Major advances have been made toward identifying specific neuronal systems that degenerate in Alzheimer's disease and related disorders; however, little is known about the molecular mechanisms underlying the neuronal degeneration itself. Our recent findings implicate proteolysis, and we propose that, in addition to mediating late stages of neuronal degeneration, altered activity of proteolytic enzyme systems may be an early neuronal sponse in metabolically compromised neurons and may underlie functional deficits in neurons before they are irreversibly injured. Our overall objectives are to investigate morphologic and molecular aspects of the two major proteolytic systems in neurons--lysosomes and the calcium-activated neutral proteinase (CANP) system--in order to clarify relationship of specific proteinase alterations to the structural changes that accompany cell atrophy and death during normal aging and in a genetic, late-onset neurodegenerative disorder in mice. Using light microscopic and electron microscopic methods, we will examine the cellular and subcellular histopathology associated with progressive stages of degeneration in mice expressing the mutation Mnd (motor neuron degeneration). Recently developed antibodies against the major brain proteinases--CANP, cathepsins B, D, G, an L--and against two specific endogenous inhibitors of CANP and a series of non-proteinase lysosomal enzymes will be used in conjunction with computerized image analysis to evaluate the content and distribution of these antigens immunocytochemically in normal-appearing and degenerating neurons within vulnerable cell populations. The activities of selected lysosomal enzymes will be determined by enzyme cytochemical techniques applied the light and ultrastructural levels. Indirect evidence for altered CANP activity in vivo will be sought using antibody probes to suspected protein substrates of CANP. Parallel studies in mice at 3-30 months of age will distinguish degenerative features from processes of normal aging and clarify the interaction of the mutation defect with age related factors. To evaluate the synthesis of these proteinases and regulatory proteins, cDNA and cRNA probes to these proteolytic components will be used to assess levels of specific mRNAs within neurons in brains of control, mutant and aged mice by in situ hybridization. In addition to increasing our understanding of the important and complex process of proteolysis in normal mature and aging neurons, these studies will provide information pertinent to the molecular basis of neuronal dysfunction and cell death in Alzheimer's disease and other late-onset human neurodegenerative disorders.
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