Experiments will be carried out to study the processes whereby neurofilaments (NF) are degraded by calcium-activated neutral protease (CANP), the nature of the NF products which are generated by this fragmentation, the fate of the NF degradative products and, especially, the possibility that NF degradative products are transported retrogradely to the perikaryon and have regulatory (""""""""feedback"""""""") effects on the transcription, translation and/or assembly (i.e., turnover) of NF proteins. The enzymatic reaction between NF and CANP will be reconstituted from purified components and compared with the same reaction occurring in situ. Attention will be directed at how this reaction is modified by different forms of CANP, by the presence of endogenous inhibitor to the enzyme and when the substrate is present as insoluble """"""""filamentous"""""""" vs soluble """"""""protofilamentous"""""""" forms. Immunohistochemical methods will be used to compare the tissue distributions of CANP and CANP-Inhibitor with that of NF proteins. Monoclonal antibodies will be employed to probe the different forms of enzyme and enzyme inhibitor. The phenomena of NF degradation and turnover will be analyzed in pulse-labeled dissociated neuronal cultures. A model system of cell-free protein synthesis will be established in order to study translation of NF proteins, CANP and CANP-Inhibitor, the characteristics of the nascent proteins and factor (e.g., nucleotide-binding fragments of NF degradation) which may modify the translation of cytoskeletal proteins. The proposed studies will probe fundamental phenomena which regulate cell size and shape, thereby serving to establish and maintain neuronal connectivity and provide for the functional integrity of neural tissues. Furthermore, NF turnover may well underlie the capability of the neuron to respond to impairment by intrinsic or extrinsic factors of injurious or diseased states. Understanding the mechanisms which regulate NF turnover may allow the effects of neuronal impairment to be minimized or the regeneration and restoration of neuronal function to be optimezed.
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