NF-kappaB-dependent production of nitric oxide by astrocytes mediates apoptosis in differentiated PC12 neurons following exposure to manganese and cytokines

Document Type

Article

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.

Medical Subject Headings

Animals; Apoptosis (physiology); Astrocytes (cytology, metabolism); Caspases (metabolism); Cell Differentiation (physiology); Cells, Cultured; Coculture Techniques; Cytokines (metabolism); Enzyme Activation; I-kappa B Proteins (genetics, metabolism); In Situ Nick-End Labeling; Manganese (metabolism); Mice; Mice, Inbred C57BL; NF-KappaB Inhibitor alpha; NF-kappa B (metabolism); Neurons (cytology, physiology); Nitric Oxide (metabolism); Nitric Oxide Synthase Type II (genetics, metabolism); PC12 Cells; Rats; S-Nitroso-N-Acetylpenicillamine (metabolism)

Publication Date

11-18-2005

Publication Title

Brain research. Molecular brain research

ISSN

0169-328X

Volume

141

Issue

1

First Page

39

Last Page

47

PubMed ID

16168523

Digital Object Identifier (DOI)

10.1016/j.molbrainres.2005.07.017

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