Neuronal degeneration is a common phenomenon associated with aging in the central nervous system. It has been hypothesized that the causes for either the degenerative changes associated with normal aging or the exaggerated damage seen in disease states such as Alzheimer's disease are linked to hyperfunction of L-glutamate as an excitatory transmitter. Such hyperactivity of glutamate neurotransmission leads to excessive intracellular calcium (Ca2+) accumulation through increased activation of glutamate receptors, especially of the N-methyl-D-aspartate (NMDA) receptors. Three hypotheses are being tested in the proposed experimental work. First, that there is a correlation between the expression of specific NMDA receptor proteins in brain neurons and the susceptibility of the same or neighboring neurons to damage induced by ROS. Second, that NO and ROS modify NMDA receptor proteins at specific sites so that the activity of the receptors is down-regulated. Third, that neurons that express high levels of NOS yet are partially protected from NMDA-induced damage may use a variety of processes to diminish the effects of NO and ROS on cell viability.
The specific aims for the proposed studies are: 1. To determine the importance of various forms of NMDA receptors in the activation of NOS and the formation of ROS in primary neuronal cultures. The ROS and NO generation in neurons will be measured following exposure to glutamate, NMDA, and various agonists and antagonists acting on NMDA receptors. The NMDA receptor subunits expressed and their role in cytotoxicity will be evaluated. 2. To characterize the specific amino acid modifications in either NMDAR1 or glutamate-binding proteins and of the rate of turnover of these proteins following generation of NO and oxygen free radicals in neurons. These studies will include the characterization of specific amino acid modifications in cloned and expressed proteins following exposure of the proteins in vitro to NO or oxoperoxynitrate (ONOO-) or superoxide anion or hydrogen peroxide. 3. To characterize the possible protective mechanisms operating in neurons which express high levels of NOS, mechanisms that may include alteration in calmodulin (CaM) activation of NOS due to oxidative modification of CaM, rapid Fe-nitrosation of a few proteins or high levels of expression of proteins such as BCL-2, BCL-X and superoxide dismutase (SOD). These studies will include the measurement of the levels of Fe-nitrosyl complex formation in neurons following activation of NMDA receptors and the determination of the comparative expression of the mRNA for MnSOD, CuZn- SOD, BCL-2, BCL-X and NOS. Two important outcomes of these studies will be a better understanding of the receptor macromolecules that are involved in the regulation of NO and ROS formation in neurons and of the mechanisms used by neurons to limit oxidative damage. If both goals are successfully accomplished, then we will have a better definition of potential targets for therapeutic intervention in neurodegenerative diseases associated with the aging process.
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