hybridization to detect the distribution of cells expressing mRNAs for AROM and ERalpha at postnatal days 5-25 (P5-25). Our findings revealed developmental regulation of both mRNAs in the neostriatum, archistriatum, hippocampus, diencephalon and midbrain. Within the vocal control circuitry, cells expressing ERalpha mRNA were found in the medial HVC (P10-25), archistriatum lateral to the RA (Ad; P25), in the ICo (P5-25), and along the fiber tract containing efferents from the RA. High levels of AROM mRNA were found in the neostriatum, including both the lateral and mMAN and along their projections to the RA and HVC, respectively, (P5-25), in the archistriatum (P18-25) and around RA (P18). Codistribution of the two mRNAs occurred along the border of the HVC suggesting that in this region, local synthesis of estrogen may be acting through its nuclear receptor to regulate gene transcription. Taken together, our findings show that the neural circuitry controlling song may be exposed to the effects of estrogen during early postnatal development. However, in most of the song control regions, these mRNAs were not expressed together either temporally or spatially, indicating that AROM may have a role in the development of the song system independent of ERalpha.

Two neuronal cell lines expressing the myelin basic protein gene display differences in their in vitro survival and in their response to glia.

We have generated two conditionally immortalized neuronal cell lines from primary cultures of embryonic day 13 (E13) and postmitotic (postnatal day 0; P0) cortical neurons transformed with the temperature-sensitive SV-40 large-T antigen. Two clonal cell lines (CN1.4 from E13 cultures and SJ3.6 from P0 cultures) were isolated and stable maintained in vitro. Both cell lines expressed a number of neuronal markers such as the neurofilaments, glutamic acid decarboxylase 67, neuron-specific enolase, and the BG21 isoform of the myelin basic protein gene. At 34 degrees C, the CN1.4 cell line had elaborated short processes, whereas the SJ3.6 cell line produced long processes that formed a delicate network. When these cell lines were cultured at 39 degrees C, some of the cellular processes grew longer, adopting a more mature neuronal morphology. Interestingly, at 39 degrees C, the in vitro survival of these cell lines differed significantly. Whereas the survival of CN1.4 cell line was greatly unaffected, SJ3.6 cells died soon after they were cultured at 39 degrees C. The cell death of SJ3.6 cells was accompanied by fragmentation and condensation of DNA in their nuclei, indicative of an apoptotic event. Under these conditions, SJ3.6 showed an upregulation of the p75 receptor. When this cell line was cocultured with oligodendrocytes, astrocytes, or glial conditioned media (GCM), there was a marked increase in survival. In contrast, little effect of glial cells or GCM was observed on the CN1.4 cell line. These lines appear to be useful models to study neuronal-glial interactions in addition to neuronal cell death and the effects of glial factors that promote the survival of neurons.

Embryonic expression of the myelin basic protein gene: identification of a promoter region that targets transgene expression to pioneer neurons.

The myelin basic protein (MBP) gene produces two families of structurally related proteins from three different promoters-the golli products, generated from the most upstream promoter, and the MBPs, produced from the two downstream promoters. In this report we describe the expression of golli proteins within some of the earliest neuronal populations of the brain, including Cajal-Retzius cells and preplate neurons of the forebrain, representing a new marker for these cells. To identify elements responsible for neuronal expression of the golli products, we generated transgenic animals from constructs containing different portions of the upstream promoter. A construct containing 1.1 kb immediately upstream of the golli transcription start site targeted expression of beta-galactosidase to preplate neurons and a subset of Cajal-Retzius cells in transgenic mice-the first reported genetic element to target expression to these pioneer cortical populations. Although expression in Cajal-Retzius cells declined with embryonic development, preplate cells continued to express the transgene after arriving at their final destination in the subplate. Interestingly, expression persisted in subplate neurons found within a distinct layer between the white matter and cortical layer VI well into postnatal life. Birth dating studies with bromodeoxyuridine indicated that these neurons were born between E10.5 and E12.5. Thus, the transgene marked subplate neurons from their birth, providing a fate marker for these cells. This work suggests a role for the MBP gene in the early developing brain long before myelination and especially in the pioneer cortical neurons important in the formation of the cortical layers.

Identification of the dopamine D3 receptor in oligodendrocyte precursors: potential role in regulating differentiation and myelin formation.

Expression of the dopamine D3 receptor (D3r) was found in primary mixed glial cultures from newborn brain and in the corpus callosum in vivo during the peak of myelination. Expression of the D3r mRNA, but not D2r mRNA, was detected as early as 5 d in vitro (DIV) by RT-PCR. Immunoblot studies revealed D3r protein was also expressed in the cultures. Double immunofluorescence analysis for the D3r and for surface markers of specific stages of oligodendrocyte development indicated that D3r expression occurred in precursors and in immature oligodendrocytes but not in mature oligodendrocytes (i.e. , A2B5(+) 007(-) 01(-) and A2B5(+) 007(+) 01(-) cells but not A2B5(-) 007(+) 01(+) cells). Confocal microscopic analysis indicated that D3r was associated with cell bodies and cell membranes but not with the processes emanating from cell somas. Immunohistochemistry of brain sections revealed the presence of D3r in some oligodendrocytes located mainly within the genu and radiato of the corpus callosum during the active period of myelination. Treatment of cultures with 20 microM quinpirole led to decreased numbers of O1(+) oligodendrocytes possessing myelin-like membranes as well as an increase in the number of precursors in 14 DIV cultures. This effect was prevented by the dopamine antagonist haloperidol. These results show that the D3r expression is not restricted to neurons but it is also expressed in differentiating oligodendrocytes before terminal maturation. It also suggests that dopamine or some other D3r ligand may play a role in oligodendrocyte differentiation and/or the formation of myelin by mature oligodendrocytes.