Functional characterization of mutants of the Alzheimer's disease gamma-secretase enzyme complex

The Alzheimer's disease-linked protein, Presenilin (PS), forms the active site of gamma-secretase. This enzyme is responsible for cleaving the beta-amyloid precursor protein (APP) to produce amyloid beta (Abeta) and AICD, and also for cleaving Notch to produce NICD. There are two PS homologues in mammals, PS1 and PS2, and gene knock-out studies in mice suggest that PS1 is the major gamma-secretase and that it contributes disproportionately to Abeta peptide generation while PS2 plays a more minor role. Based on this and other observations we hypothesized that familial Alzheimer's disease (FAD) mutations in PS2 would have a dramatic effect on function in order to have an observable effect on Abeta levels in the presence of normal PS1 alleles. In support of our hypothesis, the four verified PS2 FAD mutations cause substantial changes in the Abeta 42/40 ratio, comparable to PS1 mutations that cause very early onset FAD. Most of the PS2 mutations also cause a significant decrease in Abeta 40, AICD and NICD production suggesting that they are partial loss of function mutations.; PS is stably associated with Nicastrin (NCT), PEN-2 and APH-1 within the gamma-secretase enzyme complex. Although much research has focused on the regions of PS that are important for gamma-secretase function, less is known about NCT. To further our understanding of the role of NCT in gamma-secretase activity and complex formation, we have undertaken a systematic evaluation of a conserved region in the juxtamembrane region of the extracellular domain of NCT. Two mutants, S632A and W648A, greatly reduce gamma-secretase activity as seen by a reduction in Abeta and AICD levels. Several lines of evidence suggest that these mutations within the juxtamembrane region of the extracellular domain result in reduced gamma-secretase activity because they affect the ability of NCT to stably associate with the other gamma-secretase components. Since NCT and APH-1 must first bind in order for PS and PEN-2 to stably join the complex, we propose that S632 and W648 are essential for a stable interaction with APH-1.