application): While it is well recognized that the accumulation of extracellular amyloid is a hallmark of AD, many of the key pathological events of AD may also be directly related to the intracellular accumulation of this insoluble peptide. Recent studies in cell culture have indicated that A-beta1-42 (but not A-beta1-40), once accumulated inside cells, is resistant to degradation and can serve as a nidus for a prion-like replication model (the solid phase replication model for A-beta accumulation). Further studies in brain suggest that A-beta1-42 is the preferential form that accumulates in AD and that A-beta can be found within neurons though much less is known about its state and associated proteins. Once present in cultured cells, the aggregated amyloid induces the production of reactive oxygen species and lipid peroxidation products and ultimately results in the leakage of the lysosomal membrane. The breakdown of the lysosomal membrane may be a key pathological event, leading to the release of heparan sulfate and lysosomal hydrolysases. Taken together, these observations provide the novel view that amyloid deposits and some of the early events of amyloid pathogenesis initiate randomly within single cells in AD. This mechanism can explain some of the more enigmatic features of AD pathogenesis, like the focal nature of amyloid plaques, dystrophic neurites and neurofibrillary tangle pathology and the miscompartmentation of extracellular and cytosolic components observed in the AD brain. This project will use a combination of biochemical and light and electron microscopic, immunocytochemical approaches to study the accumulation of intracellular amyloid. The studies will be a combination of in vitro on cultured cells and in vivo studies on control and-AD brain, thereby combining the precision of in vitro studies with the need to evaluate predictions and findings in vivo. Select experiments will also be conducted on the aged canine brain that accumulates extensive amyloid and where the postmortem conditions can be precisely controlled. Preliminary data support the hypothesis that amyloid (""""""""preamyloid"""""""") accumulates in neurons in the aging and AD brain, can be shown to have a granular appearance in neurons and may be associated with a breakdown in intracellular compartmentation.
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