Sequential Proteolysis by γ-Secretase and Its Implications for Alzheimer’s Disease

Abstract

The production and aggregation of the amyloid β-peptide (Aβ) is thought to play a central role in Alzheimer’s disease (AD) pathogenesis. The presenilin (PS)-containing γ-secretase complex cleaves the amyloid β-protein precursor C-terminal fragment (APP CTFβ) to generate Aβs of 38-49 residues. Evidence suggests that these Aβs are the result of successive γ-secretase cleavages, which are thought to start at the ε sites to generate Aβ48 or Aβ49, followed by C-terminal trimming mostly every three residues to produce secreted Aβs. Specifically, two product lines have been proposed: the Aβ49-46-43-40 line and the Aβ48-45-42-38 line. An increased proportion of aggregation-prone Aβ42 compared to Aβ40 is believed to be important in AD pathogenesis. Despite the apparent relevance of the production of the Aβ C-terminus in AD, questions surround the mechanisms by which γ-secretase generates the Aβ spectrum and how familial AD-causing (FAD) mutations alter Aβ production. This dissertation first examined the C-terminal trimming function of γ-secretase and how PS FAD mutations alter this activity. We found that synthetic Aβ49, Aβ48, Aβ46, and Aβ45 are trimmed to Aβ40 and Aβ42 by γ-secretase in vitro. Moreover, our results were consistent with the two-pathway model in which Aβ49 is primarily converted to Aβ40 and Aβ48 to Aβ42, 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 ε-cleaved Aβs, particularly the trimming of Aβ49 to Aβ40. We also investigated substrate determinants for ε site endoproteolysis and C-terminal trimming of APP CTFβ by γ-secretase. The deletion of residues around the ε sites indicated that upstream sequences, and not depth within the transmembrane domain, are the determinants of ε site specificity. We also show that instability of the APP CTFβ transmembrane helix near the ε site increases endoproteolysis, and that instability near the carboxypeptidase cleavage sites facilitates C-terminal trimming by γ-secretase. Last, the potential role of Aβ45-49 in AD pathogenesis was considered. We did not detect these Aβ 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 Aβs may be neurotoxic.