FREE RADICALS AND MITOCHONDRIA IN NEURONAL APOPTOSIS

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DESCRIPTION (Adapted from applicant abstract): Half of all neurons produced during embryogenesis undergo apoptotic death shortly before or soon thereafter. Those neurons obtaining a sufficient quantity of a required neurotrophic factor escape this death. Neuronal death with characteristics similar to those seen during development also occurs after stroke and in neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Many similarities between developmental death and death caused by insult or disease suggest that comparable processes kill neurons in both situations, and that information gained about mechanisms of developmental neuronal death may aid in understanding and treating pathological death. The goal of this research proposal is to understand the role of free radical oxygen in the apoptotic death of neurons. The principal model of neuronal apoptosis that the applicant will study is that of sympathetic neurons in culture. These cells undergo apoptosis both in vivo and in vitro when deprived of nerve growth factor (NGF). Published data, and the applicant's preliminary results show that there is a dramatic increase in production of free radical oxygen (reactive oxygen species; ROS) by mitochondria in these cells soon after NGF deprivation. This ROS burst is a required component of apoptotic death. The applicant will test the hypothesis that ROS contribute to apoptosis by iron-catalyzed production of hydroxyl radicals in mitochondria. He postulates that these extremely reactive radical species directly, or indirectly, damage mitochondria and cause them to release cytochrome c, or other pro-apoptotic proteins, into the cytoplasm. He plans to use biochemical techniques, confocal microscopy, and electron microscopy to investigate this hypothesis. He also plans to test the hypothesis that the anti-apoptotic protein, Bcl-2 and the pro-apoptotic protein, Bax, promote neuronal survival or death by regulating the ROS burst or ROS effects on mitochondrial integrity. He will test these hypotheses by over-expressing Bcl-2 in neurons, by intracellular microinjection of a Bcl-2 expression vector, and by use of neurons from Bcl-2 transgenic mice or Bax-deficient mice. To determine the general importance of these findings about the role of ROS, mitochondria, and the Bcl-2 family in apoptosis of sympathetic neurons, the applicant will investigate their role in a CNS model system, cerebellar granule cells in culture. These studies will provide clear answers about the role of free radicals and mitochondria in neuronal apoptosis and of identifying ways of manipulating this death pharmacologically.