OXYGEN RADICAL TOXICITY AND CHRONIC NEURONAL LOSS

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Oxygen radicals and other radical species are toxic to cells, most often by damaging membrane lipids and DNA. Cells have multiple levels of antioxidant defenses, and superoxide dismutase is the first level of defense against superoxide radicals. The notion that a gradual decrease in these defenses might play a role in aging or neurodegenerative diseases has been considered for years, but has remained controversial. The recent discovery of mutations in the gene for Cu/Zn superoxide dismutase (SOD1) in familial ALS provides the first good evidence that a specific defect in antioxidant defenses could be responsible for a neurodegenerative disease. To determine whether decreased SOD activity could contribute to motor neuron loss, we intend to model the chronic loss of SOD1 in spinal cord organotypic cultures and then to use this model to investigate various neuroprotective strategies: (1) Toxicity Model: To develop a model of chronic oxidative toxicity in organotypic spinal cord cultures. Is chronic inhibition of superoxide dismutase toxic to motor neurons in spinal cord: We will develop a rodent spinal cord organotypic culture system, then define susceptibility of large cholinergic anterior horn cells to acute and chronic exposure to SOD1 inhibition. Significance: Creation of in vitro models allows for experimental control and pharmacologic manipulation that is impossible in patients. (2) Using a model of chronic oxidative injury, can motor neuron loss be prevented by antioxidants and other potential neuroprotectants: Significance: Current knowledge of biochemical cascades operant in programmed cell death suggest that neuronal toxicity induced by oxidative stress might be blocked with both antioxidants and neurotrophic factors. These studies will provide data on which class of drugs might be most beneficial and specific to the prevention of motor neuron death in ALS. (3) The Bcl-2 family of natural antioxidants: Can the bcl-2 proto- oncogene protect against the neurotoxicity of chronic oxidative stress? Organotypic spinal cord cultures prepared from transgenic mice in which Bcl-2 has either been knocked out or over-expressed will be used to study the role of this putative mediator of antioxidant pathways as a natural antioxidant or neuroprotectant. Significance: Bcl-2 is a unique endogenous protein that can block apoptotic death. As a natural antioxidant, it could function to prevent chronic neural degeneration due to oxidative stress. (4) Specificity: Does glutamate toxicity participate in the selective targeting of motor neurons in models of oxidative stress? Significance: Selective cell death is central to understanding the pathogenesis of ALS as well as other neurodegenerative processes. These experiments will test the overall hypothesis that chronic excess oxygen radical play an important pathophysiologic role in motor neuron death. If this were true it would have fundamental consequences for our understanding on chronic oxidative insults in neural degenerative disorders and aging, and how to approach them neuropharmacologically.