Calcium-activated neutral proteinases (CANP) are pivotal enzymes in cellular regulation and have been implicated in mechanisms of aging and memory formation. Based on recent evidence, we hypothesize that down- regulation or inhibition of calcium-mediated proteolysis in Alzheimer's disease (AD) is an early event in the pathogenesis of neurofibrillary degeneration and altered membrane cytoskeleton dynamics leading to membrane injury and cell death. Our objectives are to define the physiological functions of CANP in neuronal cells and to directly test the hypothesis that reduced CANP activity leads to alterations of cellular function relevant to AD pathogenesis. Novel procedures will be employed to selectively decrease the physiological activity of CANPs in intact NB2a/d1 cells, a new mouse subclone that expresses CANP system components and all major neuronal cytoskeletal proteins. Down- regulation of CANP activity will be achieved by several approaches: (a) microinjection of two distinct forms of purified brain calpastatin, the specific endogenous inhibitors of CANP; (b) microinjection of specific anti-CANP antibodies and (c) introduction into cells of synthetic oligodeoxyribonucleotide sequences that are complementary to selected sequences of endogenous mRNA transcripts encoding mouse brain CANPs (""""""""antisense"""""""" RNA) and are chemically modified to increase their permeability and nuclease-resistance. CANP down-regulation will be established by immunoassay of CANP and calpastatin content and radioassay of CANP activity. We will investigate the consequences of CANP down-regulation on the proteolysis of specific cytoskeleton and cytoskeleton-associated proteins in intact NB2a/d1 cells using newly developed in vivo techniques. Alterations in the ultrastructure of the fibrous and membrane cytoskeleton will be characterized at the light and electron microscope level using conventional stains and immunocytochemistry with antibodies to a range of cytoskeleton-related proteins. Cytoskeleton abnormalities and other observed consequences of CANP regulation will be related to cellular changes being characterized in separate ongoing studies of proteolysis in AD brain. These studies, the first of their kind in neuronal cells, should provide fundamental information on the cellular function of the CANP system and its role in Alzheimer's disease.
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