This application focuses on the cell and molecular biology of neuronal cell death and beta-amyloid formation in Alzheimer disease (AD) and Down syndrome (DS) and addresses specifically the role of two proteolytic systems in the disease pathogenesis. Defects in the calcium-activated neutral proteinase (calpain) system identified in AD brain and fibroblasts will be further studied using novel immunochemical approaches that reveal information on the in vivo activity of calpain in cells. The nature of the calpain system in vulnerable and resistant neuronal populations in AD and DS and the mechanisms by which altered regulation of calpains and their specific inhibitor, calpastatin, may contribute to AD neuropathology will be investigated. Human brain, genetic mouse mutant models of neuronal cell death and cultured neurons will be studied using light and EM immunocytochemistry, in situ hybridization and other molecular techniques. The activity of the calpain system in human neuroblastoma cells will be selectively up-regulated and down-regulated by intracellular loading of purified calpain system components and specific antibodies, antisense mRNA methods, and the use of new synthetic calpain inhibitors. Stably transfected neuronal cells will be engineered to promote up-regulation or down-regulation of the calpain system by driving expression of transfected calpain and calpastatin genes with strong promotors or transfecting cells with DNA constructs encoding antisense MRNA. Effects of these perturbations on downstream metabolic cascades regulated in part by calpains (e.g., second-messenger-stimulated protein phosphorylation and membrane cytoskeleton dynamics and metabolism) will be examined using pulse labeling techniques. Western blot analysis, phosphopeptide mapping etc. Using these models, we will test the hypothesis, based on preliminary evidence, that the calpain system is involved in APP processing and investigate the possibility that modulating the calpain system will protect neuronal cells against calcium-induced injury and will rescue transfected cells expressing the neurotoxic C-terminal fragment of APP (APP 643-695). Using immunochemical and molecular techniques, we will examine the molecular basis for our observations that lysosomal hydrolases greatly increase in affected neurons and are abnormally localized extracellularly with beta-amyloid in AD and DS. How lysosomal system abnormalities relate to neuronal cell death and beta-amyloid deposition will be examined in AD and DS brain. Selected genetic animal models of neuronal cell death will be used to establish the relationship of lysosome abnormalities to specific modes of neurodegeneration. A pilot project will investigate the involvement of a key membrane cytoskeletal protein, protein 4.1, in neurofibrillary tangle formation and also its regulation by phosphorylation in vivo in retinal ganglion cell neurons. Together, these studies should increase our understanding of the role of proteolytic systems in neuronal cell death and identify strategies for reducing the vulnerability of cells to degeneration in Alzheimer's disease.

National Institute of Health (NIH)
National Institute on Aging (NIA)
Unknown (R35)
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Biological and Clinical Aging Review Committee (BCA)
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Mc Lean Hospital (Belmont, MA)
United States
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Nixon, Ralph A (2004) Niemann-Pick Type C disease and Alzheimer's disease: the APP-endosome connection fattens up. Am J Pathol 164:757-61
Nixon, Ralph A (2003) The calpains in aging and aging-related diseases. Ageing Res Rev 2:407-18
Anderson, A J; Stoltzner, S; Lai, F et al. (2000) Morphological and biochemical assessment of DNA damage and apoptosis in Down syndrome and Alzheimer disease, and effect of postmortem tissue archival on TUNEL. Neurobiol Aging 21:511-24
Cataldo, A M; Peterhoff, C M; Troncoso, J C et al. (2000) Endocytic pathway abnormalities precede amyloid beta deposition in sporadic Alzheimer's disease and Down syndrome: differential effects of APOE genotype and presenilin mutations. Am J Pathol 157:277-86
Mathews, P M; Cataldo, A M; Kao, B H et al. (2000) Brain expression of presenilins in sporadic and early-onset, familial Alzheimer's disease. Mol Med 6:878-91
Lai, F; Kammann, E; Rebeck, G W et al. (1999) APOE genotype and gender effects on Alzheimer disease in 100 adults with Down syndrome. Neurology 53:331-6
Buchner, K; Adamec, E; Beermann, M L et al. (1999) Isoform-specific translocation of protein kinase C following glutamate administration in primary hippocampal neurons. Brain Res Mol Brain Res 64:222-35
Adamec, E; Beermann, M L; Nixon, R A (1998) Calpain I activation in rat hippocampal neurons in culture is NMDA receptor selective and not essential for excitotoxic cell death. Brain Res Mol Brain Res 54:35-48
Troncoso, J C; Cataldo, A M; Nixon, R A et al. (1998) Neuropathology of preclinical and clinical late-onset Alzheimer's disease. Ann Neurol 43:673-6
Shea, T B; Dahl, D C; Nixon, R A et al. (1997) Triton-soluble phosphovariants of the heavy neurofilament subunit in developing and mature mouse central nervous system. J Neurosci Res 48:515-23

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