Molecular studies of the γ-secretase complex : focus on genetic and pharmacological modulation

Abstract: γ-Secretase is a multi-subunit protease complex, composed of presenilin (PS1 or PS2), Nicastrin, Pen-2 and Aph-1, which generates the Alzheimer disease (AD) related 30-43 amino acid long amyloid β-peptide (Aβ). The complex is also crucial for important cell signaling, such as the Notch receptor pathway. More than 200 different Familial AD (FAD) causing mutations have been identified. They are all restricted to either PS1, PS2 or the amyloid β-precursor protein (APP), from which Aβ is generated, therefore proving how central γ-secretase mediated Aβ production is in AD pathogenesis. A common feature of FAD mutants is an increased Aβ42/Aβ40 ratio production. This results in a more amyloidogenic Aβ product and accelerated oligomerization and plaque formation. A number of γ-secretase inhibitors have been in clinical trials but so far there have been no major progress, due to mechanism-based side effects that is probably caused by impaired Notch signaling. It is therefore very important to develop novel therapeutic strategies targeting Aβ production without interfering with other crucial γ-secretase signaling pathwats. The aim of my thesis was to i) get a better understanding of the molecular basis behind the heterogeneous activity of γ-secretase resulting in different Aβ peptides, ii) to identify novel ways to target γ-secretase mediated Aβ production in a Notch sparing manner, iii) to explore the impact of a novel class of drugs called γ- secretase modulators (GSMs) on different γ-secretase processes. In Paper I, we specifically investigated whether the membrane integration and/or the active site of PS would be affected by different PS1 FAD mutations, which cause an increased Aβ42/Aβ40 production ratio. We found that while some FAD mutations located in hydrophobic domains around the catalytic site (TMD6, H7 and TMD7) changed the membrane integration of PS1, all FAD mutations studied affected the structure of the catalytic site of γ-secretase. In Paper II the large hydrophilic loop of PS1 was examined. Interestingly, by using a deletion mutant strategy, we found that, similar to many FAD mutants, Aβ38, Aβ39 and Aβ40 were dramatically decreased in the absence of the loop, while Aβ42 was affected to a lesser extent, resulting in a net increase in the Aβ42/Aβ40 ratio. Importantly, neither AICD nor NICD formation was impaired, suggesting that the integrity of the loop region is important for proper γ-site cleavage but not for the overall cleavage activity at the ε- site. To further study the mechanism of γ-secretase processing, we reported in Paper III the first study describing single residues in a γ-secretase component besides presenilin, such as Nicastrin, that affects the processing of γ-secretase substrates differently. In the final study, Paper IV, we studied the pharmacology of different GSMs and found that it is possible to generate in vivo potent second-generation γ-secretase-targeting modulatory compounds that are pre-selective for Aβ over Nβ production without affecting NICD formation. These findings may have major implications for the development of GSMs for AD and will be further discussed in the thesis.