Presenilin clinical mutations can affect γ‐secretase activity by different mechanisms

Abstract

Mutations in human presenilin (PS) genes cause aggressive forms of familial Alzheimer's disease. Presenilins are polytopic proteins that harbour the catalytic site of the c-secretase complex and cleave many type I transmembrane proteins including b-amyloid precursor protein (APP), Notch and syndecan 3. Contradictory results have been published concerning whether PS mutations cause 'abnormal' gain or (partial) loss of function of c-secretase. To avoid the possibility that wild-type PS confounds the interpretation of the results, we used presenilin-deficient cells to analyse the effects of different clinical mutations on APP, Notch, syndecan 3 and N-cadherin substrate processing, and on c-secretase complex formation. A loss in APP and Notch substrate processing at e and S3 cleavage sites was observed with all presenilin mutants, whereas APP processing at the c site was affected in variable ways. PS1-D9 and PS1-L166P mutations caused a reduction in b-amyloid peptide (Ab) 40 production whereas PS1-G384A mutant significantly increased Ab 42 . Interestingly PS2, a close homologue of PS1, appeared to be a less efficient producer of Ab than PS1. Finally, subtle differences in c-secretase complex assembly were observed. Overall, our results indicate that the different mutations in PS affect c-secretase structure or function in multiple ways.

Evidence that Enzyme Processivity Mediates Differential Aβ Production by PS1 and PS2

“…1B ), thereby validating our substrate and cell-based system. Interestingly, and in contradiction with observations reported by Bentahir et al [10], the same PS1- and PS2-expressing cells that generated different amounts of Aβ generated equal amounts of AICD-GVP as measured by both a neo-epitope ELISA specific for the N-terminus of AICD (Fig. 1C ) and a Luciferase reporter gene assay (Fig.…”

“…Conditioned media was then harvested for Aβ ELISA at the same time that cell lysates were harvested for AICD-GVP ELISA or Luciferase assay. Consistent with previous reports [10, 11, 13], the Aβ ELISA demonstrated that PS1 generates approximately five-fold more Aβ than PS2 (Fig. 1B ), thereby validating our substrate and cell-based system.…”

“…Again, this assay indicated that PS1 and PS2 generate equal amounts of NICD in a γ-secretase-dependent manner (Fig. 2D ), consistent with previous reports [10]. In addition, NICD levels were measured by Western blot and densitometry to further confirm that PS1 and PS2 generate approximately equal amounts of NICD product (Fig.…”

“…Previous work indicates that γ-secretase cleaves APP in a processive manner such that ε-site cleavage (resulting in AICD release) precedes γ-site cleavage (resulting in Aβ release) [25, 37]. In addition, several distinct PS1 and PS2 familial mutations influence the relative production of AICD and Aβ [10, 38, 39], supporting the notion that the production of AICD and Aβ are dissociable. Taken together, these findings suggest that processivity differences between PS1 and PS2 could account for their differential Aβ production, consistent with the finding with PS1 FAD mutations [39].…”

“…Since PS1 produces more Aβ than PS2, the above findings would suggest that PS1 also generates more AICD than PS2. However, with the exception of one report [10], little is known about the relative activity of PS1- and PS2-containing γ-secretase complexes in the initial APP substrate ε-site cleavage event leading to AICD release.…”