| The production and aggregation of the amyloid beta-peptide (Abeta) is thought to play a central role in Alzheimer's disease (AD) pathogenesis. The presenilin (PS)-containing gamma-secretase complex cleaves the amyloid beta-protein precursor C-terminal fragment (APP CTFbeta) to generate Abetas of 38-49 residues. Evidence suggests that these Abetas are the result of successive gamma-secretase cleavages, which are thought to start at the beta sites to generate Abeta48 or Abeta49, followed by C-terminal trimming mostly every three residues to produce secreted Abetas. Specifically, two product lines have been proposed: the Abeta49-46-43-40 line and the Abeta48-45-42-38 line. An increased proportion of aggregation-prone Abeta42 compared to Abeta40 is believed to be important in AD pathogenesis. Despite the apparent relevance of the production of the Abeta C-terminus in AD, questions surround the mechanisms by which gamma-secretase generates the Abeta spectrum and how familial AD-causing (FAD) mutations alter Abeta production.;This dissertation first examined the C-terminal trimming function of gamma-secretase and how PS FAD mutations alter this activity. We found that synthetic Abeta49, Abeta48, Abeta46, and Abeta45 are trimmed to Abeta40 and Abeta42 by gamma-secretase in vitro. Moreover, our results were consistent with the two-pathway model in which Abeta49 is primarily converted to Abeta40 and Abeta48 to Abeta42, but also demonstrated a small degree of crossover between the pathways. Most importantly, we found that PS1 FAD mutations dramatically reduce the efficiency of trimming of beta-cleaved Abetas, particularly the trimming of Abeta49 to Abeta40.;We also investigated substrate determinants for epsilon site endoproteolysis and C-terminal trimming of APP CTFbeta by gamma-secretase. The deletion of residues around the epsilon sites indicated that upstream sequences, and not depth within the transmembrane domain, are the determinants of epsilon site specificity. We also show that instability of the APP CTFbeta transmembrane helix near the epsilon site increases endoproteolysis, and that instability near the carboxypeptidase cleavage sites facilitates C-terminal trimming by gamma-secretase.;Last, the potential role of Abeta45-49 in AD pathogenesis was considered. We did not detect these Abeta species in AD brains by immunoprecipitation and western blot. However, we developed cellular systems to investigate their toxicity and obtained preliminary data suggesting that these Abetas may be neurotoxic. |
