This continuation application molecular events affecting the dynamics and organization of the neuronal cytoskeleton which may lead to reduced functional and regenerative capacity, atrophy, and possible death of neurons in the brain during aging. We propose continued comprehensive studies of the axonal transport, posttranslational modifications, assembly, and turnover of cytoskeletal proteins in retinal ganglion cell 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 test the hypothesis that the observed slowing of axonal transport during aging preferentially involves cytoskeleton proteins. The molecular basis of this age-related transport slowing and of accompanying changes in neurofilament (NF) and microtubule (MT) density and organization will be investigated in studies on the early assembly events, phosphorylation, and turnover of neurofilament proteins and tubulin in young adult and aged mice. Using a combination of in vivo and in vitro techniques involving two dimensional phosphopeptide map analysis, protein sequencing, and ultrastructural analyses applied to normal mature retinal neurons, we will further define the complex role of phosphorylation in regulating the behaviors of neurofilament protein and tubulin during subunit polymerization, entry into axons, and incorporation into the axonal cytoskeleton. Major regional changes in the organization of the NF-MT cytoskeleton along retinal cell axons, defined by computer assisted EM morphometric analyses, will be correlated with the phosphorylation of specific polypeptide domains on neurofilament subunits by identified protein kinases in vivo. New information about the neuronal cytoskeleton will be obtained that should be pertinent to basic neurobiological issues of developmental growth, function, and plasticity of neurons. These studies are directly relevant to the basic mechanisms of neuronal aging, the pathogenesis of neurofibrillary pathology and cell death in neuronal disorders including Alzheimer's disease, disorders affecting retinal ganglion cells such as glaucoma, and the rescue and regeneration of injured retinal neurons.
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