Effects of ATP analogues and basic fibroblast growth factor on astroglial cell differentiation in primary cultures of rat striatum

We have used primary cultures of rat striatum to study the effects of ATP analogues on the elongation of astrocytic processes, a parameter of astroglial cell differentiation. Parallel studies were performed with basic fibroblast growth factor, a known regulator of astroglial cell function. After three days in culture, both the growth factor and αβ-methylene-ATP induced dramatic increases in the mean length of astrocytic processes/cell. For both agents, effects were dose-dependent. The effect of αβ-methylene-ATP was antagonized by the trypanoside suramin and mimicked by 2-methyl-thio-ATP, suggesting the involvement of a suramin-sensitive P2-purinoceptor. Neither an additive nor a synergistic effect between αβ-methylene-ATP and basic fibroblast growth factor on the elongation of processes was detected in cultures exposed to both agents. Indeed, an inhibition with respect to the effects induced by either agent alone was recorded, suggesting that the growth factor and the purine analogue can modulate astrocytic differentiation by activation of common intracellular pathways.It is concluded that, like basic fibroblast growth factor, ATP can promote the maturation of astrocytes towards a more differentiated phenotype characterized by the presence of longer astrocytic processes. These findings might have interesting implications for astroglial cell differentiation during brain development and for ischemia- and trauma-associated hypergliosis.     

P2, P1, and P0 myelin protein expression in developing rat sixth nerve: a quantitative immunocytochemical study

Myelination and the expression of myelin proteins P2, P1, and P0 were studied quantitatively in the rat sixth cranial nerve during development. The postnatal development and growth of all myelin sheaths in this nerve have been studied morphometrically in a companion paper. Epon-embedded blocks with closely matched topography in the transverse plane were selected from rats perfused at ages 1-4, 8, 15, and 20 days. From each block, serial semithin sections were cut, etched, and immunostained according to the peroxidase-antiperoxidase method with well-characterized polyclonal antisera that reacted specifically with P0 glycoprotein and the basic proteins P1 and P2. The immunoreactivities of individual myelin sheaths were measured by densitometry. Numbers of compact myelin lamellae, myelin spiral lengths, and axon diameters were determined on electronmicrographs of adjacent thin sections. At birth anti-P0 immunoreactivity was found on sheaths with two and more compact lamellae; neither P1 nor P2 immunoreactivity was observed. On day 2, myelin sheaths with five and eight lamellae were stained respectively by anti-P1 and anti-P2. On day 3 the percentages of myelin sheaths stained were substantially higher: P0 95%, P1 78%, P2 15%. By day 4, anti-P0 and anti-P1 immunoreactivity was present in 95% of myelin sheaths; 35% were stained by anti-P2. For P2, staining intensity and percentage of myelin sheaths stained continued to increase and by day 20, 85% were anti-P2-positive. The density of immunoreactivity was not uniform in all myelin sheaths. At young ages staining varied with all three proteins. The variability decreased as myelin sheaths thickened; it persisted longest for anti-P2. We conclude that the density and distribution of immunoreactivities of P0, P1, and P2 reflect their relative concentrations during myelin sheath development and growth. We attribute lack of detectable anti-P2 immunoreactivity in some small sheaths at 20 days to their early stage of myelination and also to limitations of the method. We infer from our observations that all myelin-forming Schwann cells express P2 basic protein.     

SR13/PMP-22 expression in rat nervous system,in PC12 cells,and C6 glial cell lines

SR13/PMP-22 is a protein that was identified after screening a sciatic nerve cDNA library. Our study focused on comparing the level and pattern of expression of SR13/PMP-22 protein and RNA. Northern blot analysis revealed that although SR13/PMP-22 mRNA was present in all nervous tissues and cells studied, levels were at least seven fold higher in the sciatic nerve and the spinal cord. During sciatic nerve postnatal development and maturation, the SR13/PMP-22 mRNA was detected at 2 days after birth, reached a maximal level at day 24, and decreased to 1/3 of the maximum in adult animals. Nerve transection reduced the level of SR13/PMP-22 mRNA to less than 5% in the segment distal to the nerve injury. Experiments using in situ hybridization localized the SR13/PMP-22 mRNA in Schwann cells. Schwann cells present in the vicinity or distal to the nerve cut repressed the signal for the message. In situ hybridization experiments also demonstrated that dorsal root ganglia satellite cells contained the message for SR13/PMP-22. The SR13/PMP-22 antisera used in our study showed a complex pattern of staining. As expected, the SR13/PMP-22 antibody peptide 1 immunoreacted with the sciatic nerve sheath. However, immunocytochemistry of the dorsal root ganglia revealed that the staining was contained in the neuron's cell body and processes and also in satellite cells. We also identified immunoreactive cell bodies and fibers in the spinal cord dorsal horn. Tissue culture studies demonstrated that SR13/PMP-22 mRNA is induced in NGF treated PC12 but not in C6 glioma cell lines grown under experimental conditions that stimulated cell growth arrest. Our experiments suggest that SR13/PMP-22 may have some other function(s) in addition to its hypothesized role in peripheral myelination. © 1994 Wiley-Liss, Inc.     

Thy-1 antigen expression on rat brain cell lines

Cell lines derived from the central nervous system of rats were screened serologically for the presence of Thy-1 (theta), a cell surface differentiation antigen shared by brain and thymus of rats and mice. Both cytotoxicity absorption and indirect immunofluorescence assays were performed using a rabbit anti-rat thymocyte serum (ATS) with Thy-1 specificity. The complement-dependent cytotoxicity of ATS detected a mouse-rat cross-reacting determinant of the molecule bearing the Thy-1 antigen. Of 20 lines tested, 1 of 6 neuronal and 7 of 14 non-neuronal lines expressed Thy-1, as judged by their capacity to absorb ATS cytotoxicity for a Thy-1 positive thymoma line. Similar results were obtained in quantitative absorption assays of these lines employing a mouse anti-Thy 1.1 alloantiserum, but the xenoantiserum (ATS) was more sensitive for detecting the rat molecule bearing Thy-1. Indirect immunofluorescence, which was performed on several of the lines, yielded results in complete agreement with the cytotoxicity absorption assays, and revealed a generalized distribution of antigen in a speckled or patchy pattern over the membrane of cell bodies and processes.     

Schwann cell caveolin-1 expression increases during myelination and decreases after axotomy

The caveolins are a family of related proteins that form the structural framework of caveolae. They have been implicated in the regulation of signal transduction, cell cycle control, and cellular transport processes, particularly cholesterol trafficking. Caveolin-1 is expressed by a variety of cell types, including Schwann cells, although its expression is greatest in differentiated cell types, such as endothelial cells and adipocytes. In the present work, we characterize caveolin-1 expression both during rat sciatic nerve development and after axotomy. Schwann cells express little caveolin-1 on postnatal days 1 and 6. By P30, myelinating Schwann cells express caveolin-1, which is localized in the outer/abaxonal myelin membranes as well as intracellularly. After axotomy, Schwann cell caveolin-1 expression in the distal nerve stump decreases as Schwann cells revert to a premyelinating (p75-positive) phenotype; residual caveolin-1 within the nerve largely localizes to myelin debris and infiltrating macrophages. We speculate that caveolin-1 plays a role in the biology of myelinating Schwann cells.     

c-Kit receptor expression in normal human Schwann cells and Schwann cell lines derived from neurofibromatosis type 1 tumors

The growth factor receptor c-Kit has several well-characterized functions during the development of numerous cell types, including red blood cells, mast cells, and melanocytes. Its role in Schwann cells has been described in transformed cells derived from malignant peripheral nerve sheath tumors from patients with neurofibromatosis type 1 (NF1 MPNST; Badache et al. [1998] Oncogene 17:795-800). However, c-Kit functions have not been investigated in normal Schwann cells. We report here that neonatal rat Schwann cells express low c-Kit levels, whereas expression levels for c-Kit are high for Schwann cells derived from MPNST of NF1 patients. In addition, c-Kit expression is not detectable in normal adult human Schwann cells. Although the c-Kit ligand stem cell factor (SCF) induces the phosphorylation of protein kinase B (or Akt) and prevents apoptosis in Schwann cells, SCF has no effect on the proliferation or differentiation of Schwann cells.     

Region‐selective glutamine synthetase expression in the rat central nervous system following portocaval anastomosis

Glutamine synthetase (GS) content was investigated using immunohistochemical methods in the hippocampus, cerebellum and spinal cord of rats after long‐term portocaval anastomosis (PCA). Six months after surgery, GS content was increased in several areas of each region and decreased in others, compared with controls. In the hippocampus, the CA1–CA3 pyramidal subfields and the dentate molecular layer had a high level of GS expression; PCA reduced GS content in other hippocampal regions, such as the dentate hilus. In the cerebellum, PCA significantly increased GS immunoreactivity in the Bergmann glial processes of the molecular layer and decreased GS immunoreactivity in astrocytes of the granule cell layer. In the spinal cord, GS immunoreactivity increased in the dorsal horn and decreased in the ventral horn. Blood vessels located in zones with GS‐immunopositive perineuronal astrocytes in PCA‐exposed brains were surrounded by strongly GS‐immunostained perivascular processes. These results suggest that PCA exposure had a differential effect on GS expression in different regions of the central nervous system. The increased immunoreactivity of GS‐positive cells in PCA‐exposed brains correlates with glutamatergic areas, which may contribute to protecting neurons against extracellular glutamate and/or ammonia excess.     

Syntaxin 1 is required for DCC/Netrin‐1‐dependent chemoattraction of migrating neurons from the lower rhombic lip

Directed cell migration and axonal guidance are essential steps in neural development that share many molecular mechanisms. The guidance of developing axons and migrating neurons is likely to depend on the precise control of plasmalemma turnover in selected regions of leading edges and growth cones, respectively. Previous results provided evidence of a signaling mechanism that couples chemotropic deleted in colorectal cancer (DCC)/Netrin‐1 axonal guidance and exocytosis through Syntaxin1(Sytx1)/TI‐VAMP SNARE proteins. Here we studied whether Netrin‐1‐dependent neuronal migration relies on a similar SNARE mechanism. We show that migrating neurons in the lower rhombic lip (LRL) express several SNARE proteins, and that DCC co‐associates with Sytx1 and TI‐VAMP in these cells. We also demonstrate that cleavage of Sytx1 by botulinum toxin C1 (BoNT/C1) abolishes Netrin‐1‐dependent chemoattraction of migrating neurons, and that interference of Sytx1 functions with shRNAs or Sytx1‐dominant negatives disrupts Netrin‐1‐dependent chemoattraction of LRL neurons. These findings indicate that a Sytx1/DCC interaction is required for Netrin‐1 guidance of migrating neurons, thereby highlighting a relationship between guidance signaling and SNARE proteins that regulate membrane turnover.     

Interleukin-1 and tumor necrosis factor-mediated regulation of C3 gene expression in human astroglioma cells

In this report, we show that in the human astroglioma cell line D54-MG, both interleukin-1 (IL-1β) and tumor necrosis factor-alpha (TNF-α) enhance C3 gene expression in a time- and dose-dependent manner. Kinetic analysis demonstrates that after 96 h, C3 mRNA levels increase approximately 30-fold and 20-fold in response to IL-1β or TNF-α, respectively. C3 protein production increases proportionally, reaching levels 36-fold and 18-fold higher than untreated controls upon exposure to IL-1β or TNF-α, respectively. D54-MG cells require a minimal 1 h exposure to IL-1β in order to enhance C3 gene expression significantly, while 4 to 8 h are required for TNF-α. Simultaneous treatment of D54-MG cells with IL-1β and interferon-gamma (IFN-γ) resulted in an additive increase in both C3 mRNA and protein expression, a finding not seen with the combination of TNF-α and IFN-γ. Primary rat astrocytes also express increased C3 mRNA levels after 48 h in response to IL-1β (5.3-fold increase) and TNF-α (7-fold increase), while an additive effect was observed upon simultaneous treatment with both IL-1β and IFN-γ. In the central nervous system (CNS), endogenous complement and cytokine production by astrocytes, and enhancement by IFN-γ, a product of activated T cells often seen in the CNS in neural autoimmune disease, may contribute to the pathogenesis of inflammatory demyelinating diseases such as multiple sclerosis.     

Thy-1 cell surface antigen on cloned nerve cell lines of the rat and mouse: Amount, location and origin of the antigen on the cells

The Thy-1 antigen is a cell surface glycoprotein found on a number of tissues of the rat and mouse, including neurons. The amount of this antigen has been measured on several rat and mouse clonal cell lines of nervous tissue origin. Three of the rat cell lines (clones PC12, BN1010-3, BN1010-1) were found to contain substantial amounts of the antigen, having as many as 1.6 million Thy-1 antigenic sites per cell. This amount was independent of the cell density or growth rate. Most, if not all, of the antigen is accessible to antibody binding on the external surface of the cells, and is synthesized by the cells.     

Complement activation on human neuroblastoma cell lines in vitro: route of activation and expression of functional complement regulatory proteins

Two human neuroblastoma cell lines activated the classical pathway of complement in serum. Activation caused the opsonisation of these cells with complement fragments but with moderate cell killing. Neuroblastoma expressed regulators MCP and CD59 but did not express DAF or CR1. Neutralisation of CD59 rendered the cells susceptible to killing. Neuroblastoma also expressed C1-inhibitor, factor H, clusterin and S-protein. Expression of several regulators was enhanced by incubation with cytokines. Complement inhibition using soluble CR1 markedly reduced opsonisation and killing of neuroblastoma. Our results suggest that complement might play a role in neuronal loss and that treatment with complement inhibitors might be of therapeutic value.     

Temporal expression of osteopontin and CD44 in rat brains with experimental cryolesions

Expression of osteopontin and CD44 in the brain was studied after cryolesioning to understand how osteopontin and its receptor, CD44, are involved in processes in the brains of rats with cryolesions. Western blot analysis showed that osteopontin increased significantly at days 4 and 7 post-injury and declined slightly thereafter in cryolesioned brains in comparison with levels in sham-operated controls. An immunohistochemical study localized osteopontin in activated microglia/macrophages in the core lesions, where the majority of macrophages proliferate. Osteopontin was also detected temporarily in some neurons and a few astrocytes in the lesion periphery on days 4 and 7 post-injury, but the immunoreactivity in macrophages, neurons, and astrocytes disappeared by day 14 post-injury. There was some CD44, a receptor for osteopontin, in the brain cells of sham-operated rats. After injury, intense CD44 immunostaining was seen in the majority of macrophages and in reactive astrocytes, but not in neurons, in the ipsilateral lesions after day 4 post-injury, and this immunoreactivity remained on day 14 post-injury. These findings suggest that activated microglia/macrophages and some neurons are major sources of osteopontin during the early stage of brain damage induced by a cryolesion and that osteopontin interacts with CD44 expressed on astrocytes and activated microglia/macrophages in the damaged cerebral cortex, possibly mediating cell migration after cryolesioning in the rat brain.