The myelin basic protein gene is expressed in differentiated blood cell lineages and in hemopoietic progenitors.

Myelin basic proteins (MBP) are major constituents of the myelin sheath of oligodendrocytes and Schwann cells in the central nervous system and the peripheral nervous system, respectively. We previously showed that MBP-related transcripts are present in the bone marrow and the immune system. These mRNAs are transcribed from a region called 0', consisting of three exons, located upstream of the classical MBP exons; these three exons belong to the long MBP gene otherwise called "Golli-MBP." The most abundant of these mRNAs, now called HMBP (hemopoietic MBP), encompasses the sequence encoded by the region 0' plus exon 1 and part of intron 1 of the classic MBP gene. Antisera to recombinant HMBP proteins are immunoreactive with proteins of about 26-28 kDa in brain, thymus, and spleen. This report demonstrates that HMBP proteins are present in the vast majority (>95%) of thymic T cells, which express the corresponding transcripts, as do mature T cells from lymph nodes and spleen. HMBP mRNAs and proteins are also manifest in the majority of spleen B lymphocytes and in B cell lines. In addition to lymphoid cells, HMBP proteins are in all types of myeloid lineage cells, i.e., macrophages, dendritic cells, and granulocytes, as well as in megakaryocytes and erythroblasts. Finally, HMBP proteins are present in CD34+ bone marrow cells, and, furthermore, in highly proliferative cultures, these CD34+ cells express HMBP RNAs and proteins. Thus, MBP gene products are present both in the nervous system and in the entire hemopoietic system.

[Microglia: origin and development]. / Microglie: origine et développement.

As suggested by Del Rio Ortega a long time ago, it is now widely accepted that microglia are the resident macrophages of the central nervous system. Microglia represent about 10% of the adult brain cell population. We have previously shown that the late embryonic and adult mouse brain contain potential microglial progenitors. We report here that microglial progenitors can be detected in neural folds from embryonic day 8. They originate from the yolk sac in which macrophage progenitors are found from embryonic day 7. We also report that the bulk of microglial cells (about 95%) appear during post-natal development. A major finding is that microglia arise by an intense in situ proliferation comparable to that of neural cells. Taken together, these results show that adult mouse microglia originate from cells migrating from the yolk sac and whose progeny actively proliferates in the brain during development.

Expression of gamma-glutamyl transpeptidase in mouse perivascular astrocytes and in a protoplasmic-like astroglial cell clone.

Gamma-glutamyl transpeptidase (GGT) is known to be present in the central nervous system (CNS) but its cellular localization is still subject to controversy. In this report, we have investigated, with a specific antiserum, the immunolabelling pattern of GGT in the adult mouse CNS at the light and electron microscopic (EM) levels. At the optical level, GGT immunoreactivity ensheathes the majority of vessels in the grey matter. Immunoelectron microscopy shows that labelling is essentially due to the presence of GGT in the astrocytic endfeet which surround vessels. In addition, some pericytes and periendothelial cells are also clearly labelled. We then investigated GGT activity in astroglial cell clones which may represent the in vitro counterpart of the main astroglial cell types. The striking result is that a protoplasmic-like astroglial cell clone shows a noticeable GGT activity, while, in contrast, no activity was detected in the fibrous and the Golgi-Bergmann-like astroglial clones. Taken together, these data indicate that, in the mouse CNS, GGT is essentially present in protoplasmic astrocytes.

No evidence for transmission disequilibrium between a new marker at the myelin basic protein locus and multiple sclerosis in French patients.

The myelin basic protein (MBP) gene is a candidate locus for susceptibility to multiple sclerosis. Several groups have tested a complex (TGGA)n repeat in the 5' region of this gene for association/linkage with multiple sclerosis, with divergent results. This region of tandem repetitive sequence has been subjected to complex rearrangements, and there is a possibility that alleles of the same size have different internal structures, which reduces the interest of this marker for linkage disequilibrium studies and may at least partly explain the conflicting results obtained so far. To overcome this problem, we isolated a new polymorphic (CA)n repeat within the Golli-MBP locus. The limited number of alleles identified makes this other marker suitable for transmission disequilibrium studies. We tested this marker for linkage with multiple sclerosis, using the transmission-disequilibrium test (TDT) on a sample of 196 nuclear families in which the genotypes of both parents could be unambiguously defined. We found no evidence of transmission disequilibrium between multiple sclerosis and any of the three alleles of this marker, even when the patients were subdivided according to their HLA-DRB1*1501 status. The present data thus provide no evidence for a contribution of the MBP gene to multiple sclerosis susceptibility in French patients.

Microglia derive from progenitors, originating from the yolk sac, and which proliferate in the brain.

Microglia, the resident CNS macrophages, represent about 10% of the adult brain cell population. Although described a long time ago, their origin and developmental lineage is still debated. While del Rio-Hortega suggested that microglia originate from meningeal macrophages penetrating the brain during embryonic development, many authors claim that brain parenchymal microglia derive from circulating blood monocytes originating from bone marrow. We have previously reported that the late embryonic and adult mouse brain parenchyma contains potential microglial progenitors [F. Alliot, E. Lecain, B. Grima, B. Pessac, Microglial progenitors with a high proliferative capacity in the embryonic and the adult mouse brain, Proc. Natl. Acad. Sci. U.S.A. 88 (1991) 1541-1545]. We now report that they can be detected in the brain rudiment from embryonic day 8, after their appearance in the yolk sac and that their number increases until late gestation. We also show that microglia appear during embryonic development and that their number increases steadily during the first two postnatal weeks, when about 95% of microglia are born. Finally, the main finding of this study is that microglia is the result of in situ proliferation, as shown by the high proportion of parenchymal microglial cells that express PCNA, a marker of cell multiplication, in embryonic and postnatal brain. Taken together, our data support the hypothesis that terminally differentiated brain parenchymal microglia are derived from cells originating from the yolk sac whose progeny actively proliferates in situ during development.

Pericytes and periendothelial cells of brain parenchyma vessels co-express aminopeptidase N, aminopeptidase A, and nestin.

Within the parenchyma of the CNS, the endothelium of all vessels is surrounded by a layer of cells, pericytes in capillaries and periendothelial or intima smooth muscle cells in other vessels. The origin of these cell types, their relationship, and their role are unclear. However, it has been recently shown that genetically engineered mice that lack pericytes develop microaneurysms at late gestation and die before birth (Lindahl et al. [1997] Science 277:242-245). The goal of this study was to identify in situ molecular markers that would be common to pericytes and periendothelial cells of adult mouse brain. Immunocytochemistry experiments were carried out at the optical and electron-microscopic levels on mouse brain sections with antibodies specific for aminopeptidase N, aminopeptidase A, and the intermediate filament nestin. The results of our experiments show that in all brain parenchyma vessels of all sizes, pericytes and periendothelial cells are immunoreactive for aminopeptidase N, essentially at the plasma membrane level, and are also labeled by nestin specific antibodies, which decorate typical intermediate filaments. In addition, brain pericytes and periendothelial cells are also immunoreactive to monoclonal antibodies to aminopeptidase A. In contrast, pericytes and periendothelial cells do not express microglial markers. Taken together these data show that pericytes and periendothelial intima smooth muscle cells share common markers, suggesting a common origin or function, and are distinct from microglia.

Brain parenchyma vessels and the angiotensin system.

It is now recognized that the brain contains an autonomous angiotensin (AG) system, including the aminopeptidases A and N required for angiotensin metabolism. Using immunohistochemical techniques, we show that capillary pericytes and periendothelial cells of other vessels express aminopeptidase A (APA) and aminopeptidase N (APN) at their plasma membrane in adult mouse brain parenchyma. We therefore investigated the localization of angiotensin II(III), known as putative substrates for these enzymes, as well as that of their precursor angiotensin I. We report here the presence of immunoreactivity to angiotensin I and II(III) around most brain vessels. Angiotensins are present at the plasma membrane of brain parenchymal cells, presumably perivascular astrocytes which are also immunoreactive to AT1-receptor antibodies. The very close relationship between AGII(III) and their metabolizing enzymes APA and APN suggests a specific functional role for brain perivascular angiotensins.

Ly-6C is expressed in brain vessels endothelial cells but not in microglia of the mouse.

The Ly-6C antigen is expressed in various cell types of the immune system, including macrophages. Using the monoclonal antibody ER-MP20 which specifically recognises Ly-6C, we have investigated whether brain parenchymatous microglia express Ly-6C in vivo as well as in vitro. In brain sections from developing and adult C57/BI mice, all vessels were strongly immunolabelled. Electron microscopic immunohistochemistry showed that the endothelial cells are the cell type expressing Ly-6C. In contrast, we never observed immunoreactivity on microglia; however, microglial cells proliferating in vitro were strongly ER-MP20 positive. These data show that Ly-6C is not a marker for microglia in vivo.

Retinal dysplasia in mice lacking p56lck.

The product of the proto-oncogene p56lck is a non-receptor tyrosine kinase member of the Src family. It is found in T cells (Marth et al., 1985, 1988) and in the mouse brain (Omri et al., 1996; Van Tan et al., 1996). In this report, we describe experiments showing that Lck is present in the mouse retina neurons. Lck gene expression was identified after isolating and sequencing the specific 5' and 3' part of the cDNA obtained by RT-PCR. In adult retina Lck immunoreactivity was most abundant in photoreceptor cells and within the outer plexiform layers. Staining was also observed in the inner nuclear and plexiform layers. In transgenic mice, the disruption of the Lck gene had serious consequences on the organization of the retina causing retinal dysplasia. These mice have partial retinal detachment with infolding and rosette formation in the photoreceptor sheet. These retinal abnormalities observed in Lck deficient mice lead to the loss of normal architecture of the photoreceptor and the inner nuclear layers, and provide an important role of Lck protein in the retina development. The lack of the Lck protein produces a spectrum of retinal pathology that resembles human retinopathy of prematurity (ROP).

Myelin basic protein-related proteins in mouse brain and immune tissues.

Brain and immune system tissues express myelin basic protein (MBP) mRNAs that contain novel exons upstream of those of the classic MBPs. We have generated antisera against a recombinant protein that includes the deduced sequence for one of the predicted species of the new MBP-related protein, hemopoietic HMBPR1. We report here the presence of multiple proteins reactive to the antisera in a range of tissues. The principal finding is that the antisera recognize a family of proteins of approximately 25 kDa that are restricted to brain, thymus, and spleen, the only tissues in which the new MBP-related transcripts are present. These antisera also detect other proteins of apparent molecular mass consistent with other isoforms of predicted MBP-related proteins. The expression of MBP-related proteins in immune system tissues may be important for self-tolerance to CNS MBPs and the initiation of immune-associated demyelinating diseases.

The Lck tyrosine kinase is expressed in brain neurons.

The lck gene product, p56lck, is a member of the src-related family of protein tyrosine kinases. It is known as lymphocyte specific and involved in thymocyte development and in the immune response mediated by the T cell receptor. We report that the lck gene is also expressed in adult mouse CNS and that brain p56lck is similar to the thymus protein. In situ hybridization and immunohistochemistry show that the lck gene is expressed in neurons throughout the brain in distinct regions, including hippocampus and cerebellum. In primary cultures from fetal mouse brain, neuronal cells are immunoreactive to Lck antiserum. This suggests that the lck gene product might be involved in a new signal transduction pathway in mouse brain.

Nectinepsin: a new extracellular matrix protein of the pexin family. Characterization of a novel cDNA encoding a protein with an RGD cell binding motif.

We report the isolation and characterization of a novel cDNA from quail neuroretina encoding a putative protein named nectinepsin. The nectinepsin cDNA identifies a major 2.2-kilobase mRNA that is detected from ED 5 in neuroretina and is increasingly abundant during embryonic development. A nectinepsin mRNA is also found in quail liver, brain, and intestine and in mouse retina. The deduced nectinepsin amino acid sequence contains the RGD cell binding motif of integrin ligands. Furthermore, nectinepsin shares substantial homologies with vitronectin and structural protein similarities with most of the matricial metalloproteases. However, the presence of a specific sequence and the lack of heparin and collagen binding domains of the vitronectin indicate that nectinepsin is a new extracellular matrix protein. Furthermore, genomic Southern blot studies suggest that nectinepsin and vitronectin are encoded by different genes. Western blot analysis with an anti-human vitronectin antiserum revealed, in addition to the 65- and 70-kDa vitronectin bands, an immunoreactive protein of about 54 kDa in all tissues containing nectinepsin mRNA. It seems likely that the form of vitronectin found in chick egg yolk plasma by Nagano et al. ((1992) J. Biol. Chem. 267, 24863-24870) is the protein that corresponds to the nectinepsin cDNA. This new protein could be an important molecule involved in the early steps of the development.

A spontaneously immortalized mouse microglial cell line expressing CD4.

We have derived a microglial clone, named C8-B4, from the 8-day mouse cerebellum organ culture which gave rise to distinct astroglial cell lines as previously reported. Indeed, the C8-B4 clone expresses classical microglial markers (MAC1, F4/80, 2-4G2) and appears to be derived from a committed microglial precursor since it does not express differentiation antigens present during the early stage of the monocytic lineage. This microglial clone expresses two characteristics not previously reported for microglial cell lines: it synthesizes the CD4 molecule and produces and releases large amounts of glutamate.

Retinal engineering: engrafted neural cell lines locate in appropriate layers.

A major question in central nervous system development, including the neuroretina, is whether migrating cells express cues to find their way and settle at specific locations. We have transplanted quail neuroretinal cell lines QNR/D, a putative amacrine or ganglion cell, and QNR/K2, a putative Müller cell into chicken embryo eyes. Implanted QNR/D cells migrate only to the retinal ganglion and amacrine cell layers and project neurites in the plane of retina; in contrast, QNR/K2 cells migrate through the ganglion and amacrine layers, locate in the inner nuclear layer, and project processes across the retina. These data show that QNR/D and QNR/K2 cell lines represent distinct neural cell types, suggesting that migrating neural cells express distinct address cues. Furthermore, our results raise the possibility that immortalized cell lines can be used for replacement of specific cell types and for the transport of genes to given locations in neuroretina.

A novel glial fibrillary acidic protein mRNA lacking exon 1.

Glial fibrillary acidic protein (GFAP) is the major intermediate filament protein in the mature astrocytes. We have assayed for the presence of GFAP mRNA gene in mouse tissues outside the nervous system. Nuclease S1 protection experiments show that RNAs lacking exon 1 are transcribed in bone marrow. From a mouse bone marrow cDNA library we isolated GFAP cDNAs which start in the 3' part of intron 1 and contain all the downstream GFAP exons. The new GFAP mRNAs, which we call GFAP gamma mRNAs, are already present in the brain at embryonic day 15 and in adult forebrain and cerebellum. Their presence in astrocytic cell lines suggests that astrocytes may be the site of in vivo expression of these mRNAs. In addition we have detected GFAP gamma mRNAs in mouse spleen. Furthermore in human an analogous GFAP mRNA containing the 3' part of intron 1 and lacking the exon 1 is also present in adult brain. These results suggest a new regulation of the GFAP gene expression.

Identification of new human myelin basic protein transcripts in the immune and central nervous systems.

Five new myelin basic protein (MBP) transcripts were identified which each have preferential sites of expression in adult human brain and immune system. They contain a novel 5' coding region which presents extensive sequence similarity to the mouse 0' region. One of these ribonucleic acid (RNA) species, HMBPR1, is found essentially, if not only, in haemopoietic and immune cells. Two alternatively spliced transcripts called MBP2a and c are only expressed in the central nervous system (CNS). In addition, the two other transcripts are expressed in both immune and nervous systems. Thus, the MBP locus can generate multiple forms of RNA, whose start sites and splicing depend on the tissue in which they are expressed. The presence of an MBP transcript specifically expressed in the adult human immune system suggests previously unsuspected functions related to the pathogenesis of multiple sclerosis.

CD4 expression in neurons of the central nervous system.

CD4 is a member of the Ig gene super family expressed on the surface of many thymocytes and of a subset of T lymphocytes. Human CD4 is the receptor for HIV envelope glycoprotein gp120. Human and mouse CD4 transcripts are expressed in human and mouse central nervous system (CNS), but no corresponding proteins have been reported yet. We have analyzed mRNA expression and carried out immunological experiments on adult mouse brain with probes specific for the long and short CD4 transcripts and with antibodies monospecific for mouse CD4. The main result of these experiments is that the full length CD4 transcript and the CD4 protein are expressed coordinately in neurons throughout the adult mouse brain. CD4 immunoreactivity is also present in brain small vessel walls, ependymal cells, and choroid plexus. The brain mouse CD4 protein is indistinguishable from the thymus protein. In addition, we show that neuronal cells in primary cultures from human fetal CNS are immunoreactive to human CD4 mAbs.

A novel cDNA corresponding to transcripts expressed in retina post-mitotic neurons.

The long term objective of this study is to isolate genes specifically expressed at the onset of neuronal cell cycle withdrawal. As an experimental paradigm we have used a quail neuroretinal cell clone (clone K2) immortalized by a thermosensitive mutant of Rous Sarcoma Virus. K2 cells proliferate at 36 degrees C but stop synthesizing DNA after a shift to 41.5 degrees C. We have constructed a cDNA library from K2 cells transferred to 41.5 degrees C and autosubtracted with RNAs from K2 cells maintained at 36 degrees C. This strategy has led to the isolation of cDNAs which recognize mRNAs expressed in quail neuroretina (NR) during development. We report here one of these cDNAs, cDNA QN1, that hybridizes with transcripts expressed in retina neurons, in parallel with their withdrawal from the cell cycle. QN1 ORF codes for a 138 kDa polypeptide corresponding to the protein observed in Western blot analysis. A role of QN1 product(s) on neuronal quiescence is suggested by the positive effect of an antisense oligonucleotide on DNA synthesis of K2 cells.

A new family of transcripts of the myelin basic protein gene: expression in brain and in immune system.

A cDNA clone (MBP2) corresponding to a novel mouse myelin basic protein (MBP) mRNA has been isolated from an adult mouse bone marrow cDNA library. It contains the MBP exons 1a-7 except exon 5. Using PCR experiments we have determined that this MBP2 mRNA belongs to a new MBP mRNA family initiated upstream from exon 1b. Their 5' end extends into exon 1a and/or the region 0' previously described. These mRNAs are generated by alternative splicing of the primary transcript involving excision of exon 1a, 1b, 2, 5, or 6. Thus, these new mRNAs are produced from a promoter(s) located upstream from the major promoter 1b. They are expressed in brain (at least from embryonic day 15), in bone marrow, and in other hemolymphopoietic tissues, particularly in macrophage cells. As their expression is not restricted to myelinating cells, the function of these novel MBP mRNAs and putative proteins might not be related to myelination.