The mechanism of amyloid beta peptide (beta/A4) accumulation in familial Alzheimers disease (FAD) and Down's syndrome is unknown and probably heterogeneous. The discovery of point mutations in exon 17 of betaPP770 in affected members of several families with AD is evidence that an abnormal beta amyloid precursor protein (betaAPP) can cause the disease in some cases. No genetic defect has been reported in the large Canadian and Italian FAD pedigrees which have known linkage to chromosome 21. Gene duplication is implicated in Down's syndrome. Our goal to elucidate the cause of beta/A4 accumulation in each of these conditions -will be addressed by the following specific experiments: 1) To examine the possibility of altered transcription of the betaAPP gene between affected and unaffected members of the Canadian and Italian families. We ask whether differences in the levels betaAPP MRNA or protein in FAD occur and if so, whether this reflects altered functioning of the promotor region or changes in MRNA stability. Total endogenous MRNA in dividing and stressed fibroblast cell lines from family members will be correlated with betaAPP protein levels by immunoblot. The same cell lines will also be transiently transfected with a full length betaApp promotor-reporter gene construct. 2) To examine the question, of how betaAPP levels are regulated as a function of gene dosage, we will address the unmet need to analyze rigorously the level of betaAPP transcripts in cells from living patients with trisomy 21 and controls. Total betaAPP MRNA and protein levels in lymphocyte lysates will be quantitated and compound to other gene products on or off chromosome 21. 30 To determine whether betaAPP exon 17 mutations a) favor an alternative intracellular cleavage event that generates carboxyl terminal fragment(s) containing the intact betaA4 or b) cause the appearance of such fragments only under special cellular conditions (e.g. stress). Pulse chase experiments and immunoprecipitations with various betaAPP antibodies of cells transfected with the mutant betaAPP CDNA'S will be conducted. 40 To examine the hypothesis that a) the extracellular portion of the integral membrane glycoprotein, betaAPP, functions as a substrate adhesion molecule receptor, similar to the Integrins; and b) the intracellular portion binds to the cytoskeleton. Cellular ligands that bind to betaAPP under normal conditions will be identified as co-precipitating proteins on SDS- PAGE after cells in culture are metabolic labeled with 35 S methionine. The effect of the exon 17 mutations on transfected cell adhesion to the substratum will also be studied . An understanding of betaAPP gene transcription, MRNA stability, protein degradation and its normal function is critical to the prevention of beta/A4 accumulation.