Astrocyte-derived nitric oxide in manganese neurotoxicity: from cellular and molecular mechanisms underlying selective neuronal vulnerability in the basal ganglia to potential therapeutic modalities

Abstract

Chronic exposure to manganese (Mn) causes the neurodegenerative movement disorder, manganism. A mouse model was developed to elucidate mechanisms involved in the etiology and progression of injury. Twelve-week old female C57Bl/6J mice were exposed to MnCl2 (100 mg/kg/day) by oral gavage daily for 8 weeks. After the experiment striatal dopamine (DA) content was decreased with the manifestation of hypoactivity. A distinct population of neurons was vulnerable to the effects of Mn, including enkephalin (ENK)-positive projection neurons, interneurons expressing neuronal nitric oxide synthetase (nNOS/NOS1), and choline acetyltransferase (ChAT)-expressing interneurons. Activation of surrounding astrocytes occurred with expression of inducible nitric oxide synthase (iNOS/NOS2) and production of nitric oxide (NO)/peroxynitrite (ONOO-). Activated astrocytes were detected primarily near the microvasculature in both the striatum and globus pallidus (GP). It is suggested that Mn exposure may damage the blood-brain barrier (BBB) and induce astrocytosis and NOS2 expression, subsequent NO production may cause the death of adjacent neurons. This hypothesis was also tested in an in vitro co-culture model. Differentiated pheochromocytoma cells (PC12 cells) were co-cultured with primary astrocytes and exposed to Mn and inflammatory cytokines. Mn and cytokines induced NOS2 expression and NO production in astrocytes, which correlated with apoptosis of PC12 cells. Apoptosis of PC12 cells was prevented by overexpression of a phosphorylation-deficient mutant of IκBα that inhibited NOS2 expression in astrocytes. It is concluded that Mn-and cytokine-dependent apoptosis in PC12 cells requires astrocyte-derived NO and nuclear factor κB (NF-κB)-dependent expression of NOS2. To explore possible means of interdicting this inflammatory process in astrocytes, a noval pharmacologic ligands of the peroxisome proliferator-activated receptor gamma (PPARγ) agonist, 1,1-Bis(3'-indolyl)-1-(p-trifluoromethylphenyl) methane (DIM-C-pPhCF3) were used in the same co-culture system. DIM-C-pPhCF3 protected PC12 cells from apoptosis through inhibition of NOS2 expression in astrocytes after Mn and cytokines exposure. By contrast, the PPARγ antagonist, 2-chloro-5-nitrobenzanilide (GW9622), had the opposite effect, increasing both NO production in astrocytes and neuronal injury. It is concluded that PPARγ is involved in the regulation of NOS2 expression in astrocytes and that agonists of PPARγ may represent a potential treatment method for Mn neurotoxicity.