title: NF-kappaB-dependent production of nitric oxide by astrocytes mediates apoptosis in differentiated PC12 neurons following exposure to manganese and cytokines.
abstract: Neuronal injury in manganism is accompanied by activation of astroglia within the basal ganglia that is thought to increase production of inflammatory mediators such as nitric oxide (NO). The present studies postulated that astroglial-derived NO mediates neuronal apoptosis induced by manganese (Mn) and pro-inflammatory cytokines. Pheochromocytoma (PC12) cells differentiated with nerve growth factor (NGF) were co-cultured with primary astrocytes and exposed to Mn and tumor necrosis factor-alpha (TNF-alpha) plus interferon-gamma (IFN-gamma). Mn enhanced cytokine-induced expression of inducible nitric oxide synthase (NOS2, EC 1.14.13.39) and production of NO in astrocytes that correlated with apoptosis in co-cultured neurons, as determined by caspase activity, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL), and nuclear morphology. Apoptosis in PC12 neurons required the presence of astrocytes and was blocked by overexpression of a phosphorylation-deficient mutant of IkappaBalpha (S32/36A) in astrocytes that prevented induction of NOS2. Pharmacologic inhibition of NOS2 with (+/-)-2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT) significantly reduced neuronal apoptosis, and the addition of low concentrations of the NO donor, S-nitroso-N-acetylpenicillamine (SNAP), to neurons cultured without astrocytes was sufficient to recover the apoptotic phenotype following exposure to Mn and TNF-alpha/IFN-gamma. It is concluded that Mn- and cytokine-dependent apoptosis in PC12 neurons requires astroglial-derived NO and NF-kappaB-dependent expression of NOS2.
authors.affiliation: Molecular, Cellular, and Integrative Neuroscience, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, 1680 Campus Delivery, Fort Collins, CO 80526-1680, USA.
authors.forename: Xuhong
authors.initials: X
authors.lastname: Liu
authors.forename: Julie A
authors.initials: JA
authors.lastname: Buffington
authors.forename: Ronald B
authors.initials: RB
authors.lastname: Tjalkens
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D001253:Astrocytes / Q000166:cytology / Q000378:metabolism*; D020169:Caspases / Q000378:metabolism; D002454:Cell Differentiation / Q000502:physiology; D002478:Cells, Cultured; D018920:Coculture Techniques; D016207:Cytokines / Q000378:metabolism*; D004789:Enzyme Activation; D020672:I-kappa B Proteins / Q000235:genetics / Q000378:metabolism; D020287:In Situ Nick-End Labeling; D008345:Manganese / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism*; D009474:Neurons / Q000166:cytology / Q000502:physiology*; D009569:Nitric Oxide / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism; D016716:PC12 Cells; D051381:Rats; D026423:S-Nitroso-N-Acetylpenicillamine / Q000378:metabolism
chemical_list: D016207:Cytokines; D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606205:Nfkbia protein, mouse; C000606206:Nfkbia protein, rat; D000072000:NF-KappaB Inhibitor alpha; D009569:Nitric Oxide; D008345:Manganese; D026423:S-Nitroso-N-Acetylpenicillamine; D052247:Nitric Oxide Synthase Type II; D020169:Caspases