Proteome analysis of primary neurons and astrocytes from rat cerebellum

Neurons and astrocytes are predominant cell types in brain and have distinguished morphological and functional features. Although several proteomics studies were carried out on the brain, work on individual brain cells is limited. Generating individual proteomes of neurons and astrocytes, however, is mandatory to assign protein expression to cell types rather than to tissues. We aimed to provide maps of rat primary neurons and astrocytes using two-dimensional gel electrophoresis with subsequent in-gel digestion, followed by MALDI-TOF/TOF. 428 protein spots corresponding to 226 individual proteins in neurons and 406 protein spots representing 228 proteins in astrocytes were unambiguously identified. Proteome data include proteins from several cascades differentially expressed in neurons and astrocytes, and specific expressional patterns of antioxidant, signaling, chaperone, cytoskeleton, nucleic acid binding, proteasomal, and metabolic proteins are demonstrated. We herein present a reference database of primary rat primary neuron and astrocyte proteomes and provide an analytical tool for these structures. The concomitant expressional patterns of several protein classes are given and potential neuronal and astrocytic marker candidates are presented.     

Neuromodulin (GAP43): a neuronal protein kinase C substrate is also present in 0-2A glial cell lineage. Characterization of neuromodulin in secondary cultures of oligodendrocytes and comparison with the neuronal antigen

Neuromodulin (also called GAP43, G50, F1, pp46), a neural-specific calmodulin binding protein, is a major protein kinase C substrate found in developing and regenerating neurons. Here, we report the immunocytochemical characterization of neuromodulin in cultured 0-2A bipotential glial precursor cells obtained from newborn rat brain. Neuromodulin is also present in oligodendrocytes and type 2 astrocytes (stellate-shaped astrocytes), which are both derived from the bipotential glial 0-2A progenitor cells, but is absent of type 1 astrocytes (flat protoplasmic astrocytes). These results support the hypothesis of a common cell lineage for neurons and bipotential 0-2A progenitor cells and suggest that neuromodulin plays a more general role in plasticity during development of the central nervous system. The expression of neuromodulin in secondary cultures of newborn rat oligodendrocytes and its absence in type 1 astrocytes was confirmed by Northern blot analysis of isolated total RNA from these different types of cells using a cDNA probe for the neuromodulin mRNA and by Western blot analysis of the cell extracts using polyclonal antibodies against neuromodulin. The properties of the neuromodulin protein in cultured oligodendrocytes and neuronal cells have been compared. Although neuromodulin in oligodendrocytes is soluble in 2.5% perchloric acid like the neuronal counterpart it migrates essentially as a single protein spot on two-dimensional gel electrophoresis whereas the neuronal antigen can be resolved into at least three distinct protein spots. To obtain precise alignments of the different neuromodulin spots from these two cell types, oligodendrocyte and neuronal cell extracts were mixed together and run on the same two-dimensional gel electrophoresis system. Oligodendroglial neuromodulin migrates with a pI identical to the basic forms of the neuronal protein in isoelectric focusing gel. However, the glial neuromodulin shows a slightly lower mobility in the second dimensional lithium dodecyl sulfate-PAGE than its neuronal counterpart. As measured by 32Pi incorporation, neuromodulin phosphorylation in oligodendrocytes is dramatically increased after short-term phorbol ester treatments, which activate protein kinase C, and is totally inhibited by long-term phorbol ester treatments, which downregulates protein kinase C, thus confirming its probable specific in vivo phosphorylation by protein kinase C. In primary cultures of neuronal cells, two of the three neuromodulin spots were observed to be phosphorylated with an apparent preferential phosphorylation of the more acid forms.     

Laminin and fibronectin in normal and malignant neuroectodermal cells

Laminin and fibronectin, the major noncollagenous matrix glycoproteins, were studied in connection with normal brain cells and neuroectodermal cell lines. Laminin, a Mr 900,000 dalton matrix glycoprotein and an essential component of basement membranes, was found to be produced by cultured cells of several malignant cell lines of neuroectodermal origin. In cultured mouse C1300 neuroblastoma line cells laminin was localized, by immunoelectron microscopy, to the rough endoplasmic reticulum and, to sites of cell-to-cell and cell-to-substratum adhesion. Further experiments on the intracellular transport of this glycoprotein in C1300 cells confirmed that laminin is, at least partially, transported through the Golgi pathway. These results favor a role for laminin in attachment and cellular interactions of malignant neuronal cells. Laminin was also found in connection with neurons and glial cells from mammalian brain. In primary cultures from developing rat brain the vast majority of non-neuronal cells (80%) expressed immunoreactivity for the glial fibrillary acidic protein, a cytoskeletal protein specific for astrocytes. During the first week in culture all the glial fibrillary acidic protein-positive cells, with the exception of mature-looking star-shaped astrocytes, exhibited immunoreactivity for laminin. The intracellular laminin disappeared gradually after a few weeks in culture, but an extensive laminin matrix persisted and seemed to be localized on the upper surface of the non-neuronal cells. The neurofilament-positive neurons were negative for laminin. Pretreatment of the cultures with the ionophore monensin, caused accumulation of laminin-immunoreactivity within the Golgi region, which confirmed that laminin is, indeed, produced by cultured astrocytes and secreted through the Golgi complex. No fibronectin immunoreactivity was found in the majority of glial cells. However, under culture conditions where fibronectin was omitted from the culture medium there was, in the primary cultures, a minor population of glial fibrillary acidic protein-positive flat glial cells that exhibited intracytoplasmic immunofluorescence for fibronectin. In the presence of fibronectin in culture medium no fibronectin-positive glial cells could be detected. It thus appears that laminin, and to a minor extent fibronectin, are proteins that normal glial cells are capable of producing under specific conditions. Laminin and fibronectin were localized in adult rat brain in capillary and meningeal structures.(ABSTRACT TRUNCATED AT 400 WORDS)     

Neurons and astrocytes exhibit lower activities of global genome nucleotide excision repair than do fibroblasts

Nucleotide excision repair (NER) is a DNA repair pathway, which eliminates various types of helix-distorting DNA damage including some forms of oxidative damage and UV-induced photoproducts. To understand why patients with NER-defective disorders develop progressive neurological abnormalities, we investigated NER capabilities in neural cells. Primary cultured neurons and astrocytes derived from rat embryonic brains were prepared in mixed-cell cultures, and fibroblasts from the same embryos were cultured for comparison. Neurons in culture were unable to proliferate, while cultured astrocytes maintained that capacity. Determination of (6-4) photoproducts in situ using antibodies against those DNA lesions was used to measure NER capabilities in individual neural cells, which were identified by staining with specific cell markers. The results demonstrate that both neurons and astrocytes have significantly lower NER capabilities than fibroblasts. That result was consistent with the finding that levels of an NER-related protein (proliferating cell nuclear antigen, PCNA) recruited at the localized UV-damage sites were lower in neurons and in astrocytes than in fibroblasts. Interestingly, the degrees of NER deterioration in those neural cells were almost equivalent to those found in NER-defective human fibroblasts (TTD2VI) that show an increased sensitivity to UV. Thus, the present study suggests that an attenuated NER capacity is not specific to post-mitotic neurons, but may be common to neural cells constituting the central nervous system regardless of their residual proliferative capacity. Although the reduced but substantial NER capability of neural cells is indispensable to preventing progressive neurological abnormalities, that low NER capability might have implications for brain ageing.     

TWO FORMS OF NEURONAL ACTIN

Abstract— —Cultures of neurons essentially free of non-neuronal cells were prepared from chick sympathetic neurons and from sensory neurons that had been enriched on a simple density gradient. The proteins of these cultures were examined by two-dimensional gel electrophoresis and two species found in each type of nerve cell that ran close to, but not precisely with, muscle actin. They comigrated with the β and γ actins previously seen in developing myoblasts (W halen et al. , 1976). Peptide patterns obtained from the two neuronal proteins by limited papain digestion, as well as from three analogous proteins of cultured fibroblasts and purified chicken muscle actin, were extremely similar. The same two species, in similar amounts, were found in soluble and residual fractions of cultured neurons produced by brief detergent treatment; in fractions enriched for neuronal processes or cell soma from cultured sensory ganglia; and in purified actin recovered from material released upon gentle homogenisation of embryonic chick brains.     

Immunocytochemical demonstration of vimentin in astrocytes and ependymal cells of developing and adult mouse nervous system

The occurrence of vimentin, a specific intermediate filament protein, has been studied by immunoflourescence microscopy in tissue of adult and embryonic brain as well as in cell cultures from nervous tissue. By double imminofluorescence labeling, the distribution of vimentin has been compared with that of subunit proteins of other types of intermediate filaments (glial fibrillary acidic [GFA] protein, neurofilament protein, prekeratin) and other cell-type specific markers (fibronectin, tetanus toxin receptor, 04 antigen). In adult brain tissue, vimentin is found not only in fibroblasts and cells of larger blood vessels but also in ependymal cells and astrocytes. In embryonic brain tissue, vimentin is detectable as early as embryonic day 11, the earliest stage tested, and is located in radial fibers spanning the neural tube, in ventricular cells, and in blood vessels. At all stages tested, oligodendrocytes and neurons do not express detectable amounts of vimentin. In primary cultures of early postnatal mouse cerebellum, a coincident location of vimentin and GFA protein is seen in astrocytes, and both types of filament proteins are included in the perinuclear aggregates formed upon exposure of the cells to colcemid. In cerebellar cell cultures of embryonic-day-13 mice, vimentin is seen in various cell types of epithelioid or fibroblastlike morphology but is absent from cells expressing tetanus toxin receptors. Among these embryonic, vimentin-positive cells, a certain cell type reacting neither with tetanus toxin nor with antibodies to fibronectin or GFA protein has been tentatively identified as precursor to more mature astrocytes. The results show that, in the neuroectoderm, vimentin is a specific marker for astrocytes and ependymal cells. It is expressed in the mouse in astrocytes and glial precursors well before the onset of GFA protein expression and might therefore serve as an early marker of glial differentiation. Our results show that vimentin and GFA protein coexist in one cell type not only in primary cultures in vitro but also in the intact tissue in situ.     

Highly Basic 30- and 32-Kilodalton Proteins Associated with Synapse Formation on Polylysine-Coated Beads in Enriched Neuronal Cell Cultures

Neuronal proteins involved in axonal outgrowth and synapse formation were examined in an enriched neuronal cell culture system of the cerebellum. In rat cerebellar cell cultures, 98.9% of the cells are neurons and the remaining 1.1% of the cells are flat nonneuronal cells. These enriched neuronal cultures, examined with two-dimensional gel electrophoresis, showed protein patterns similar to those of neonatal cerebellum, but very different patterns from glial enriched cultures. High levels of a neuronal membrane acidic 29-kilodalton (kD) protein were found. It has been shown previously that neuronal cultures incubated with polylysine-coated beads will develop numerous presynaptic elements on the bead surface. We report here that isolation of the beads from enriched neuronal cell cultures incubated with [35S]methionine showed, with two-dimensional nonequilibrium pH gradient gel electrophoresis (2D-NEPHGE), levels of a basic 32-kD protein (pI 8) note detected in cultures alone, and increased levels of a 30-kD protein (pI 10). When culture medium was examined with 2D-NEPHGE, three acidic proteins were identified that were secreted by the cultured neurons. In summary, a neuronal enriched cell culture system was used with isolated polylysine-coated beads to identify basic 30-kD and 32-kD proteins that may be involved in synapse formation.     

Proteomic study of neuron and astrocyte cultures from senescence-accelerated mouse SAMP8 reveals degenerative changes

Senescence-accelerated prone (SAMP) strain 8 mice suffer an earlier development of cognitive age-related pathologies and a shorter life span than conventional mice. Protein alterations in astrocytes, in addition to those in neurons, may contribute to neurodegenerative damage. We applied proteomics techniques to study cell-specific early markers of brain aging-related degeneration in SAMP8. The two-dimensional protein expression patterns of the SAMP8 neuron and astrocyte cultures were compared with those obtained from senescence-accelerated resistant mouse strain 1 cultures. Differentially expressed spots were identified by matrix-assisted laser desorption/ionization–time of flight peptide map fingerprinting and database search. Proteins belonged to cell pathways of energy metabolism, biosynthesis, cell transduction and signaling, stress response, and the maintenance of cytoskeletal functions. Most of the changes were cell type specific. However, there was a general increase in cell transduction, signaling, and stress-related proteins and a decrease in cytoskeletal proteins. In addition, neurons showed an increased expression of proteins involved in biosynthetic pathways. A number of the protein alterations have been previously reported in the brain tissue proteome of SAMP8, aged brain or Alzheimer's disease brain. Alterations in neuron and astrocyte proteoma indicated that both cell types are involved in the brain degenerative changes of SAMP8 mice. However, network analysis suggests that neuronal changes are more complex and have a greater influence.     

Enzyme levels in cultured astrocytes,oligodendrocytes and Schwann cells,and neurons from the cerebral cortex and superior cervical ganglia of the rat

Data are presented for 16 enzymes from 8 metabolic systems in cell cultures consisting of approximately 95% astrocytes and 5% oligodendrocytes. Nine of these enzymes were also measured in cultures of oligodendrocytes, Schwann cells, and neurons prepared from both cerebral cortex and superior cervical ganglia. Activities, in mature astrocyte cultures, expressed as percentage of their activity in brain, ranged from 9% for glycerol-3-phosphate dehydrogenase to over 300% for glucose-6-phosphate dehydrogenase. Creatine phosphokinase activity in astrocytes was about the same as in brain, half as high in oligodendrocytes, but 7% or less of the brain level in Schwann cells and superior cervical ganglion neurons and only 16% of brain in cortical neurons. Three enzymes which generate NADPH, the dehydrogenases for glucose-6-phosphate and 6-phosphogluconate, and the NADP-requiring isocitrate dehydrogenase, were present in astrocytes at levels at least twice that of brain. Oligodendrocytes had enzyme levels only 30% to 70% of those of astrocytes. Schwann cells had much higher lactate dehydrogenase and 6-phosphogluconate dehydrogenase activities than oligodendrocytes, but showed a remarkable similarity in enzyme pattern to those of cortical and superior cervical ganglion neurons.Special issue dedicated to Dr. Lewis Sokoloff.     

Immunocytochemical and RT-PCR analysis of connexin36 in cultures of mammalian glial cells

The expression of connexin36 (Cx36) was studied in primary cultures of rat brain glial cells: mature astrocytes, ameboid and ramified microglia and immature oligodendrocytes (at middle period of myelinogenesis). The data from these cells were compared with those obtained from cultures of neocortical and hypothalamic neurons. mRNA encoding Cx36 was investigated by RT-PCR, the Cx36 protein by immunocytochemistry using a polyclonal antibody against Cx36 in cells characterized by antibodies specific for the single cell types. The Cx36 was found in oligodendrocytes, both ameboid and ramified microglial cells and in neurons. Astrocytes showed no detectable expression of the Cx36. The expression of Cx36 in oligodendrocytes and microglial cells suggests an involvement of the direct cell-cell communication channels formed by Cx36 in myelin formation and in brain development, damage and repair processes.     

Differential expression of gap junctions in neurons and astrocytes derived from P19 embryonal carcinoma cells

The P19 embryonal carcinoma cell line represents a pluripotential stem cell that can differentiate along the neural or muscle cell lineage when exposed to different environments. Exposure to retinoic acid induces P19 cells to differentiate into neurons and astrocytes that express similar developmental markers as their embryonic counterparts. We examined the expression of gap junction genes during differentiation of these stem cells into neurons and astrocytes. Untreated P19 cells express at least two gap junction proteins, connexins 26 and 43. Connexin32 could not be detected in these cells. Treatment for 96 hr with 0.3 mM retinoic acid induced the P19 cells to differentiate first into neurons followed by astrocytes. Retinoic acid produced a decrease in connexin43 mRNA, protein, and functional gap junctions. Connexin26 message was not affected by retinoic acid treatment. The neurons that developed consisted of small round cell bodies extending two to three neurites and expressed MAP2. Connexin26 was detected at sites of cell–cell and cell–neurite contact within 3 days following differentiation with retinoic acid. The astrocytes were examined for production of their intermediate filament marker, glial fibrillary acidic protein (GFAP). GFAP was first detected at 8 days by Western blotting. In culture, astrocytes co-expressed GFAP and connexin43 similar to primary cultures of mouse brain astrocytes. These results suggest that differentiation of neurons and glial cells involves specific connexin expression in each cell type. The P19 cell line will provide a valuable model with which to examine the role gap junctions play during differentiation events of developing neurons and astrocytes. Dev. Genet. 21:187–200, 1997. © 1997 Wiley-Liss, Inc.     

Expression of the mRNA for τ Proteins During Brain Development and in Cultured Neurons and Astroglial Cells

Two tau cDNA probes of 1.6 and 0.3 kilobases (kb) have been used to study the expression of the tau mRNAs during mouse brain development and in highly homogeneous primary cultures of neurons and astrocytes. (1) Whatever the stage, a 6-kb mRNA was detected with the two probes. In the astrocytes a 6-kb mRNA hybridized clearly only with the 1.6-kb probe. (2) During brain development the abundance of tau mRNA increases from a late fetal stage (-4 days) until birth, remains high until 6 days postnatal, and then markedly decreases to reach very low values in adulthood. Such a marked decrease in the abundance of tau mRNA parallels that of alpha-tubulin mRNA. These data suggest that: (1) depending on the stage of development and on the cell type (neurons or astrocytes) tau mRNAs of the same size encode several tau proteins differing in molecular weight: several tau proteins are expressed either during early stages of development (juvenile tau proteins of 48 kilodaltons) or in adulthood (mature tau proteins of 50-70 kilodaltons) or are specific of the astrocyte (83 kilodaltons). (2) The expression of the two major components of axonal microtubules, tubulin and tau proteins, seems to be developmentally coordinated.     

Characterisation and metabolism of astroglia-rich primary cultures from cathepsin K-deficient mice

Abstract Cathepsin K is important for the brain, because its deficiency in mice is associated with a marked decrease in differentiated astrocytes and changes in neuronal patterning in the hippocampus as well as with learning and memory deficits. As cathepsin K activity is most prominent in hippocampal regions of wild type animals, we hypothesised alterations in astrocyte-mediated support of neurons as a potential mechanism underlying the impaired brain functions in cathepsin K-deficient mice. To address this hypothesis, we have generated and characterised astroglia-rich primary cell cultures from cathepsin K-deficient and wild type mice and compared these cultures for possible changes in metabolic support functions and cell composition. Interestingly, cells expressing the oligodendrocytic markers myelin-associated glycoprotein and myelin basic protein were more frequent in astroglia-rich cultures from cathepsin K-deficient mice. However, cell cultures from both genotypes were morphologically comparable and similar with respect to glucose metabolism. In addition, specific glutathione content, glutathione export and γ-glutamyl-transpeptidase activity remained unchanged, whereas the specific activities of glutathione reductase and glutathione-S-transferase were increased by around 50% in cathepsin K-deficient cultures. Thus, lack of cathepsin K in astroglia-rich cultures appears not to affect metabolic supply functions of astrocytes but to facilitate the maturation of oligodendrocytes.     

Macro- and microvascular endothelial cells in vitro: Maintenance of biochemical heterogeneity despite loss of ultrastructural characteristics

Summary Microvascular endothelial cells from bovine adrenal medulla and brain and macrovessel endothelial cells from bovine aorta were isolated and cultured under similar conditions in order to determine morphologic and biochemical heterogeneity in vitro. All three cell types exhibited nearly identical ultrastructural morphology and two-dimensional gel protein patterns of³⁵S-methionine-labeled whole cells. Two-dimensional gel analysis of³⁵S-methionine-labeled plasma membrane proteins however, revealed two-dimensional gel protein patterns unique to the tissue type from which the endothelial cells were isolated. This suggests that the functional significance of these specific endothelial cell types is manifested primarily in surface-associated proteins and that many of the differences are sustained in culture. To determine the potential of aorta, brain, and adrenal medulla endothelial cell (EC) cultures to respond to developmentally significant signals, morphology, growth pattern, and cell surface proteins were monitored in the presence and absence of growth factors. A 17 to 26% increase in cell density as well as an increase in the number of elongated and overlapping cells resulted when all three EC types were exposed to a mitogenic medium. Additionally, expression of specific glycoprotein profiles, as determined by Concanavalin A Western blotting of two-dimensional gels, was dependent on the presence or absence of growth factors in the medium. The ability to induce this morphologic and biochemical variation in the three endothelial cell types was maintained into later passage. Taken together, these data imply that endothelial cells isolated from different tissues exhibit and maintain biochemical heterogeneity and do not completely dedifferentiate into a common endothelial cell type in culture. Furthermore, expression of specific subsets of cell surface proteins is dependent on environmental conditions, and in some cases is both cell-type and media-type dependent. Thus, even though endothelial cells are considered terminally differentiated cells, there exists additional or “latent” heterogeneity in the ability of these different cells to respond to “developmental signals” (i.e. mitogenic medium) in vitro. This work was supported in part by a grant from the American Heart Association (860929), NIH (GM29127), and a Biomedical Research Grant from the University of Massachusetts.     

Vitamin E, Ascorbate, Glutathione, Glutathicne Disulfide, and Enzymes of Glutathione Metabolism in Cultures of Chick Astrocytes and Neurons: Evidence that Astrocytes Play an Important Role in Antioxidative Processes in the Brain

Abstract: GSH, GSSG, vitamin E, and ascorbate were measured in 14-day cultures of chick astrocytes and neurons and compared with levels in the forebrains of chick embryos of comparable age. Activities of enzymes involved in GSH metabolism were also measured. These included -γ-glutamylcysteine synthetase, GSH synthetase, γ-glutamyl cyclotransferase, γ-glutamyltranspeptidase, glutathione transferase (GST), GSH peroxidase, and GSSG reductase. The concentration of lipid-soluble vitamin E in the cultured neurons was found to be comparable with that in the forebrain. On the other hand, the concentration of vitamin E in the astrocytes was significantly greater in the cultured astrocytes than in the neurons, suggesting that the astrocytes are able to accumulate exogenous vitamin E more extensively than neurons. The concentrations of major fatty acids were higher in the cell membranes of cultured neurons than those in the astrocytes. Ascorbate was not detected in cultured cells although the chick forebrains contained appreciable levels of this antioxidant. GSH, total glutathione (i.e., GSH and GSSG), and GST activity were much higher in cultured astrocytes than in neurons. γ-Glutamylcysteine synthetase activity was higher in the cultured astrocytes than in the cultured neurons. GSH reductase and GSH peroxidase activities were roughly comparable in cultured astrocytes and neurons. The high levels of GSH and GST in cultured astrocytes appears to reflect the situation in vivo. The data suggest that astrocytes are resistant to reactive oxygen species (and potentially toxic xenobiotics) and may play a protective role in the brain. Because enzymes of GSH metabolism are generally well represented in cultured astrocytes and neurons these cells may be ideally suited as probes for manipulating GSH levels in neural tissues in vitro. Cultured astrocytes and neurons should be amenable to the study of the effects of various metabolic insults on the GSH system. Such studies may provide insights into the design of therapeutic strategies to combat oxidative and xenobiotic stresses.     

Effects of cell differentiation on replication of A/WS/33, WSN, and A/PR/8/34 influenza viruses in mouse brain cell cultures: biological and immunological characterization of products.

The responses of mouse embryo brain (MEB) cell cultures and of Madin-Darby canine kidney cells and chicken embryo fibroblasts to infection with A/PR/8/34 (PR8), A/WS/33 (WS), or the neurovirulent WSN variant were compared in terms of (i) single-cycle yields of hemagglutinating and associated neuraminidase (NA) activities and plaque-forming particles, the latter with or without trypsin activation [PFU(TR++) or PFU(TR--), respectively], and (ii) expression of nucleoprotein (NP), M1, and NS1 protein, determined for specific cell types by immunostaining, for whole culture lysates by Western blot analysis of NP and M1. Primary MEB cultures grown in serum-enriched medium were infected after 6 days (young), when none of the cells reacted specifically and exclusively with any of the nerve cell marker antibodies used, or after greater than or equal to 21 days (aged), when astrocytes (the predominant cell type), neurons, and oligodendrocytes were morphologically and immunologically mature. Secondary astrocyte-enriched cultures were used when they contained 90 to 99% of their cells as astrocytes at an early stage of differentiation. By all criteria, young MEB cultures were only marginally less permissive for each of the three viruses than were chicken embryo fibroblasts or Madin-Darby canine kidney cells. Aged MEB cultures, by comparison, produced undiminished NP, hemagglutinin, and neuraminidase, but yields of PFU(TR++) and expression of M1 protein (relative to NP) were reduced for all three viruses, most for PR8 and least for WSN; relative reduction of NS1 protein was demonstrable only in PR8-infected aged cultures. Immunostaining revealed low levels of M1 and NS1 expression only in astrocytes, not in oligodendrocytes and neurons. In PR8-infected mature astrocytes, NP accumulated in the nucleus; it persisted in some cells for at least 8 weeks after infection. The presence of NP did not seem to interfere with cell division. Secondary MEB cultures containing 90 to 99% immature astrocytes were less restricted than were aged primary cultures. Thus, it appears that reduced permissivity of nerve cell cultures, as measured in this study, is most closely correlated with advancing differentiation and maturity of astroglial cells. Assembled virions, including those that score as PFU(TR++) in restricted cultures (e.g., PR8-infected aged MEB), may be mainly products of mature oligodendrocytes and neurons.     

Marked Regional Heterogeniety of 125I-Bolton Hunter Substance P Binding and Substance P-Induced Activation of Phospholipase C in Astrocyte Cultures from the Embryonic or Newborn Rat

The specific binding of 125I-Bolton Hunter substance P (125I-BHSP) was estimated on 4- to 5-week-old primary cultures of astrocytes from several brain structures and the spinal cord of 16-day-old embryonic or newborn rats. In both cases, high levels of binding of 125I-BHSP were found on intact astrocytes from the brainstem, but this binding was low or negligible on cells from the cerebral cortex, striatum, hypothalamus, and mesencephalon. In addition, hippocampal astrocytes from newborn rats were also devoid of 125I-BHSP binding sites, while a binding of 125I-BHSP (half that of brainstem cells) was observed on astrocytes from the cerebellum and spinal cord. It was also shown that this regional heterogeneity in 125I-BHSP binding was not linked to differences in the inactivation of the ligand, cell plating density. or eventual cell contaminants. Five-day-old cultures from 16-day-old embryos were used to estimate 125I-BHSP binding on neuron-enriched cultures. Specific 125I-BHSP binding was found on cells from the brainstem, mesencephalon, and hypothalamus, but neurons from the cerebral cortex or the striatum contained low or negligible amounts of 125I-BHSP binding sites. Competition studies using tachykinins and SP analogues indicated that 125I-BHSP binding sites on brainstem astrocytes (16-day-old embryos) have the pharmacological profile expected for NK1 binding sites. SP (1 microM) stimulated phosphoinositide breakdown in cells rich in 125I-BHSP binding sites (brainstem) but not in those devoid of 125I-BHSP binding (striatum).(ABSTRACT TRUNCATED AT 250 WORDS)     

Asymmetry of glutamine transporters in cultured neural cells

Transfer of glutamine between astrocytes and neurons is an essential part of the glutamate-glutamine cycle in the brain. Transport of glutamine was investigated in primary cultures of astrocytes and neurons and compared to glutamine transport in cell lines with glial and neuronal properties. Glutamine uptake in astrocytes was mainly mediated by general amino acid transporters with properties similar to ASCT2, LAT1, LAT2, SN1 and y(+)LAT2. In cultured neurons, transport activities were detected consistent with the presence of LAT1, LAT2 and y(+)LAT2, but the most prominent activity was a novel Na(+)-dependent glutamine transporter that could be inhibited by D-aspartate. The mRNA for system A isoforms ATA1 and ATA2 was detected in both neurons and astrocytes, but system A activity was only detected in neurons. ASCT2 on the other hand appeared to be astrocyte-specific. The cell lines F98 and 108CC-15, having astroglial and neuronal properties, respectively, expressed sets of glutamine transporters that were unrelated to those of the corresponding primary culture and are thus of limited use as models to study transfer of glutamine between astrocytes and neurons.