cdw17-antigen and Spinal-Cord-Injuries

The present study describes the role of glycosphingolipids in neuroinflammatory disease and investigates tumor necrosis factor alpha (TNFalpha)-induced astrogliosis following spinal cord injury. Astrogliosis is the hallmark of neuroinflammation and is characterized by proliferation of astrocytes and increased glial fibrillary acidic protein (GFAP) gene expression. In primary astrocytes, TNFalpha stimulation increased the intracellular levels of lactosylceramide (LacCer) and induced GFAP expression and astrocyte proliferation. D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol.HCl (PDMP), a glucosylceramide synthase and LacCer synthase (GalT-2) inhibitor, inhibited astrocyte proliferation and GFAP expression, which were reversed by exogenous supplementation of LacCer but not by other glycosphingolipids. TNFalpha caused a rapid increase in the activity of GalT-2 and synthesis of LacCer. Silencing of GalT-2 gene using antisense oligonucleotides also attenuated the proliferation of astrocytes and GFAP expression. The PDMP and antisense-mediated inhibition of proliferation and GFAP expression was well correlated with decreased Ras/ERK1/2 pathway activation. Furthermore, TNFalpha-mediated astrocyte proliferation and GFAP expression was also inhibited by LY294002, a phosphatidylinositol 3-kinase inhibitor, which was reversed by exogenous LacCer. LY294002 also inhibited TNFalpha-induced GalT-2 activation and LacCer synthesis, suggesting a phosphatidylinositol 3-kinase-mediated regulation of GalT-2. In vivo, PDMP treatment attenuated chronic ERK1/2 activation and spinal cord injury (SCI)-induced astrocyte proliferation with improved functional recovery post-SCI. Therefore, the in vivo studies support the conclusions drawn from cell culture studies and provide evidence for the role of LacCer in TNFalpha-induced astrogliosis in a rat model of SCI. To our knowledge, this is the first report demonstrating the role of LacCer in the regulation of TNFalpha-induced proliferation and reactivity of primary astrocytes.

Topics: Animals; Antigens, CD; Astrocytes; Cell Proliferation; Cells, Cultured; Chromones; Enzyme Activation; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Galactosyltransferases; Glial Fibrillary Acidic Protein; Lactosylceramides; Morpholines; Oligonucleotides, Antisense; Phosphatidylinositol 3-Kinases; ras Proteins; Rats; Rats, Sprague-Dawley; Signal Transduction; Spinal Cord Injuries; Tumor Necrosis Factor-alpha

In the present study a possible role of glycosphingolipids (GSLs) in inducible nitric oxide synthase (iNOS) gene expression and nitric oxide (NO) production after spinal cord injury (SCI) in rats has been established. In primary rat astrocytes lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) treatment increased the intracellular levels of lactosylceramide (LacCer) and induced iNOS gene expression. d-Threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol.HCI (PDMP), a glucosylceramide synthase and LacCer synthase (galactosyltransferase, GalT-2) inhibitor, inhibited LPS/IFN-gamma induced iNOS expression, which was reversed by exogenously supplied LacCer, but not by other glycosphingolipids. LPS/IFN-gamma caused a rapid increase in the activity of GalT-2 and synthesis of LacCer. Silencing of GalT-2 gene with the use of antisense oligonucleotides resulted in decreased LPS/IFN-gamma-induced iNOS, TNF-alpha, and IL-1beta gene expression. The PDMP-mediated reduction in LacCer production and inhibition of iNOS expression correlated with decreased Ras and ERK1/2 activation along with decreased IkappaB phosphorylation, NF-kappaB DNA binding activity, and NF-kappaB-luciferase reporter activity. LacCer-mediated Ras activation was redox-mediated and was attenuated by antioxidants N-acetyl cysteine (NAC) and pyrrolidine dithiocarbamate (PDTC). In vivo administration of PDMP after SCI resulted in improved functional outcome (Basso, Beattie, Bresnahan score); inhibition of iNOS, TNF-alpha, and IL-1beta expression; decreased neuronal apoptosis; and decreased tissue necrosis and demyelination. The in vivo studies supported the conclusions drawn from cell culture studies and provided evidence for the possible role of GalT-2 and LacCer in SCI-induced inflammation and pathology. To our knowledge this is the first report of a role of LacCer in iNOS expression and the advantage of GSL depletion in attenuating post-SCI inflammation to improve the outcome of SCI.

Topics: Acetylcysteine; Animals; Antigens, CD; Antioxidants; Apoptosis; Astrocytes; Cells, Cultured; Demyelinating Diseases; Drug Evaluation, Preclinical; Enzyme Induction; Enzyme Inhibitors; Fatty Acids; Female; Gait Disorders, Neurologic; Galactosyltransferases; I-kappa B Proteins; Inflammation; Interferon-gamma; Lactosylceramides; Lipopolysaccharides; Morpholines; Nerve Tissue Proteins; NF-kappa B; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Oxidation-Reduction; Phosphorylation; Proline; Protein Processing, Post-Translational; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Signal Transduction; Spinal Cord Injuries; Thiocarbamates; Transfection