BBS4 protein has basal body/ciliary localization in sensory organs but extra-ciliary localization in oligodendrocytes during human development.

Bardet-Biedl syndrome protein 4 (BBS4) localization has been studied in human embryos/fetuses from Carnegie stage 15 to 37 gestational weeks in neurosensory organs and brain, underlying the major clinical signs of BBS. We observed a correlation between the differentiation of the neurosensory cells (hair cells, photoreceptors, olfactory neurons) and the presence of a punctate BBS4 immunostaining in their apical cytoplasm. In the brain, BBS4 was localized in oligodendrocytes and myelinated tracts. In individual myelinated fibers, BBS4 immunolabelling was discontinuous, predominantly at the periphery of the myelin sheath. BBS4 immunolabelling was confirmed in postnatal developing white matter tracts in mouse as well as in mouse oligodendrocytes cultures. In neuroblasts/neurons, BBS4 was only present in reelin-expressing Cajal-Retzius cells. Our results show that BBS4, a protein of the BBSome, has both basal body/ciliary localization in neurosensory organs but extra-ciliary localization in oligodendrocytes. The presence of BBS4 in developing oligodendrocytes and myelin described in the present paper might attribute a new role to this protein, requiring further investigation in the field of myelin formation.

Oligodendrocyte gene expression is reduced by and influences effects of chronic social stress in mice.

Oligodendrocyte gene expression is downregulated in stress-related neuropsychiatric disorders, including depression. In mice, chronic social stress (CSS) leads to depression-relevant changes in brain and emotional behavior, and the present study shows the involvement of oligodendrocytes in this model. In C57BL/6 (BL/6) mice, RNA-sequencing (RNA-Seq) was conducted with prefrontal cortex, amygdala and hippocampus from CSS and controls; a gene enrichment database for neurons, astrocytes and oligodendrocytes was used to identify cell origin of deregulated genes, and cell deconvolution was applied. To assess the potential causal contribution of reduced oligodendrocyte gene expression to CSS effects, mice heterozygous for the oligodendrocyte gene cyclic nucleotide phosphodiesterase (Cnp1) on a BL/6 background were studied; a 2 genotype (wildtype, Cnp1+/- ) × 2 environment (control, CSS) design was used to investigate effects on emotional behavior and amygdala microglia. In BL/6 mice, in prefrontal cortex and amygdala tissue comprising gray and white matter, CSS downregulated expression of multiple oligodendroycte genes encoding myelin and myelin-axon-integrity proteins, and cell deconvolution identified a lower proportion of oligodendrocytes in amygdala. Quantification of oligodendrocyte proteins in amygdala gray matter did not yield evidence for reduced translation, suggesting that CSS impacts primarily on white matter oligodendrocytes or the myelin transcriptome. In Cnp1 mice, social interaction was reduced by CSS in Cnp1+/- mice specifically; using ionized calcium-binding adaptor molecule 1 (IBA1) expression, microglia activity was increased additively by Cnp1+/- and CSS in amygdala gray and white matter. This study provides back-translational evidence that oligodendrocyte changes are relevant to the pathophysiology and potentially the treatment of stress-related neuropsychiatric disorders.

The myelin proteolipid protein gene modulates apoptosis in neural and non-neural tissues.

Mutations of the myelin proteolipid protein gene (Plp) are associated with excessive programmed cell death (PCD) of oligodendrocytes. We show for the first time that PLP is a molecule ubiquitously expressed in non-neural tissues during normal development, and that the level of native PLP modulates the level of PCD. We analyze three non-neural tissues, and show that native PLP is expressed in trophoblasts, spermatogonia, and cells of interdigital webbing. The non-neural cells that express high levels of native PLP also undergo PCD. The level of PLP expression modulates the level of PCD because mice that overexpress native PLP have increased PCD and mice deficient in PLP have decreased PCD. We show that overexpression of native PLP causes a dramatic acidification of extracellular fluid that, in turn, causes increased PCD. These studies show that the level of native PLP modulates the amount of PCD during normal development via a pH-dependent mechanism.

Oligodendrocytes in female carriers of the jimpy gene make more myelin than normal oligodendrocytes.

The female carrier of the jimpy (jp) gene is a model system to study the plasticity of neuroglial cells and the mechanisms they use to compensate for a temporary deficit in myelin. Myelin in the female carriers is reduced 30-40% during the first postnatal month but is normal in adults. We hypothesized that the number of oligodendrocytes (OLs) in the female carriers is increased, based upon previous data showing OL proliferation is increased but the number of dying OLs is only slightly elevated in development. To test this hypothesis, antibodies to carbonic anhydrase (CA)II, an OL-specific marker, were used to quantify the number of OLs in the spinal cords of 1-month-old and adult female carriers. Contrary to expectations, the number of OLs is significantly reduced in the dorsal funiculus and grey matter by 21% in adult female carriers compared to controls. A reduction of lesser magnitude is present in the 1-month-old animals. Electron microscopic montages prepared from normal and carrier dorsal funiculus were used to count total numbers of glia. Ultrastructural quantification shows a similar reduction in the number of OLs and confirms the validity of the CAII immunostaining as a means to quantify OLs. These data show that there are 21% fewer OLs in the central nervous system (CNS) of adult female carriers but normal amounts of myelin. Presumably, some OLs in the carrier CNS are maintaining more myelin than their counterparts in normal CNS would. These findings demonstrate that (1) a reduction in number of OLs does not necessarily involve a reduction in the amount of myelin, and (2) OLs have considerable flexibility in the amount of myelin they can make.

Radial glia and astrocytes in developing and adult telencephalon of the lizard Gallotia galloti as revealed by immunohistochemistry with anti-GFAP and anti-vimentin antibodies.

The development of radial glia and astrocytes in the telencephalon of the lizard Gallotia galloti was studied by immunohistochemistry with anti-vimentin and anti-GFAP antibodies. Vimentin appears at embryonic stage 32 (E32) in the proliferative zone of the lateral ventricle and subpial end-feet in the marginal zone. At E34-35 the staining intensity for vimentin in all radial glia is maximal. It then decreases and disappears in most structures in adult animals. GFAP appears at E35 in the end-feet in the marginal zone and its intensity increases until adulthood, particularly in radial and sinuous fibers and in fibers that originate from the sulci and invade the ventral striatum and the septum. In contrast, the reaction is weak in the cortex, in the anterior dorso-ventricular ridge, and in the amygdala nuclei. Radial glia is still present in the adult, and the composition of its intermediate filaments changes during development from vimentin to GFAP. No GFA-positive cell bodies except those of ependymal glia were detected in telencephalon.

Glial fibrillary acidic protein and vimentin immunohistochemistry in the developing and adult midbrain of the lizard Gallotia galloti.

The distribution of glial fibrillary acidic protein (GFAP)- and vimentin-containing cells was studied by immunohistochemistry in the midbrain of the lizard Gallotia galloti. At embryonic stage 32 (E32), vimentin immunoreactivity appeared first in cell bodies located in the ventricular walls, in radial fibers, and subpial end-feet and increased in these structures until E34/E35. Faint GFAP immunoreactivity gradually appeared in the same structures between E34 and E37, and this increased until adulthood, whereas vimentin immunoreactivity decreased after E35, becoming limited to a few end-feet and fibers in the adult, mainly in the tegmentum. Thus, in developing Gallotia midbrain a shift from vimentin-containing to GFAP-containing intermediate filaments begins around E36 or E37. At E40, in addition to the cell bodies in the ependymal area, dispersed GFAP-positive cells, possibly immature astrocytes appeared. These cells showed the same shift. In the adult lizard, GFAP-positive radial glia are still present and coexist with GFAP-positive astrocytes, which are prefentially located in the marginal optic tract and the oculomotor nuclei, but are absent in the fasciculus longitudinalis medialis. Optic tectum, pretectum, tegmentum, and isthmic nuclei are the areas richest in GFAP-positive radial fibers: these were much less abundant in the deep mesencephalic nuclei. Thus, in this lizard, GFAP-positive astrocytes display a clear cut regional distribution: they are present in mesencephalon, whereas they are absent in telencephalon.

Immunoelectron microscopy of alpha 2-glycoprotein. An astrocyte-specific antigen.

The cellular and fine structural localization of the soluble brain-specific acidic alpha 2-glycoprotein was investigated using the indirect immunohistochemical method. The electron microscope was used to unambiguously identify cells containing the antigen. A single type of cell, the astrocyte, was found to be labelled with specific antisera directed against alpha 2-glycoprotein. Immunoperoxidase reaction product was found in astrocyte perikarya, their processes and perivascular end feet. It was found to be apparently associated with the cytoplasmic surface of mitochondria, reticular membranes and the plasma membrane. No specific labelling of neurones, oligodendrocytes, myelin or capillary endothelial cells was observed. The data is discussed in relation to the immunological properties of alpha 2-glycoprotein already reported.

Antigenic conservation of brain guanylate cyclase during evolution.

A comparative study of brain guanylate cyclase from different animal species (including man, bird, fish and amphibian) has been performed using a specific antibody directed against soluble rat brain guanylate cyclase. Analyses were performed on supernatant fractions by the double-immunodiffusion test, by the protein blotting technique after SDS-polyacrylamide gel electrophoresis and by analytical isoelectric focusing on agarose allowing specific immunodetection of isoelectric patterns. Membrane-bound guanylate cyclase from rat brain and soluble guanylate cyclase from several rat tissues cross-reacted with the antibody. All the brain enzymes tested were found to be identical by double-immunodiffusion. The electrophoretic and isoelectrophoretic profiles of the different brain guanylate cyclases were found to exhibit many common features with some differences between mammalian and non-mammalian enzymes. In human brain, guanylate cyclase has been localized in glial and neuronal cells by immunohistochemistry. The results demonstrate that guanylate cyclase has been well conserved during the course of evolution and are consistent with the involvement of guanylate cyclase and cyclic GMP in basic cellular function.

A monoclonal antibody recognizing subpopulations of neurones in mouse brain.

A monoclonal antibody, designated C138, was raised against a particulate fraction from young postnatal mouse cerebellum. In sections from adult mouse brain and cerebellum, this antibody stained a sub-population of neurons. Cell bodies of large neurones and fibre tracts were negative in all brain regions examined, whereas neuropil was heavily labelled. Immune-electron microscopy confirmed the specificity of the antibody for certain types of neurones and indicated that the antigen may be associated with microtubular structures. Immunofluorescence studies on dissociated cerebellar cultures showed that the antigen was expressed inside all tetanus toxin-positive neurones but not by non-neuronal cells.

A developmentally modified neurone-specific marker in rodent cerebellum.

Out of several monoclonal antibodies secreted by hybridomas resulting from the fusion of a mouse myeloma cell line with spleen cells from mice immunized with cerebellar membranes from 12 day old rats, one, called 11.9, produced an unusual immunolabelling pattern when tested on sections of rat cerebellum. The cerebellar distribution of the antigenic sites recognized by this antibody using an immunoperoxidase technique at the optical and ultrastructural levels is described in detail in this report. The immunoreaction product was found in the adult rat to be associated with the microtubules and the zone immediately beneath the plasma membrane of parallel fibres. In young animals the density of immunostaining appears to be higher than in the adult, and the staining is detectable in addition in the perikaryal cytoplasm of granule cells. Biochemical studies using the Western immunoblot technique demonstrate that the antigens consist of two polypeptides of molecular weights 120 and 185 kD. The possible relation of the antigens to cytoskeletal structures is discussed and the labelling pattern is compared with that produced by other known monoclonal antibodies.

Cellular localization of the brain specific alpha 2-glycoprotein in rat cerebellum: an immunohistological study.

The cellular localization of the brain-specific, soluble, acidic alpha 2-glycoprotein was studied in rat cerebellum by using the immunoperoxidase technique at the light-and electron-microscopy levels with monospecific immune serum directed against this glycoprotein. Only astrocytes, their processes, and their end feet (subpial or perivascular) contained heavy immunoperoxidase reaction product. Cerebellar neurones, oligodendrocytes, myelin and blood vessel endothelia did not stain. Thus it appears that alpha 2-glycoprotein is an astrocyte marker.

Characterization of ependymal cells in hypothalamic and choroidal primary cultures.

Long-term primary cultures derived from fetal mouse or rat hypothalamus and choroid plexus were obtained in serum-supplemented and chemically defined media. In order to identify and characterize cell types growing in our cultures, we used morphological features provided by phase-contrast, scanning and transmission electron microscopy. Immunological criteria were recognized, using antibodies against intermediate filament proteins (vimentin, gliofibrillar acid protein, cytokeratin, desmin, neurofilament proteins), actin, myosin, ciliary rootlets, laminin and fibronectin in single or double immunostaining, and monoclonal antibodies known to detect epitopes of ependymal or endothelial cells. Minor cell types such as astrocytes, fibroblasts and endothelial cells were distinguished. Ependymal cells, which exceeded 75% of the cultured cells, were identified by their cell shape and epithelial organization revealed by phase-contrast and transmission electron microscopy, by their apical differentiation evidenced by scanning and transmission electron microscopy, and by certain molecular markers (e.g. gliofibrillar acid or ciliary rootlet proteins) detected by immunofluorescence. Four ependymal cell types were recognized: choroidal ependymocytes, ciliated and unciliated ependymal cells, and tanycytes. All these cultured ependymal cell types showed a remarkable resemblance to in vivo ependymocytes, in terms of marker expression and ultrastructural features.

A new brain cell surface glycoprotein identified by monoclonal antibody.

Of 207 monoclonal antibodies produced against cultured mouse cerebellar cells, 16 reacted with cerebellar cell surfaces and 4 reacted with glycoproteins. One of them, called an anti-BSP-3 (Brain cell Surface Protein-3) defines a 48,000 molecular weight protein which can be iodinated at the surface of cultured cerebellar cells. Lectin-binding and sugar incorporation studies established the glycoprotein nature of the antigen. Astroglia (glial fibrillary acidic protein-positive cells) in primary cerebellar cultures were labelled intensely for this antigen by the indirect immunofluorescence method while neuronal cells and their processes were more weakly labelled. Fibronectin-positive cells were negative for BSP-3. In cerebellar sections using the immunoperoxidase method at both the optical and electron microscope levels, the difference in staining intensity between astrocytes and neuronal cells was not significant: in Purkinje cells and in the large neurones present in the deep cerebellar nuclei the immunoperoxidase percipitate was confined to the plasma, membrane while in both astrocytes and granule cells cytoplasmic labelling was also observed. Oligodendrocytes do not appear to react with the anti-BSP-3 monoclonal antibody; neither do endothelial or leptomeningeal cells. The availability of a monoclonal antibody produced by a stable hybridoma line will be a powerful tool in attempts to purify the BSP-3 antigen and to elucidate its function.

Double labeling immunohistochemical technique provides evidence of the specificity of glial cell markers.

Sequential treatment of rat cerebellar slices with two antisera, each specific for a different glial cell marker, revealed, by indirect immunofluroescence combined with the immunoperoxidase method on the same tissue section, that carbonic anhydrase isoenzyme II (CA II) is exclusively localized in oligodendrocytes while the glial fibrillary acidic protein (GFA) is, as well established, a specific marker for astrocytes.

Oligodendrocyte cell surface recognized by a novel monoclonal antibody specific to sulfatide.

A rat monoclonal antibody (OL-1) was obtained by in-vitro immunization of rat spleenocytes with paraformaldehyde-fixed primary cultured glial cells derived from newborn rat brain and subsequent fusion with a rat myeloma cell line. The antibody secreted by the hybridoma immunostains live rat and mouse oligodendrocytes in primary and secondary cultures. The antibody binds specifically to oligodendrocytes and myelin structures in-situ. Radioimmunolabelling assays with a number of purified glycolipids offer thin layer chromatography separation show that OL-1 antibody binds strongly to sulfatide and to a lesser extent to galactosyl diglyceride.

Molecular heterogeneity and structural evolution during cerebellar ontogeny detected by monoclonal antibody of the mouse cell surface antigen BSP-2.

The monoclonal antibody anti-BSP-2 defines a set of glycoproteins present on the neuronal cell surface in dissociated mouse cerebellar cultures and on neurons and astrocytes in sections of the mouse cerebellum. This antibody was used in the present study to characterize the antigens recognized in cerebellar cultures and in the developing and adult mouse cerebellum in vivo. In extracts from cerebellar cultures and from late postnatal or adult cerebellum, the anti-BSP-2 antibody reacted with a triplet of glycosylated polypeptide chains of 180,000, 140,000 and 120,000 mol. wt. Early postnatal cerebellum contained a different form of BSP-2 antigen which migrated as one broad or several closely spaced diffuse bands in the 190,000-250,000 mol. wt. region of SDS polyacrylamide gels. During cerebellar ontogeny, the adult pattern emerged gradually between postnatal days 5 and 13. The cellular expression of the BSP-2 antigen was studied by immunohistochemistry on sections of the developing cerebellum. At postnatal day 3, the antigen was found mainly on cell bodies and fibers of the Bergmann glia and on astrocytes of the granular layer. Immature granule cells of the outer zone of the external granular layer lacked the antigen, but they appeared to acquire the antigen during their migration to the internal granular layer. At postnatal day 13, the immunofluorescence pattern was not different from the one seen in the adult. These results suggest that the neonatal 190,000-250,000 mol. wt. form of BSP-2 may at least in part be expressed by astroglial cells and they show a close correlation between the emergence of the adult forms of the antigen and the appearance of labeled granule cells in the internal granular layer. In vitro degradation implying cleavage of sialic acid residues, but probably also proteolysis and/or cleavage of different glycans converted the neonatal form of BSP-2 into the triplet pattern and ultimately into a p120 component. Neuraminidase digestion of the adult antigens produced small molecular weight shifts without converting one band into the other, but endogenous enzyme activities were capable of degrading the p180 and p140 bands by converting them into the p120 protein. Our findings support the idea that distinct, but structurally similar surface glycoproteins created by post-translational modifications from a common precursor molecule may be expressed by different cell types or during different developmental stages. As shown by sequential immunoprecipitation experiments, BSP-2 and the rat neuronal membrane protein D2 may belong to the same family of surface glycoproteins.

Glutamine synthetase and energy metabolism enzymes in cultivated chick neurons and astrocytes: modulation by serum and hydrocortisone.

Primary cultures of astroglial cells and of neurons obtained from chick embryos were grown in culture medium with and without serum added. The expression of glutamine synthetase (GS) in the cultured nerve cells was investigated immunocytochemically and biochemically. The cellular localization of GS in cerebellar tissue sections and in cerebral cortex of chick embryos was investigated by immunohistochemical staining. In tissue sections the enzyme is only present in astrocytes and their processes; neurons and their structures do not express the enzyme. In contrast, in pure neuronal primary cultures, a high level of GS was detected by biochemical and immunochemical methods. Thus, our results clearly indicate the presence of GS in pure neuronal cell cultures and its absence in this type of cells in vivo. Removal of serum from the culture medium enhanced GS levels in primary astrocyte cultures, but was without effect on GS activity in neurons. Addition of calf serum to the culture medium induces a two-fold increase of cellular lactate dehydrogenase (LDH) activity in neurons by increasing specifically the M subunit containing isoenzymes. The sensitivity of chick astroglial cells and neurons toward the GS inducing effect of hydrocortisone and modulation of its effect by serum was also investigated. Differences in the sensitivity of the two types of nerve cells in culture toward the GS inducing effect of hydrocortisone, and the effect of serum could be demonstrated.

Abnormal Ca(2+) regulation in oligodendrocytes from the dysmyelinating jimpy mouse.

Jimpy (jp) is a point mutation in the gene on the X chromosome which codes for the major myelin proteolipid protein. Most oligodendrocytes (OLs) in the jp mouse undergo cell death at the time when they should be actively myelinating. Loss of mature OLs results in severe CNS dysmyelination. Dying jp OLs have the morphology of apoptotic cells but it is not clear how the mutation activates biochemical pathways which lead to programmed death of OLs in jp CNS. There is compelling evidence from a number of systems that high levels of intracellular Ca(2+) ([Ca2+]i) can activate downstream processes which result in both apoptotic and necrotic cell death. To determine whether [Ca2+](i) dysregulation might be involved in the death of jp OLs, we used ratiometric imaging to determine levels of [Ca2+](i) in OLs cultured from jp and normal CNS and in immortalized cell lines derived from jp and normal OLs. Immortalized jp OLs and OLs isolated directly from jp brain both showed a similar elevation in [Ca2+](i) ranging from 60% to 150% over control values. A higher baseline [Ca2+](i) in jp OLs might increase their vulnerability to other insults due to abnormal protein processing or changes in signaling pathways which act as a final trigger for cell death.

Postnatal development of rat cerebellum: massive and transient accumulation of concanavalin A binding glycoproteins in parallel fiber axolemma.

Modifications of protein-bound sugars during postnatal development of rat cerebellum were studied. Glycoprotein-bound mannose accumulates, in the particulate fractions, at an earlier age than the bulk of glycoprotein sugar. This corresponds to a transient and massive accumulation of glycoproteins which bind to Concanavalin A (Con A). These glycoproteins were localized by using fluorescent Con A and the horseradish peroxidase-Con A method. Cerebellar white matter and the molecular layer bind massive amounts of Con A. The binding in the molecular layer is transient. It follows the same time course as the Con A-binding glycoproteins of particulate fractions, and it is largely confined to the axolemma of parallel fibers. Only growing or newly formed parallel fibers bind Con A. The disappearance of the binding is simultaneous with the maturation of parallel fibers and their synapse formation. These phenomena can be related to fiber growth and maturation and, also, to synapse formation. The possibility of a specific role of Con A-binding glycoproteins is discussed.

Developing rat cerebellum: Glutamine and glutamate influx correlated to the cellular distribution of glutamine synthetase.

Glutamate has been suggested to be the neurotransmitter of the granule cells in cerebellar cortex. Autoradiographic studies using very low concentrations (1µM range) of 2-3-[(3)H]l-glutamate or 2-3-[(3)H]l-glutamine have shown that both amino acids were preferentially taken up in the molecular layer of the cerebellar cortex of adult rats, but [(3)H]Glu accumulated essentially in glial cells, while Gln did not show a cellular preference (de Barry et al., Neuroscience 7,1289-1297,1982). In this paper we show that during development the preferential accumulation of [(3)H]Glu and [(3)H]Gln are the same as in the adult but, in addition, at young ages (7-10 days) [(3)H]Gln accumulated in replicating cells of the external granular layer (EGL). The absence of glutamine synthetase in the EGL cells suggests that this accumulation of exogenous Gln might be used for the synthesis of purines and pyrimidines required for the active multiplication of these cells and is not correlated to neurotransmission. The metabolism of [(3)H]Gln was slow at young ages and changed during development. The metabolism of the Glu taken up was constant throughout development which is consistent with the hypothesis that high affinity Glu uptake is mainly a glial cell property and that the increasing accumulation rate during development reflects glial maturation.