Alzheimer’s Disease and γ-Secretase Cleavage:
The Effect of PS1-FAD Mutations on γ-Secretase Cleavage of Notch and APP

Alzheimer’s disease (AD) is a devastating disorder of the elderly, which affects the ability to remember, to think and to understand - all the things that we cherish most about being human. It is the most common neurodegenerative disease, affecting 2 million Americans. AD is ultimately fatal, with an average duration of 6-10 years. Currently available therapeutic interventions do not prevent the disease, but instead offer limited symptomatic improvement. These facts add to the importance of developing new therapeutic approaches in order to prevent Alzheimer’s disease.

Although it is predominantly a disease of late life, there are families in which Alzheimer’s disease is inherited as an autosomal dominant disorder of mid-life. Three genes have been implicated in this form of the disease (Familial Alzheimer’s Disease, FAD): the β-amyloid precursor protein (APP), presenilin1 (PS1) and presenilin 2 (PS2). Interestingly, almost all mutations in these three genes increase the production of the longer amyloid β-peptide (Aβ₄₂), which is considered to be a central event in the pathogenesis of Alzheimer’s disease (AD). Aβ is generated from the β-amyloid precursor protein (APP) during its processing by a cascade of the events involving the cleavage of APP by β-secretase and γ-secretase. The latter involves an unusual intramembraneous cleavage of APP, which under normal conditions generates Aβ, containing 40 amino acids in length (80% Aβ₄₀); whereas, the production of the longer, more amyloidogenic peptide Aβ42 is favored when FAD-mutations are present in APP, PS1 or PS2 genes. However, the molecular mechanism(s) of this event is still not understood, and it is still unclear how almost all mutations in APP, PS1 and PS2 genes exert a unique effect on γ-secretase cleavage of APP, increasing the production of Aβ₄₂.

-secretase is a multiprotein complex. Several reports show that γ-secretase cleavage depends critically on the presenilins, suggesting that presenilins could represent part of the γ-secretase catalytic center. Interestingly, the majority of FAD-mutations occur in PS1 gene and the generation of Aβ is mainly PS1-dependent, suggesting that PS1 has an important function for g-secretase activity and specificity towards cleavage of APP. In addition to γ-secretase cleavage of APP, several other proteins have recently been discovered to be cleaved by the γ-secretase. Notch, a type I transmembrane protein that is important for development, is cleaved by γ-secretase, generating a C-terminal fragment called NICD (Notch IntraCellular Domain). However, γ-secretase cleavage of Notch is different from that at the Ab site of APP. It occurs more C-terminal to the cytoplasmic membrane boundary and is assumed to involve a single cleavage , in contrast to multiple cleavages at the A’ sites in APP. Recently, an additional γ-secretase cleavage within the transmembrane domain of APP has been found that resembles the γ-secretase cleavage of Notch. It is also presenilin-dependent and generates a CTFγ/AICD fragment (APP IntraCellular Domain) that is suggested to have an analogous role to NICD, mediating signaling in the nucleus. These findings suggest that γ-secretase complexes that cleave APP and Notch may be different.

Preliminary data in Dr. Goate’s laboratory on “-secretase cleavage of APP and Notch showed that some PS1-FAD mutations, while causing a unique effect on the increase in Aβ₄₂ generation, show completely opposing effects on the production of CTFg and NICD. These findings indicated that the γ-secretase complex that cleaves APP at the A’ site may be different from the γ-secretase that generates CTFγ from APP or NICD from Notch. For example, PS1-FAD mutation G209V was found to completely block the production of CTFg and NICD, while causing a FAD effect on A’ production (increase in A’42 levels). These results suggested that a single mutation in the PS1 gene can cause a loss of function of the γ-secretase towards generation of CTFγ and NICD but at the same time can cause a gain of function of the γ-secretase cleavage at the Aβ site of APP. The aim of this project was to analyze in more detail the effect of PS1-FAD mutations on “-secretase cleavage of APP and Notch. In particular, we generated additional FAD-mutations at the G209 site of PS1 (G209R and G209E) by site-directed mutagenesis. After transient cotransfection of the PS1-/-PS2-/- mouse embryonic fibroblast with either C99 or NotchDE and PS1 cDNA constructs, we detected a similar levels of CTFg and NICD produced by PS1-G209R and PS1-G209E FAD-mutations. These results indicated that PS1-FAD mutations affect CTFg and NICD generation in a similar manner, in contrast to Aβ, supporting our hypothesis that γ-secretase complexes that generate CTFγ/NICD and Ab may be different. We speculate that these cleavage events may be dependent on different γ-secretase cofactors.