Rapid progress has been made during the last two years on the two major structural lesions of Alzheimer disease (AD): neurofibrillary tangles (NFT) and amyloid. It has been shown that the abnormal antigens of NFT are also present in neurites, the delicate processes of neurons. Ultrastructural immunocytochemistry has demonstrated that the antigens shared with the NFT are located in 15nm thick straight filaments (SF), that are different from the paired helical filaments (PHF), the main components of the NFT of AD. Evidence suggests that this neurite change represents a primary alteration or an earlier stage in the formation of PHF. A second important finding is the evidence that ubiquitin (Ub), a protein that in association with other heat shock proteins (HSP) is thought to protect the cell by forming conjugates with abnormal proteins, plays a role in AD and in other neurodegenerative disease characterized by alteration of the cytoskeleton. Critical breakthroughs have been made on amyloid opening the way to the study of the b amyloid precursor protein (APP). We propose three sets of studies that extend our current work but take also into account the recent advances in AD. In the first set of studies we will treat PHF- and SF-enriched fractions with a series of solvents in order to identify extractable and insoluble PHF- and SF protein components. The second set of studies deals with Ub and HSP. It is designed to determine a) the expression of Ub, Ub-conjugates and HSP under normal and stress conditions, in fibroblasts from donors with sporadic and familial AD and Down syndrome as well as in neurons of mice with trisomy 16, the mouse model of Down syndrome; b) the cellular distribution of Ub-conjugates, and the identity of the Ub- acceptor proteins in these conjugates; c) the functional role that Ub and HSP have in neurons as demonstrated by blocking translational expression of Ub and 70kDa HSP. In the third set of studies, various aspects of the APP are investigated: a) the amount of APP mRNA will be determined under normal and stress conditions in cell from donors with sporadic and familial AD or Down syndrome as well as in neurons from mice with trisomy 16; b) the same cell systems will be assessed in fibroblasts and eventually neuroblastoma cells by blocking APP expression in antisense APP mutant cells. These three sets of studies are expected to provide significant information on a) the nature and mode of formation of PHF and SF in AD; b) the role of UB and HSP in AD and other neurodegenerative diseases; c) the identity and the basic features of APP which are needed to clarify the process of amyloid formation.
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