The primary objective of this proposal is to investigate the underlying mechanisms of organophosphorus compound-induced delayed neurotoxicity (OPIDN). OPIDN is a distal axonopathy of the peripheral and central nervous systems and is characterized by axonal swellings containing aggregates of cytoskeletal proteins i.e., neurofilaments and tubulin. Within the past decade we have demonstrated that pathognomonic of OPIDN is an aberrant increased autophosphorylation of calcium/calmodulin kinase II (CaM Kinase II) and increased phosphorylation of cytoskeletal proteins i.e., MAP 2, tubulin, neurofilament triplet proteins, and myelin basic protein. Concurrent with increased phosphorylation is an enhancement in calmodulin binding to CaM Kinase II. In this proposal we specifically propose to 1) study the regulation of CaM Kinase II autophosphorylation and the phosphorylation of its substrate cytoskeletal proteins; 2) characterize the protein makeup of the axonal swellings; 3) correlate the phosphorylation of cytoskeletal proteins with axonal degeneration. To determine the regulatory mechanisms involved in protein phosphorylation, the in vivo phosphorylation state of CaM Kinase II and its various substrate cytoskeletal proteins in 0P treated and control hens will be determined. To determine the extent of isoforms of CaM Kinase present and their involvement in the development of OPIDN, we plan to construct a cDNA library to the hen enzyme to probe for alterations in the alpha and beta mRNA's upon 0P treatment. Secondly, we will determine the nature of the cytoskeletal aggregates with respect to their phosphorylation state and time and spatial distribution. This will be accomplished by implementing immunohistochemical techniques. Lastly, we will test the hypothesis that aberrant increased phosphorylation of neurofilaments decreases their axonal transport rate which results in their accumulation at distal parts of the axons. We will determine the rates of axonal transport of specific cytoskeletal proteins in the presence or absence of OPs. [3H]Proline and [32p]orthophosphate will be used to radiolabel the cytoskeletal proteins and visualize them electrophoretically following segmental analysis. Cumulatively, our goal is to demonstrate that functional changes in CaM Kinase II lead to increased phosphorylation and aggregation of cytoskeletal elements resulting in axonal degeneration.
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