Modulation of CD4 antigen on macrophages and microglia in rat brain.

Mononuclear phagocytes which express the HIV entry receptor CD4 have been implicated as possible sites of virus replication in brain, but there is still considerable uncertainty as to which cells in the CNS express CD4 Ag. Although it is not susceptible to HIV infection the rat provides a model to define expression of the CD4 Ag on MO in brain. We report that the CD4 epitopes W3/25 and OX35 are found only on monocytes, MO, microglia, and occasional lymphocytes and not on neurons, other glia, or endothelium. CD4 Ag levels are modulated during microglial differentiation, after reactivation after local inflammation, and within the intact blood brain barrier. MO and microglia also express other potential plasma membrane binding and entry sites for HIV viz Fc and complement receptors that are regulated independently of CD4.

The cellular receptor (CD4) of the human immunodeficiency virus is expressed on neurons and glial cells in human brain.

The expression of the CD4 antigen in normal human brain was investigated in parallel by immunohistochemical and Northern blot analyses. With anti-CD4 antibodies detecting different epitopes of the molecule, CD4+ neurons were defined in the cerebellum, thalamus, and pons. CD4+ glial cells were identified in the thalamus and pons. CD4-specific mRNA was detected in all three subareas and in the hippocampus, while other subareas were negative. The CD4+ cells were negative with anti-T cell antibodies (anti-CD2 and anti-CD8), as well as with antimonocyte antibodies (M-M 522 and M-M 42).

Intermediate filaments in nervous tissues.

Intermediate filaments have been isolated from rabbit intradural spinal nerve roots by the axonal flotation method. This method was modified to avoid exposure of axons to low ionic strength medium. The purified filaments are morphologically 75-80 percent pure. The gel electrophoretogram shows four major bands migrating at 200,000, 145,000, 68,000, and 60,000 daltons, respectively. A similar preparation from rabbit brain shows four major polypeptides with mol wt of 200,000 145,000, 68,000, and 51,000 daltons. These results indicate that the neurofilament is composed of a triplet of polypepetides with mol wt of 200,000, 145,000, and 68,000 daltons. The 51,000-dalton band that appears in brain filament preparations as the major polypeptide seems to be of glial origin. The significance of the 60,000- dalton band in the nerve root filament preparation is unclear at this time. Antibodies raised against two of the triplet proteins isolated from calf brain localize by immunofluorescence to neurons in central and peripheral nerve. On the other hand, an antibody to the 51,000-dalton polypeptide gives only glial staining in the brain, and very weak peripheral nerve staining. Prolonged exposure of axons to low ionic strength medium solubilizes almost all of the triplet polypeptides, leaving behind only the 51,000- dalton component. This would indicate that the neurofilament is soluble at low ionic strength, whereas the glial filament is not. These results indicate that neurofilaments and glial filaments are composed of different polypeptides and have different solubility characteristics.

Calcium-containing structures in vertebrate glial cells. Ultrastructural and microprobe analysis.

Electron probe microanalysis has revealed that vesicular or cisternal structures containing electron-dense material in frog ependymal glial cells contain deposits of calcium and phosphorus. The so-called "osmiophilic particles" in human astrocytes also contain calcium. It is suggested that these organelles are storage sites of calcium.

Growth cone localization of neural cell adhesion molecule on central nervous system neurons in vitro.

Ultrastructural analysis of colloidal gold immunocytochemical staining and immunofluorescence microscopy has been used to study the presence of neural cell adhesion molecule (NCAM) on the surface of neuronal growth cones. The studies were carried out with cultures of rat hypothalamic and ventral mesencephalic cells, using morphology and expression of tyrosine hydroxylase, neurofilaments, and glial fibrillary acidic protein as differential markers for neurons and glia. NCAM was found on all plasmalemmal surfaces of neurons including perikarya and neurites. The density of NCAM varied for different neurons growing in the same culture dish, and neurons had at least 25 times more colloidal gold particles on their plasmalemmal membranes than astroglia. Of particular interest in the present study was a strong labeling for NCAM on all parts of neuritic growth cones, including the lamellar and filopodial processes that extend from the tip of the axon. The density of NCAM was similar on different filopodia of the same growth cone. Therefore, in situations where homophilic (NCAM-NCAM) binding might contribute to axon pathfinding, a choice in direction is more likely to reflect differences in the NCAM content of the environment, rather than the distribution of NCAM within a growth cone. On the other hand, the variation in NCAM levels between single neurons in culture was significant and could provide a basis for selective responses of growing neurites.

A monoclonal antibody that blocks the activity of a neurite regeneration-promoting factor: studies on the binding site and its localization in vivo.

Work from several laboratories has identified a proteoglycan complex secreted by a variety of non-neuronal cells that can promote neurite regeneration when applied to the surface of culture dishes. Using a novel immunization protocol, a monoclonal antibody (INO) was produced that blocks the activity of this outgrowth-promoting factor (Matthew, W. D., and P. H. Patterson, 1983, Cold Spring Harbor Symp. Quant. Biol. 48:625-631). We have used the antibody to analyze the components of the active site and to localize the complex in vivo. INO binding is lost when the complex is dissociated; if its components are selectively reassociated, INO binds only to a complex containing two different molecular weight species. These are likely to be laminin and heparan sulfate proteoglycan, respectively. On frozen sections of adult rat tissues, INO binding is present on the surfaces of glial cells of the peripheral, but not the central, nervous system. INO also binds to the basement membrane surrounding cardiac and skeletal muscle cells, and binding to the latter greatly increases after denervation. In the adrenal gland and kidney, INO selectively reacts with areas rich in basement membranes, staining a subset of structures that are immunoreactive for both laminin and heparan sulfate proteoglycan. In general, the outgrowth-blocking antibody binds to areas known to promote axonal regeneration and is absent from areas known to lack this ability. This suggests that this complex, which is active in culture, may be the physiological substrate supporting nerve regeneration in vivo.

The distribution of tau in the mammalian central nervous system.

We have determined the biochemical and immunocytochemical localization of the heterogeneous microtubule-associated protein tau using a monoclonal antibody that binds to all of the tau polypeptides in both bovine and rat brain. Using immunoblot assays and competitive enzyme-linked immunosorbent assays, we have shown tau to be more abundant in bovine white matter extracts and microtubules than in extracts and microtubules from an enriched gray matter region of the brain. On a per mole basis, twice-cycled microtubules from white matter contained three times more tau than did twice-cycled microtubules from gray matter. Immunohistochemical studies that compared the localization of tau with that of MAP2 and tubulin demonstrated that tau was restricted to axons, extending the results of the biochemical studies. Tau localization was not observed in glia, which indicated that, at least in brain, tau is neuron specific. These observations indicate that tau may help define a subpopulation of microtubules that is restricted to axons. Furthermore, the monoclonal antibody described in this report should prove very useful to investigators studying axonal sprouting and growth because it is an exclusive axonal marker.

Ultrastructural localization of glial fibrillary acidic protein in mouse cerebellum by immunoperoxidase labeling.

Glial fibrillary acidic protein was localized at the electron microscope level in the cerebellum of adult mice by indirect immunoperoxidase histology. In confirmation of previous studies at the light microscope level, the antigen was detectable in astrocytes and their processes, but not in neurons or their processes, or in oligodendroglia. Astrocytic processes were stained in white matter, in the granular layet surrounding synaptic glomerular complexes, and in the molecular layer in the form of radially oriented fibers and of sheaths surrounding Purkinje cell dendrites. Astrocytic endfeet impinging on meninges and perivascular membranes were also antigen positive. In astrocytic perikarya and processes, the immunohistochemical reaction product appears both as a diffuse cytoplasmic label and as elongated strands, which by their distribution and frequency could be considered glial filaments.

Posttranslational modifications of alpha-tubulin: acetylated and detyrosinated forms in axons of rat cerebellum.

The distribution of acetylated alpha-tubulin in rat cerebellum was examined and compared with that of total alpha-tubulin and tyrosinated alpha-tubulin. From immunoperoxidase-stained vibratome sections of rat cerebellum it was found that acetylated alpha-tubulin, detectable with monoclonal 6-11B-1, was preferentially enriched in axons compared with dendrites. Parallel fiber axons, in particular, were labeled with 6-11B-1 yet unstained by an antibody recognizing tyrosinated alpha-tubulin, indicating that parallel fibers contain alpha-tubulin that is acetylated and detyrosinated. Axonal microtubules are known to be highly stable and the distribution of acetylated alpha-tubulin in other classes of stable microtubules suggests that acetylation and possibly detyrosination may play a role in the maintenance of stable populations of microtubules.

Characterization of the myosin-phosphorylating system in normal murine astrocytes and derivative sv40 wild-type and A-mutant transformant.

Myosin and myosin light-chain kinase have been isolated and characterized from small quantities of normal and SV40-transformed, murine astrocytic neuroglial cells in culture and from intact normal mouse brain. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the astrocyte myosins revealed a heavy chain of 200,000 daltons and two light chains of 20,000 and 15,000 daltons. These myosins are similar to other cytyplasmic myosins. The astrocyte 20,000-dalton light chain can be phosphorylated by an endogenous myosin light-chain kinase which has properties similar to those of the myosin light-chain kinase found in human platelets. No differences were detected in either the astrocyte myosins or myosin light-chain kinases between (a) the normal and transformed cells, (b) the transformed cells grown at the permissive and nonpermissive temperatures, or (c) the SV40 wild-type and A-mutant transformants.

Analysis of the calcium-modulated proteins, S100 and calmodulin, and their target proteins during C6 glioma cell differentiation.

We have analyzed the levels, subcellular distribution, and target proteins of two calcium-modulated proteins, S100 and calmodulin, in differentiated and undifferentiated rat C6 glioma cells. Undifferentiated and differentiated C6 cells express primarily the S100 beta polypeptide, and the S100 beta levels are four-fold higher in differentiated compared to undifferentiated cells. Double fluorescent labeling studies of undifferentiated cells demonstrated that S100 beta staining localized to a small region of the perinuclear cytoplasm and colocalized with the microtubule organizing center and Golgi apparatus. Analysis of differentiated C6 cells demonstrated that S100 beta distribution and S100 beta-binding protein profile changed significantly upon differentiation. In addition, the brain-specific isozyme of one S100-binding protein, fructose-1,6-bisphosphate aldolase C, can be detected in differentiated but not undifferentiated C6 cells. While changes in the subcellular distribution of calmodulin were not observed during differentiation, calmodulin levels and calmodulin-binding protein profiles did change. Altogether these data suggest that S100 beta and calmodulin regulate different processes in glial cells and that the regulation of the expression, subcellular distribution, and target proteins of S100 beta and calmodulin during differentiation is a complex process which involves multiple mechanisms.

A phosphatidylinositol-linked peanut agglutinin-binding glycoprotein in central nervous system myelin and on oligodendrocytes.

Here we report the isolation and initial biochemical characterization of a 120-kD peanut agglutinin-binding glycoprotein from the adult human central nervous system (CNS), which is anchored to membranes through a phosphatidylinositol linkage. Myelin incubated with phosphatidylinositol-specific phospholipase C released the protein as a soluble polypeptide of 105 kD, which was isolated with peanut agglutinin-agarose affinity chromatography. The protein was found to be highly glycosylated. The protein appears to be confined to the CNS, where its developmental expression is region specific and parallels myelination. It is in greater quantity in white matter than in gray matter and it is in isolated human CNS myelin. Furthermore, ovine oligodendrocytes in culture contain the protein on their surfaces and release it into the supernatant as a soluble 105-kD form. We call this protein the oligodendrocyte-myelin protein.

Vasoactive intestinal polypeptide: abundant immunoreactivity in neural cell lines and normal nervous tissue.

Vasoactive intestinal polypeptide immunoreactivity is present in high concentrations in clonal lines of neuronal and glial origin. The central nervous system and sympathetic ganglia are also rich in the peptide. The findings suggest that this peptide, hitherto thought limited to the gastrointestinal tract, is widely distributed in neural tissue and may have broad physiological significance.

Expression of glial fibrillary acidic protein in immature oligodendroglia.

In the human fetal spinal cord at 15 to 16 weeks, glial fibrillary acidic protein (GFAP) was demonstrated within the cytoplasm and processes of cells having the cytological, ultrastructural, and immunocytochemical features of oligodendrocytes--including processes that extend into and contribute to the formation of myelin sheaths. By 17 to 18 weeks, however, GFAP immunoreactivity was no longer evident within such cells. Thus GFAP is expressed by myelin-forming oligodendroglia early in their development.

Cell and fiber-type distribution of dystrophin.

Duchenne muscular dystrophy is the result of dystrophin deficiency. We have determined the cell types likely to express the pathogenic effects of this neuromuscular disease by determining the pattern of dystrophin expression in normal cells. We find that all physiological types of muscle cells express dystrophin at similar levels, and that the dystrophin content of various tissues correlates with the myogenic cell population of each tissue. The dystrophin content of brain and spinal cord, however, is found not to correlate with any type of muscle cell, and it is suggested that neurons express dystrophin. The potential involvement of striated muscle fibers, the vasculature, and the nervous system in the etiology of Duchenne muscular dystrophy makes it likely that the disease is a complex disorder of combined pathogenesis. We also find that the dystrophic chicken does not represent an animal model for dystrophin deficiency.

A clonal analysis of glial lineages in neonatal forebrain development in vitro.

Retrovirus-mediated gene transfer combined with triple immunostaining for astro- and oligodendroglial markers (antibodies to glial fibrillary acidic protein, GD3 ganglioside, and galactocerebroside, and the O4 antibody) was used to study clonal aspects of glial lineage in primary cultures of the neonatal rat striatum. We found two major clonal populations: astrocyte clones containing GFAP+, but GD3-, O4-, and GC- cells, and oligodendrocyte clones containing cells expressing various combinations of GD3, O4, and GC, with rare GFAP+ cells. These results indicate that astrocytes and oligodendrocytes belong to separate lineages in forebrain postnatal development.

Lineage, migration, and morphogenesis of longitudinal glia in the Drosophila CNS as revealed by a molecular lineage marker.

Previous studies described three different classes of glial cells in the developing CNS of the early Drosophila embryo that prefigure and ensheath the major CNS axon tracts. Among these are 6 longitudinal glial cells on each side of each segment that overlie the longitudinal axon tracts. Here we use transformant lines carrying a P element containing a 130 bp sequence from the fushi tarazu gene in front of the lacZ reporter gene to direct beta-galactosidase expression in the longitudinal glia. Using this molecular lineage marker, we show that 1 of the "neuroblasts" in each hemisegment is actually a glioblast, which divides once symmetrically, in contrast to the typical asymmetric neuroblast divisions, producing 2 glial cells, which migrate medially and divide to generate the 6 longitudinal glial cells. As with neuroblasts, mutations in Notch and other neurogenic genes lead to supernumerary glioblasts. The results indicate that the glioblast is similar to other neuroblasts; however, the positionally specified fate of this blast cell is to generate a specific lineage of glia rather than a specific family of neurons.

A type II keratin is expressed in glial cells of the goldfish visual pathway.

The predominant intermediate filament proteins of the goldfish visual pathway consist of neuronal and non-neuronal isoelectric variants (58 kd). We have isolated a cDNA clone for the glial intermediate filament protein (ON3) from an optic nerve expression library. The predicted amino acid sequence of this clone reveals that it codes for a type II keratin representing the goldfish equivalent of mammalian keratin K8. K8 has been shown to be associated with embryogenesis and development. Unlike the mammalian visual system, the goldfish visual pathway displays a remarkable capacity for functional regeneration. The expression of K8, a protein not usually expressed in glial cells but shown to be associated with development, in the goldfish optic nerve may be involved with the processes of growth and regeneration in the goldfish visual pathway.

Apolipoprotein E associated with astrocytic glia of the central nervous system and with nonmyelinating glia of the peripheral nervous system.

The plasma protein apolipoprotein (apo) E is an important determinant of lipid transport and metabolism in mammals. In the present study, immunocytochemistry has been used to identify apo E in specific cells of the central and peripheral nervous systems of the rat. Light microscopic examination revealed that all astrocytes, including specialized astrocytic cells (Bergmann glia of the cerebellum, tanycytes of the third ventricle, pituicytes of the neurohypophysis, and Müller cells of the retina), possessed significant concentrations of apo E. In all of the major subdivisions of the central nervous system, the perinuclear region of astrocytic cells, as well as their cell processes that end on basement membranes at either the pial surface or along blood vessels, were found to be rich in apo E. Extracellular apo E was present along many of these same surfaces. The impression that apo E is secreted by astrocytic cells was confirmed by electron microscopic immunocytochemical studies, which demonstrated the presence of apo E in the Golgi apparatus. Apo E was not present in neurons, oligodendroglia, microglia, ependymal cells, and choroidal cells. In the peripheral nervous system, apo E was present within the glia surrounding sensory and motor neurons; satellite cells of the dorsal root ganglia and superior cervical sympathetic ganglion as well as the enteric glia of the intestinal ganglia were reactive. Apo E was also present within the non-myelinating Schwann cells but not within the myelinating Schwann cells of peripheral nerves. These results suggest that apo E has an important, previously unsuspected role in the physiology of nervous tissue.

Subcellular localization of an intermediate filament protein and its mRNA in glial cells.

Eucaryotic mRNAs are generally localized in the cell body, where most protein synthesis occurs. We have found that mRNAs encoding the glial intermediate filament protein are spatially distributed in the glial cell cytoplasm close to the location of the glial filaments. Whereas the glial filament protein mRNA was located predominantly in the distal process, actin mRNA was found almost exclusively in the apical portion of the glial cell. This pattern of mRNA localization might provide a mechanism for synthesis of proteins in specific subcellular compartments by mRNA translation locally.