This application addresses molecular events affecting the dynamics and organization of the neuronal cytoskeleton which may lead to the reduced regenerative capacity, atrophy and possible death of neurons in the brain during aging. We propose a comprehensive study of the axonal transport, posttranslational modification, deposition, and turnover of cytoskeletal proteins in retinal ganglion cell (RGC) neurons and the importance of these processes as determinants of neuronal cytoskeletal ultrastructure in adult and aged mice. New in vivo approaches and investigations of previously unstudied aspects of neuronal cytoskeletal dynamics are emphasized. We will establish during aging (a) changes in the transport rates and the extent of deposition of specific major and microheterogeneous forms of neurofilament proteins (NFP), tubulins, fodrin, and other cytoskeletal proteins along RGC axons using quantitative one- and two-dimensional PAGE analyses; (b) changes in the relative contribution of the transported and deposited protein pools to the total axonal content of these proteins measured chemically; and (c) changes in the in vivo and in vitro activities of posttranslational processing mechanisms that are active toward neuronal cytoskeletal proteins including protein kinases and phosphatases, a 145K NFP-specific calcium-activated neutral proteinase (CANP), and a recently discovered 200K NFP-modifying process. The in vivo turnover rates of transported and deposited neuronal cytoskeletal proteins will be determined by new quantitative single- and double-isotope techniques. The breakdown of cytoskeletal proteins and the possible in vivo action of specific neuronal proteinases, including CANPs, will be analyzed by a novel immunoblot approach. By computerized EM morphometry, we will examine the impact of age-related changes in these processes on ultrastructural features of the axon including axon caliber size, and the densities and spatial organization of neurofilaments, microtubules and other organelles along axons. New information about the formation maintenance and dynamic functions of the neuronal cytoskeleton will be obtained that should be pertinent to basic neurobiological issues of developmental growth, regenerative capacity and functional plasticity of neurons. These studies are directly relevant to the basic mechanisms of neuronal aging, to the pathogenesis of neurofibrillary pathology and cell death in neuronal disorders including Alzheimer's disease and to the rescue and regeneration of injured retinal neurons.
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