γ-secretase is an intramembrane protease complex composed of presenilin, nicastrin, Aph-1, and Pen-2 that together cleave an ever increasing list of type I transmembrane proteins. Cleavage of one substrate, the Amyloid Precursor Protein (APP), generates the AP peptide which plays a critical role in the pathogenesis of Alzheimer's disease (AD). In this regard, γ-secretase has emerged as a promising therapeutic target for the treatment of AD; however, the exact mechanism of how γ-secretase cleaves and the function of APP processing remain unclear. My thesis was focused on these two important issues.

In the first part of my thesis, I wanted to determine which component within γ-secretase functioned as a substrate docking site. I identified Aph-1 as a potential candidate as it associated with both full-length and ectodomain-shed substrates. This was observed in overexpressed systems and validated under endogenous expression levels in cell lines and animal tissues with two unrelated γ-secretase substrates, APP and Jagged-1.

Next, I performed mutagenesis on Aph-1 to identify the regions within the protein that may play a critical role in the binding of substrates and association with the other γ-components. I performed both truncation and domain-swapped mutagenesis, where the transmembrane domains of Aph-1 were replaced with that of an unrelated protein. While these techniques have worked successfully with Presenilin, all Aph-1 mutants generated were unstable or failed to incorporate into γ-secretase.

In the final part of my dissertation, I examined the putative function of the APP intracellular domain (AICD) in transcriptional regulation. A Notch-AICD chimera was generated to allow for inducible generation of AICD via addition of the Notch ligand, Delta, or calcium depletion. As a control for AICD regulated transcription, I examined the Aβ degrading enzyme, neprilysin. Using the same cell lines and techniques employed by the original authors that identified this target, I was unable to reproduce their findings. In light of my negative data, multiple independent reports have also been unable to find a role of AICD in transcriptional regulation shedding considerable doubt on the role of AICD in gene regulation.