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)     

Fibronectin is expressed by astrocytes cultured from embryonic and early postnatal rat brain

In early primary cultures from newborn rat brain, few glial fibrillary acidic protein (GFAP)-positive glial cells expressed intracytoplasmic immunoreactivity for fibronectin. After the second week in culture, however, fibronectin was expressed by a distinct population of GFAP-positive flat astrocytes, irrespective of which brain region was studied. In cerebellar cultures, these cells were more abundant than in cortical or neostriatal cultures and often formed a major population of the GFAP-positive cells. The difference in fibronectin expression between cerebellum and the other areas studied was statistically significant. When cultures were started from 9-day-old postnatal rat brain, fibronectin-positive astrocytes appeared earlier than in those from newborn animals, in all areas studied. Further, especially in the case of cerebellum, the number of fibronectin-positive astrocytes increased as a function of time in culture. In cultures started from whole brains of 12-day-old rat embryos, fibronectin was expressed within 24 h in culture by all the cells with morphology of flat astrocytes, positive for vimentin but negative for GFAP. These results indicate that astrocytes cultured from newborn and early postnatal rat brain are a heterogeneous population of cells: depending on the brain region studied and also depending on the age of brain tissue or the time in culture, less than 1-60% of the GFAP-positive flat astrocytes expressed fibronectin. This, together with the fact that fibronectin was present in early embryonic brain cells in culture, suggests that fibronectin may be a prerequisite for the development or interactions of brain cells.     

Immortalization of fetal mouse brain glial cells by human papillomavirus type 16 E7 genes.

Fetal mouse brain glial cells in primary cultures were immortalized by recombinant retroviruses containing human papillomavirus type 16 E7 genes, and named VR-2g cells. The presence and expression of E7 genes in VR-2g cells were demonstrated by the Southern and the Northern blot analyses. VR-2g cells did not form colonies in soft agar culture, indicating that VR-2g cells have no transforming phenotypes. By the karyotype analysis, VR-2g cells consisted of two cell populations, the pseudo-diploid and the pseudo-tetraploid. VR-2g cells were positive in immunostaining with mono- and polyclonal antibodies against glial fibrillary acidic proteins (glial-specific intermediate filaments). In addition, VR-2g cells secreted neurotrophic factors as assayed with primary cultures of fetal rat striatum neurons, although molecular characterization of the factor(s) was not yet determined. These results indicate that the present method for cell immortalization will be useful for establishing untransformed cell lines from primary cultures of fetal brain cells.     

Antigenic and functional characterization of a rat central nervous system-derived cell line immortalized by a retroviral vector

We have immortalized rat central nervous system (CNS) cells of primary cultures of rat optic nerve with murine leukemia virus psi-2,SV-40-6, which is defective in assembly and contains the SV-40 large T antigen and neomycin resistance genes, to produce a cell line that we named A7. After drug selection, greater than 90% of the growing cells expressed nuclear SV-40 large T cells and a fraction of these contained the astrocyte-specific marker, glial fibrillary acidic protein. The majority of these cells also expressed surface marker A4 (specific for neural tube derivatives), Ran 2, p185 (the 185-kD phosphoprotein product of the neu oncogene), and fibronectin, but did not express the astrocyte enzymes glutamine synthetase and monoamine oxidase B. Surface markers characteristic of glial progenitors (A2B5) and oligodendrocytes (galactocerebroside) were not detected. After two rounds of cell cloning, subclone A7.6-3 expressed Ran 2, fibronectin, and the neural cell adhesion molecule (N-CAM) but not glial fibrillary acidic protein and A4. The A7 cell line and subclones also displayed certain functions of type 1 astrocytes: the conditioned medium of these cells had a potent mitogenic activity for glial progenitor cells which could be neutralized by anti-platelet-derived growth factor antibodies and monolayers of these cells supported the growth of embryonic hypothalamic neurons. We conclude that a retrovirus containing SV-40 large T antigen can immortalize rat CNS cells and that such immortalized glial cells retain at least two important functions of type 1 astrocytes: the ability to secrete platelet-derived growth factor and to support the growth of embryonic CNS neurons. Moreover, such stable immortalized clonal cell lines can be used to study gene regulation in glial cells.     

Fibronectin associated with the glial component of embryonic brain cell cultures

In a basic approach to investigations of neuronal–glial interactions during both normal brain development and its pathogenesis, embryonic brain cell populations were fractionated into purified neuronal and glial components. Using separation procedures based on differential adhesion and cytotoxicity, the isolated neuronal and glial phenotypes could be identified by distinct morphological and biochemical characteristics, including the visualization of glial fibrillary acid protein (GFA) within glial cells in immunohistochemical assays with monospecific anti-GFA serum. When unfractionated cerebrum cells dissociated from 10-day chick or 14-day mouse embryos were plated as monolayers and cultured for 1-14 days, monospecific antiserum against fibronectin (LETS glycoprotein) was found to react with many, but not all, of the cells as revealed by indirect immunofluorescence microscopy. The isolated neuronal and glial components of these populations were used to determine whether the appearance of membrane-associated fibronectin was characteristic of one cell type or the other, or both, and if neuronal–glial cell interaction was required for its expression. It was found that the surfaces of glial cells, completely isolated from neurons, showed an intense fluorescent reaction to the anti-fibronectin serum. In contrast, the purified neuronal cultures showed no fluorescence with either the anti-GFA or anti-fibronectin sera. These results demonstrate fibronectin as a cell surface protein associated primarily with glial cells and independent of neuronal–glial cell interaction for its expression. Furthermore, the results indicate that the fibronectin observed on glial cell surfaces in these cultures is produced endogenously and is not due to the preferential binding of fibronectin present in the culture medium. The role of fibronectin as an adhesive molecule in neuronal–glial interactions is discussed.     

Widespread immunoreactivity for neuronal nuclei in cultured human and rodent astrocytes

The monoclonal antibody (mAb) neuronal nuclei (NeuN) labels the nuclei of mature neurons in vivo in vertebrates. NeuN has also been used to define post-mitotic neurons or differentiating neuronal precursors in vitro . In this study, we demonstrate that the NeuN mAb labels the nuclei of astrocytes cultured from fetal and adult human, newborn rat, and embryonic mouse brain tissue. A non-neuronal fibroblast cell line (3T3) also displayed NeuN immunoreactivity. We confirmed that NeuN labels neurons but not astrocytes in sections of P10 rat brain. Western blot analysis of NeuN immunoreactive species revealed a distribution of bands in nucleus-enriched fractions derived from the different cell lines that was similar, but not identical to adult rat brain homogenates. We then examined the hypothesis that the glial fibrillary acidic protein/NeuN-double positive population of cells might correspond to neuronal precursors. Although the NeuN-positive astrocytes were proliferating, no evidence of neurogenesis was detected. Furthermore, expression of additional neuronal precursor markers was not detected. Our results indicate that primary astrocytes derived from mouse, rat, and human brain express NeuN. Our findings are consistent with NeuN being a selective marker of neurons in vivo , but indicate that studies utilizing NeuN-immunoreactivity as a definitive marker of post-mitotic neurons in vitro should be interpreted with caution.     

Biochemical and ultrastructural studies of cultured rat astroglial cells: effect of brain extract and dibutyryl cyclic AMP on glial fibrillary acidic protein and glial filaments

The growth of astroglial cells in primary cultures derived from newborn rat cerebral hemispheres was investigated in the absence and in the presence of newborn rat brain extract or dBcAMP. The parameters chosen were the content of DNA, total protein, and glial fibrillary acidic protein (GFA) as well as the morphologic development of gliofilaments. During the entire culture period the DNA content increased in control culture indicating a continuous cell division, whereas the cells stopped dividing after 14 or 4 days of treatment with either brain extract or dBcAMP respectively. In contrast, a constant increase of total protein was found in both control and treated cultures. Since cell divisions had stopped in treated cultures, the increase in total protein in these cultures indicates growth of the individual cells. The GFA levels increased progressively and similarly in control cultures and in cultures treated with brain extract. The values in the treated cultures remained slightly higher than those in controls. Conversely, immediately after the addition of dBcAMP a sudden increase in GFA protein occurred and the amounts were statistically significantly different from those of the controls. The GFA levels were expressed relative to total protein indicating that GFA constitutes an increasing amount of the total protein of the individual cells during culture. The changes in the amount of GFA was shown to parallel the morphologic development of gliofilaments. Indeed, when the level of GFA increased a progressive accumulation of gliofilaments was observed. The results obtained were discussed in relation to the astrocytic maturation.     

Biochemical and Ultrastructural Studies of Cultured Rat Astroglial Cells

The growth of astroglial cells in primary cultures derived from newborn rat cerebral hemispheres was investigated in the absence and in the presence of newborn rat brain extract or dBcAMP. The parameters chosen were the content of DNA, total protein, and glial fibrillary acidic protein (OFA) as well as the morphologic development of gliofilaments. During the entire culture period the DNA content increased in control culture indicating a continuous cell division, whereas the cells stopped dividing after 14 or 4 days of treatment with either brain extract or dBcAMP respectively. In contrast, a constant increase of total protein was found in both control and treated cultures. Since cell divisions had stopped in treated cultures, the increase in total protein in these cultures indicates growth of the individual cells. The GFA levels increased progressively and similarly in control cultures and in cultures treated with brain extract. The values in the treated cultures remained slightly higher than those in controls. Conversely, immediately after the addition of dBcAMP a sudden increase in GFA protein occurred and the amounts were statistically significantly different from those of the controls. The GFA levels were expressed relative to total protein indicating that GFA constitutes an increasing amount of the total protein of the individual cells during culture. The changes in the amount of GFA was shown to parallel the morphologic development of gliofilaments. Indeed, when the level of GFA increased a progressive accumulation of gliofilaments was observed. The results obtained were discussed in relation to the astrocytic maturation.     

Common myofibroblastic features of newborn rat astrocytes and cirrhotic rat liver stellate cells in early cultures and in vivo.

Double-immunolabelling techniques were employed to investigate the distribution of smooth muscle alpha-actin (actin) in glial fibrillary acidic protein (GFAP)-positive cells in rat brain during early postnatal development and maturation and in glial primary culture derived from newborn rat brain. In addition the expression of desmin was studied in the glial primary cultures as a function of the differentiation of the cells. Comparison of the cultured astroglial cells at an early age with hepatic stellate cells derived from CCl4-induced cirrhotic rat liver, revealed features of the astrocytic cytoskeleton characteristic of myofibroblastic cells, i.e., strong expression of both myofibroblastic markers, actin and desmin. In astroglial cells with an initial morphology reminiscent of fibroblasts the non-filamentous perinuclear immunoreaction of GFAP increased with time at the expense of actin and, partially, desmin. GFAP filaments were spread throughout the cytoplasm of the cells which acquired stellate morphology. The alterations in the morphology of the cells and the distribution and intensity of staining for GFAP and actin during the differentiation of astrocytes in culture were similar to those observed in astrocytes during the maturation of the brain. In astrocytes from a newborn brain as well as in cirrhotic hepatic stellate cells, the area of immunoreaction of GFAP was reduced and confined mainly to the nuclear region. In contrast, the cells expressed actin throughout the cytoplasm. These findings may hint at a similar function of these regionally specialized perivascular myofibroblastic cells in a normal brain and diseased liver and at inverse organ-specific functions which the cells fulfill under non-pathological conditions in vivo.     

Absence of ganglioside GM1 in astroglial cells from newborn rat brain

An immunohistochemical method utilizing anti-ganglioside GM1 antiserum combined with the peroxidase-antiperoxidase technique was applied to a mixed cell population in primary cultures of newborn rat brain. Ganglioside GM1 was demonstrated to be present in neurons and oligodendroglia, but was absent in astroglia. This demonstration was confirmed using a newly developed biotinylated choleragen-avidin-peroxidase procedure. Primary cultures from newborn rat brain cells that had been subjected to a single treatment with trypsin (first passage) and then cultured for 14 days were predominately (95%) composed of astrocytes that stained positively for glial fibrillary acidic protein but were negative for GM1 ganglioside. This preparation contained only 0.34 nmol ganglioside NeuNAc per mg protein compared to 23.9 nmol gangliosidic NeuNAc/mg protein for a five day culture of newborn rat brain mixed cell culture that had not been subjected to passage. Prolongation of culture time from 5 to 21 days in the latter preparation reduced the ganglioside NeuNAc content to 4.9 nmol gangliosidic NeuNAc/mg protein as the proportion of astrocytes in the culture increased. Ganglioside GM1 could not be detected by TLC analysis of the lipid extract obtained from the “pure” astrocyte culture, although small amounts of GM3 and some polysialogangliosides were detected. About half of the label incorporated upon 24 h incubation of astrocytes in the presence of $\text{N-[}{}^3\text{H]acetylmannosammine}$ appeared in ganglioside GM3. It is concluded that astrocytes in mixed cell primary cultures from newborn rat brain, as well as astrocytes in astroglial preparations derived from such cultures, do not contain ganglioside GM1.     

Expression of Hyaluronectin by C-6 Glial Cells at High Density

Hyaluronectin, a brain glycoprotein that has been localized to the nodes of Ranvier in vivo and to oligodendrocytes in primary cultures of neonatal rat brain cells, was shown by using an unlabeled immunoperoxidase method to be present in C-6 glial cells grown to high density. The density-dependent expression of this glycoprotein is in accordance with the known properties of the glial stem cells, i.e., induction of differentiated properties such as 2',3'-cyclic nucleotide-3'-phosphohydrolase, glutamine synthetase, S-100 protein, and glial fibrillary acidic protein.     

Fetal Calf Spinal Cords as Enriched Source of mRNA Encoding Glial Fibrillary Acidic Protein

Abstract: Total poly(A)⁺ RNA was isolated from fetal calf spinal cord, adult rat spinal cord, and young rat brain, and was translated using the rabbit reticulocyte lysate system. The amount of glial fibrillary acidic protein in the translation products was measured by immunoprecipitation with antiserum against glial fibrillary acidic protein. RNA from fetal calf spinal cord could direct glial fibrillary acidic protein synthesis such that this protein comprised approximately 1.4% of the total products. RNAs from adult rat spinal cord and brain could direct glial fibrillary acidic protein synthesis much less efficiently, with this protein comprising <0.3% of the total products. These results suggest that the gene for glial fibrillary acidic protein is strongly expressed in fetal calf spinal cord and that this tissue is an enriched source of mRNA encoding glial fibrillary acidic protein.     

Enhanced survival of cultured cerebellar Purkinje cells by plating on antibody to Thy-1

Survival of newborn mouse cerebellar Purkinje cells in dissociated monolayer cultures was substantially enhanced when the dissociated cells were plated on a substrate containing antibody to the Purkinje cell surface antigen Thy-1. Cells were identified by size, asymmetric arbors, uptake of gamma-aminobutyric acid (GABA), and failure to express glial fibrillary acidic protein. This selective enhancement was specific for antibody to the Thy-1.2 allele.     

Rat brain glial cells in culture: Effects of brain extracts on the development of oligodendroglia-like cells

Cells dissociated from brains of newborn rats and grown on plastic surfaces develop into a glial culture, composed of at least three morphologically different cell types. The predominating cell type consists of astroglial cells, which form a monolayer. The second cell type, rarely observed, consists of ependymal cells. The third type consists of small cells scattered upon the astroglial layer. After 3 weeks very few of these small cells remain and the glial culture develops into a more homogenous appearance, mainly composed of astroglial cells. The effects of various brain extracts on the development of the small cell type was investigated. The treatment by either rat or chick brain extracts caused an increase in the number of these cells, which were seen to form clusters. Brain extracts from older animals have a stronger effect than brain extracts from younger animals. These data suggest that factors contained in the brain during and after the myelination period influence the development of this cell type in dissociated cultures. The small cells were tentatively identified as oligodendroglial cells by ultrastructural and histochemical criteria. They did not contain acetylcholinesterase (AChE) and did not bind tetanus toxin. Furthermore, they did not contain glial fibrillary acidic (GFA) protein. But carbonic anhydrase II (CAII) was found in them at light and electron-microscopical level. CAII was found to be localized essentially on the plasmic membrane and on the endoplasmic reticulum of these cultured oligodendroglial cells.     

A novel human astrocyte cell line (A735) with astrocyte-specific neurotransmitter function

Summary Studies of brain cell function and physiology are hampered by the limited availability of imortal human brain-derived cell lines, as a result of the technical difficulties encountered in establishing immortal human cells in culture. In this study, we demonstrate the application of recombinant DNA vectors expressing SV40 T antigen for the development of immortal human cell cultures, with morphological, growth, and functional properties of astrocytes. Primary human astrocytes were transfected with the SV40 T antigen expression vectors, pSV3neo or p735.6, and cultures were established with an extended lifespan. One of these cultures gave rise to an immortal cell line, designated A735. All the human SV40-derived lines retained morphological features and growth properties of type 1 astrocytes. Immunohistochemical studies and Western blot analysis of the intermediate filament proteins and glutamine synthetase demonstrated a differentiated but immature astrocyte phenotype. Transport of γ-amino butyric acid and glutamate were examined and found to be by a glial-specific mechanism, consistent with the cell lines’ retaining aspects of normal glial function. We conclude that methods based on the use of SV40 T antigen can successfully immortalize human astrocytes, retaining key astrocyte functions, but T antigen-induced proliferation appeared to interfere with expression of glial fibrillary acidic protein. We believe A735 is the first documented nontumor-derived human glial cell line which is immortal.     

Immunocytochemical localization of cytochrome P-450scc in cultured rat oligodendrocytes

Primary cultures of glial cells from newborn rat forebrain were tested after 3 to 4 weeks. Oligodendrocytes and astrocytes were characterized by immunofluorescence with monoclonal antibodies to galactocerebroside and glial fibrillary acidic protein, respectively. The cytoplasm of oligodendrocytes was specifically and intensely immunostained with monospecific polyclonal antibodies to the cytochrome P-450scc involved in the synthesis of pregnenolone from cholesterol. This observation brings additional support to the concept of "neurosteroids".     

Individual neural cell types express immunologically distinct N-CAM forms

The neural cell adhesion molecules, or N-CAMs, are a group of structurally and immunologically related glycoproteins found in vertebrate neural tissues. Adult brain N-CAMs have apparent molecular weights of 180,000, 140,000, and 120,000. In this article we identify, using monoclonal antibody (Mab) 3G6.41, an immunologically distinct adult rat N-CAM form and show that this form is selectively expressed by some clonal neural cell lines. Consecutive immunoprecipitation experiments indicate that rabbit anti-N-CAM can remove from solubilized cerebellar neuron primary cultures all 180,000- and 140,000-mol-wt N-CAM molecules that react with Mab 3G6.41. However Mab 3G6.41 cannot remove all N-CAM molecules that react with rabbit anti-N-CAM. Rabbit anti-N-CAM binds to and immunoprecipitates N-CAM forms from the rat neuronal cell lines B35, B65, and B104, the glial lines B12 and C6, and L6 myoblasts. Mab 3G6.41 does not bind to or immunoprecipitate N-CAM from the B12 and B65 lines but does react with the other four lines by both criteria. Many cells in primary cultures of postnatal rat that express glial fibrillary acidic protein also bind Mab 3G6.41. Thus a unique form of rat N-CAM recognized by Mab 3G6.41 is found on some but not all neuronal, glial, and muscle cells.     

Subclass of astroglia in mouse cerebellum recognized by monoclonal antibody

A monoclonal antibody was detected that distinguishes astrocyte subclasses in mouse cerebellum. This antibody, designated anti-M1, is the product of a hybridoma that arose from the fusion of NS1 myeloma cells and splenocytes derived from a rat immunized with crude membranes from early postnatal mouse cerebella. The distribution and regulation of M1 antigen expression in vivo were examined by indirect immunofluorescence on frozen thin sections of mouse brain. M1 expression shows differing age dependencies within subpopulations of astroglia. M1 is first detectable around postnatal day 7 in white matter astrocytes and persists in this cell type throughout adulthood. By postnatal day 10, M1 is additionally detected in Bergmann glial fibers and in granule layer astrocytes. M1 expression in these latter astrocytic cell types is transient and cannot be detected after the fourth postnatal week. Cerebella of adult neurological mutant weaver mice show abnormal persistence of M1 antigen expression in Bergmann glial fibers. In monolayer cultures of early postnatal cerebella, M1 antigen is detected in a subpopulation of the glial fibrillary acidic protein positive astrocytes. M1 antigen can be detected only in fixed cultured cells which allow intracellular penetration of the antibody. The developmental regulation of M1 expression and the abnormal expression of M1 in weaver mutant cerebella suggest that M1 may be a useful marker for astroglial maturation and differentiation.     

Cultured Rat Astrocytes Give Rise to Neural Stem Cells

Previously, we reported the occurrence of neural stem cells (NSCs) around an area of damage after rat traumatic brain injury (TBI), but it was unclear if this was due to blastgenesis in astrocytes, or to NSCs migrating from the subventricular zone (SVZ). In this study, NSCs were isolated and cultured from cultured type 1 astrocytes taken from newborn rat cortex in which the subventricular zone and hippocampus had been discarded. All cultured type 1 astrocytes showed glial fibrillary acidic protein (GFAP) immunopositivity. Nestin immunopositive spheres were isolated from type 1 astrocytes and cultured in the presence of bFGF and EGF in the medium. Neurospheres differentiated into Tuj1-, GFAP- and A2B5-positive cells after 4 days of culture without bFGF and EGF. These results indicate that isolated neurospheres from brain cortex astrocytes can differentiate into neurons and glia and might contribute to neurogenesis and neuroplasticity.