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Background: Neuromelanin-containing neurons of the substantia nigra are highly vulnerable to degenerate in Parkinson’s disease. Inhibition of the respiratory chain or formation of reactive oxygen species (ROS) by intracellular neuromelanin and triggering of inflammatory processes by extracellular neuromelanin emanating from melanized neurons after their demise are thought to be causally implicated in the high vulnerability of melanized neurons. Objective: We addressed the direct effect of purified neuromelanin on mitochondrial complex I activity, and its influence on ROS production and survival of primary mesencephalic neurons in the presence or absence of glia. Methods: Neuromelanin was isolated from midbrain of postmortem human brains. The content in iron and other elements was measured by inductively coupled mass spectrometry. The effect of neuromelanin on mitochondrial complex I activity was analyzed in post-nuclear extracts. Primary neuronal enriched and neuron-glia mixed cultures from midbrain were treated with different concentrations of neuromelanin. The generation of ROS was determined by fluorochrome detection. MAP2-positive and TH-positive neuronal viability was analyzed. Results: Neuromelanin did not affect complex I activity, but concentration-dependently increased ROS production in neurons and reduced the number of MAP2-positive and TH-positive cultured neurons. Glia protected neurons against the neuromelanin toxicity. Conclusion: Extracellular neuromelanin is detrimental to neurons implicating a mechanism of intracellular ROS production, but not complex I inhibition. ROS formation may be catalyzed by iron, which was sensitively identified in purified neuromelanin (3.3 mg/g). Importantly, we demonstrate that glial cells have the potential to mitigate the neurotoxic effect of neuromelanin. Copyright © 2007 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]