Cytoskeletal proteins play crucial roles in maintaining neuronal form and function. The calcium-activated neutral proteases, calpains appear to regulate the concentration and function of some proteins. Much remains unknown about the normal interactions between calpains and cytoskeletal proteins, but alterations of these interactions are likely to impair neuronal function, and to play a role in certain neurodegenerative disorders, especially Alzheimer's disease. The first goal of this proposed investigation are 1) to characterize the calpain-induced degradation of the heat-stable microtubule-associated proteins (MAPs), MAP-2 and tau and determine the role of phosphorylation in modulating the proteolysis, 2) to determine how the phosphorylation states of MAP-2 and tau regulate their binding to microtubules and 3) to assess the effects of in vivo excitatory amino acid administration on the proteolysis of MAP-2 and tau. The importance of this study lies in the more complete delineation of the role of phosphorylation in modulating the metabolism and function of MAP-2 and tau and is accomplished by using a unique combination of methods recently applied in this laboratory. These techniques include: focussed- beam microwave irradiation (a procedures that rapidly inactivates brain enzymes in situ( to sacrifice animals prior to the isolation of the heat- stable MAPs, quantitative immunoblot analysis to determine the calpain- induced proteolysis rates of MAP-2 and tau and quantitative binding assay to determine the affinities of MAP-2 and tau for microtubules. Subsequent to the characterization of these processes in normal adult rats, age-related changes in these processes will be identified using Fischer 344 rats aged 3, 12, 18, 24, 30 mo. The results obtained will provide crucial information about the regulatory role if phosphorylation in the metabolism and function of MAP-2 and tau, the changes in these processes associated with aging and will help to clarify the role of aberrant cytoskeletal phosphorylation in pathological conditions.
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