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The article presents an abstract of the medical research paper "Mouse Brain Neurons and Oligodendrocytes Express a Novel PLP/DM-20-Related Product." This paper is to be discussed in the Seventeenth Biennial Meeting of the International Society for Neurochemistry (ISN) and the Thirteenth General Meeting of the European Society for Neurochemistry (ESN) to be held at Berlin, Germany from August 8-14, 1999. This paper informs that PLP/DM-20 gene appears to be expressed in selected neurons.

The article presents an abstract of a medical research related to the expression of a novel PLP/DM20-related product in mouse brain neurons and oligodendrocytes. There are two new mRNA products that have been cloned. The products encode proteins that contain a novel 12 amino acid sequence at their N-termini linked to either the classic PLP or DM20 proteins.

The myelin basic protein (MBP) gene encodes two families of proteins: the classic MBP constituents of myelin and the golli-MBPs, the function of which is less well understood. Previous work suggests that golli proteins may play a role in Ca2+ homeostasis in oligodendrocytes (OLs) and in T-cells. Overexpression of golli in OL cell lines induces elaboration of sheets and processes. Live imaging of these cells revealed a rapid retraction of the processes and sheets after depolarization with high K+. This phenomenon was associated with a significant increase in [Ca2+]int without changes in cell viability. The results indicated that golli produced its effect through Ca2+ influx, rather than Ca2+ release from intracellular stores. Furthermore, a specific [Ca2+]int chelator (BAPTA) or Cd2+, a specific blocker of voltage-operated Ca2+ channels, abolished the ability of golli to promote process extension in a dose-dependent manner. Analysis of the golli protein identified a myristoylation site at the C terminus of the golli domain, which was essential for the action of golli on Ca2+ influx, suggesting that binding of golli to the plasma membrane is important for modulating Ca2+ homeostasis. High-resolution spatiotemporal analysis along N19 processes revealed higher-amplitude local Ca2+ influx in regions with elevated levels of golli. These findings suggest a key role for golli proteins in regulating voltage-gated Ca2+ channels in OLs during process remodeling. Our observations are consistent with the hypothesis that golli proteins, as a part of a protein complex, modulate Ca2+ influx at the plasma membrane and along OL processes.

The first postmitotic neurons in the developing neocortex establish the preplate layer. These early-born neurons have a significant influence on the circuitry of the developing cortex. However, the exact timing and trajectory of their projections, between cortical hemispheres and intra- and extra-cortical regions, remain unresolved. Here, we describe the creation of a transgenic mouse using a 1.3 kb golli promoter element of the myelin basic protein gene to target expression of a tau-green fluorescent protein (GFP) fusion protein in the cell bodies and processes of pioneer cortical neurons. During embryonic and early neonatal development, the timing and patterning of process extension from these neurons was examined. Analysis of tau-GFP fluorescent fibers revealed that progression of early labeled projections was interrupted unexpectedly by transient pauses at the corticostriatal and telencephalic-diencephalic boundaries before invading the thalamus just prior to birth. After birth the pioneering projections differentially invaded the thalamus, excluding some nuclei, e.g. medial and lateral geniculate, until postnatal days 10-14. Early labeled projections were also found to cross to the contralateral hemisphere as well as to the superior colliculus. These results indicate that early corticothalamic projections appear to pause before invading specific subcortical regions during development, that there is developmental regulation of innervation of individual thalamic nuclei, and that these early-generated neurons also establish early projections to commissural and subcortical targets.

The myelin basic protein (MBP) gene encodes two families of proteins, the classic MBP constituents of myelin and the golli-MBPs, the function of which is less well understood. In this study, targeted ablation of the golli-MBPs, but not the classic MBPs, resulted in a distinct phenotype unlike that of knock-outs (KOs) of the classic MBPs or other myelin proteins. Although the golli KO animals did not display an overt dysmyelinating phenotype, they did exhibit delayed and/or hypomyelination in selected areas of the brain, such as the visual cortex and the optic nerve, as determined by Northern and Western blots and immunohistochemical analysis with myelin protein markers. Hypomyelination in some areas, such as the visual cortex, persisted into adulthood. Ultrastructural analysis of the KOs confirmed both the delay and hypomyelination and revealed abnormalities in myelin structure and in some oligodendrocytes. Abnormal visual-evoked potentials indicated that the hypomyelination in the visual cortex had functional consequences in the golli KO brain. Evidence that the abnormal myelination in these animals was a consequence of intrinsic problems with the oligodendrocyte was indicated by an impaired ability of oligodendrocytes to form myelin sheets in culture and by the presence of abnormal Ca2+ transients in purified cortical oligodendrocytes studied in vitro. The Ca2+ results reported in this study complement previous results implicating golli proteins in modulating intracellular signaling in T-cells. Together, all these findings suggest a role for golli proteins in oligodendrocyte differentiation, migration, and/or myelin elaboration in the brain.

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., (Copyright 2001 Wiley-Liss, Inc.)

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., (Copyright 2002 S. Karger AG, Basel)

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.

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.

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.

The myelin basic protein (MBP) gene locus is composed of two overlapping transcription units that share all of the MBP exons. One of these transcription units expresses the MBPs and the other expresses a family of proteins structurally related to the MBPs. This second transcription unit is called the Golli gene, and the entire complex is called the Golli-mbp gene. In this study, the expression of the Golli gene was examined in the human fetal central nervous system (CNS). By using reverse transcriptase-polymerase chain reaction cloning we have identified eight new members of the Golli gene family of transcripts expressed in the human CNS. Golli gene expression was examined by in situ hybridization and immunohistochemistry, and surprisingly, Golli products were found to be expressed in neurons as well as oligodendrocytes. Furthermore, the subcellular distribution of Golli immunoreactivity in fetal spinal cord interneurons shifted between the various laminae. Golli protein was localized within the nuclei of interneurons in the posterior horn, but was found in the cell bodies and processes of interneurons in the anterior horn. Within oligodendrocytes, Golli protein was detected in the cell bodies and processes, including processes which were wrapping axonal segments. Golli mRNA expression was also observed in neurons within the cerebral cortex between 18 and 20 weeks postconception, prior to myelination of this brain region. During this period, there was a striking developmental increase in the numbers and in the locations of neurons expressing Golli mRNAs within the cortical plate. The diverse distribution of Golli proteins within neurons and oligodendrocytes indicates that their function is quite different from that of the MBPs to which they are closely related.

A cDNA was isolated from a zebra finch telencephalon cDNA library that encodes the myelin proteolipid protein. The clone was 2874 nucleotides long containing an open reading frame of 831 nucleotides that encoded a 277 amino acid myelin proteolipid protein. The 5'- and 3' untranslated regions were 112 and 1931 nucleotides, respectively. In Northern blots the clone hybridized to 3 bands of 3.5, 2.4 and 1.5 Kb in mouse brain RNA, but to only a single band of 3.0 kb in zebra finch brain RNA, suggesting the lack of alternative polyadenylation sites within the 3' untranslated region of the zebra finch PLP mRNAs. There was a small degree of homology between the zebra finch and chicken PLP 5' untranslated regions, but relatively little homology of the 5' untranslated regions of the zebra finch PLP cDNA clone with the homologous regions of PLP cDNAs of many mammalian species. Except for a small stretch of considerable homology, there was little overall homology with the 3' untranslated regions of mammalian PLP mRNAs. Approximately 10% (i.e. 28) of the amino acids in the zebra finch PLP differed from mammalian PLP, with most of these changes located within exon 3. There were 16 amino acid changes between zebra finch and chicken, suggesting that greater sequence variation in PLP structure is tolerated among avian species than among mammalian species.

The brain regions controlling song are much larger in male than in female zebra finches. This sex difference is thought to arise developmentally when the male's brain is exposed to higher levels of estrogen. The synthesis of estrogen from androgen is catalyzed by aromatase, a key enzyme implicated in song development in the zebra finch. To study the role of estrogen synthesis in the organization of brain regions responsible for song, a cDNA encoding aromatase was isolated from a zebra finch ovarian cDNA library. The 3188 bp cDNA contains a 1527 bp open reading frame with 5' and 3' untranslated regions of 116 bp and 1545 bp, respectively. The deduced polypeptide is 509 amino acids in length, and is highly homologous to aromatases reported for chicken (92%), human (72%), mouse (70%), rat (69%) and trout (53%). Northern blot analysis revealed 5.4 kb, 4.8 kb and 3.2 kb aromatase mRNAs in brain and ovary. In situ hybridization histochemistry revealed the expression of aromatase mRNA in ovarian thecal cells of some, but not all, follicles, suggesting that aromatase gene expression is regulated during follicular maturation. In the adult brain, the distribution of aromatase mRNA was surprisingly widespread, and included the preoptic area, hypothalamus, hippocampus and neostriatum. By contrast, little aromatase mRNA expression was noted in the song nuclei (HVC, RA, area X). This study, the first description of aromatase mRNA expression by in situ hybridization in the brain of any species, identifies a surprisingly large number of cells that express aromatase mRNA in the zebra finch telencephalon. This pattern may be a unique feature of all songbirds.

We have identified a novel transcription unit of 105 kilobases (called the Golli-mbp gene) that encompasses the mouse myelin basic protein (MBP) gene. Three unique exons within this gene are alternatively spliced into MBP exons and introns to produce a family of MBP gene-related mRNAs that are under individual developmental regulation. These mRNAs are temporally expressed within cells of the oligodendrocyte lineage at progressive stages of differentiation. Thus, the MBP gene is a part of a more complex gene structure, the products of which may play a role in oligodendrocyte differentiation prior to myelination. One Golli-mbp mRNA that encodes a protein antigenically related to MBP is also expressed in the spleen and other non-neural tissues.

An immunosorbent column specific for the myelin basic protein (BP) was prepared by coupling purified anti-BP antibodies to cyanogen bromide (BrCN)-activated Sepharose 4B. The BP-immunosorbent column bound BP between pH 4.5 and pH 6.8. In its working range the column bound approximately 400-475 microgram of BP at pH 6.8 and 250 microgram at pH 4.5 with recoveries of 72-77%. The BP-immunosorbent column could effectively separate BP from simple mixtures of BP and proteins of similar size and charge and from acid extracts of bovine brain. The results indicate that the BP-immunosorbent column can be used to isolate BP from a mixture of proteins and may be adapted for use in the small-scale purification of the myelin basic proteins involving a minimum number of steps.

The major myelin proteins fall into two classes, the basic proteins and the proteolipid proteins. In mice, five forms of the myelin basic protein (MBP) have been identified with apparent molecular masses of 21.5 kD, 18.5 kD, 17 kD and 14 kD. The 17 kD MBP variant consists of two molecular forms with similar molecular masses but different amino acid sequences. Cell-free translation studies and analyses of MBP cDNAs have shown that each of the MBP variants is encoded by a separate mRNA of approximately 2 000 bp. The five mouse MBP mRNAs appear to be derived by alternative splicing of exons 2, 5, and 6 of the MBP gene. cDNAs encoding four forms of MBP have been isolated from a human fetal spinal cord library. The mRNAs corresponding to these cDNAs are probably derived by alternative splicing of exons 2 and 5 of the human MBP gene. Proteolipid protein (PLP) cDNAs have been isolated from several species and used to establish that the size of the major PLP mRNA is approximately 3 kb. Multiple size classes of the PLP mRNAs exist in mice and rats whereas the 3 kb mRNA is the predominant form in the developing human spinal cord. In normal mice, maximal expression of the PLP gene lags behind that of the MBP gene by several days. Studies on dysmyelinating mutants have determined some of the molecular defects with respect to these two classes of myelin proteins. For example, there is a deletion of a portion of the MBP gene in the shiverer mutant. In the quaking mutant, the expression of both classes of myelin proteins is significantly reduced prior to 3 weeks. However, after 3 weeks, MBP expression approaches normal levels but the newly synthesized protein fails to be incorporated into myelin. In the jimpy mutant, although the expression of both classes of proteins is reduced, PLP expression is most severely affected.

The interaction of free and immobilized myelin basic protein (MBP) with sodium deoxycholate (DOC) and sodium dodecyl sulfate (NaDodSO4) was studied under a variety of conditions. Free MBP formed insoluble complexes with both detergents. Analysis of the insoluble complexes revealed that the molar ratio of detergent/MBP in the precipitate increased in a systematic fashion with increasing detergent concentration until the complex became soluble. At pH 4.8, equilibrium dialysis studies indicated that approximately 15 mol of NaDodSO4 could bind to the protein without precipitation occurring. Regardless of the surfactant, however, minimum protein solubility occurred when the net charge on the protein-detergent complex was between +18 and -9. Complete equilibrium binding isotherms of both detergents to the protein were obtained by using MBP immobilized on agarose. The bulk of the binding of both detergents was highly cooperative and occurred at or above the critical micelle concentration. At I = 0.1, saturation levels of 2.09 +/- 0.15 g of NaDodSO4/g of protein and 1.03 /+- 0.40 g of DOC/g of protein were obtained. Below pH 7.0 the NaDodSO4 binding isotherms revealed an additional cooperative transition corresponding to the binding of 15-20 mol of NaDodSO4/mol of protein. Affinity chromatography studies indicated that, in the presence of NaDodSO4 (but not in its absence), [125I]MBP interacted with agarose-immobilized histone, lysozyme, and MBP but did not interact with ovalbumin-agarose. These data support a model in which the detergent cross-links and causes precipitation of MBP-anionic detergent complexes. Cross-linking may occur through hydrophobic interaction between detergents electrostatically bound to different MBP molecules.

The accumulation and distribution of proteolipid proteins in rat brain and selected brain regions (cerebellum, cerebral cortex, basal ganglia, and hippocampus) were studied during early postnatal development. In whole brain an eightfold increase of proteolipid was observed between ten and 33 days after birth. This was reflected in the separate regions examined where the proteolipid protein content increased six- to ten-fold during the same period. The basal ganglia and cerebral cortex contributed the greatest amount to the total proteolipid present. However, at 28-33 days the greatest concentration (mg/g tissue) was observed in the basal ganglia and hippocampus. When the proteolipid protein preparations were examined by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, distinctive, heterogeneous patterns for each brain region were obtained. Proteolipid from basal ganglia (the region richest in white matter) consisted primarily of two major protein bands with apparent molecular weights of approximately 21,500 and 26,000. Both of these bands dramatically increased in quantity during myelination, and the larger protein coelectrophoresed with isolated myelin proteolipid protein. Both bands were also found present in proteolipid preparations from the other brain regions but in varying amounts relative to the total. The data suggest that the increase in proteolipid observed during this developmental period was due in large measure to the accumulation of myelin-specific proteolipids, but also that a significant proportion of the increase was due to the accumulation of nonmyelin components.