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gamma-Secretase is a large enzyme complex comprising presenilin, nicastrin, presenilin enhancer 2, and anterior pharynx-defective 1 that mediates the intramembrane proteolysis of a large number of proteins including amyloid precursor protein and Notch. Recently, a novel gamma-secretase activating protein (GSAP) was identified that interacts with gamma-secretase and the C-terminal fragment of amyloid precursor protein to selectively increase amyloid-beta production. In this study we have further characterized the role of endogenous and exogenous GSAP in the regulation of gamma-secretase activity and amyloid-beta production in vitro. Knockdown of GSAP expression in N2a cells decreased amyloid-beta levels. In contrast, overexpression of GSAP in HEK cells expressing amyloid precursor protein or in N2a cells had no overt effect on amyloid-beta generation. Likewise, purified recombinant GSAP had no effect on amyloid-beta generation in two distinct in vitro gamma-secretase assays. In subsequent cellular studies with imatinib, a kinase inhibitor that reportedly prevents the interaction of GSAP with the C-terminal fragment of amyloid precursor protein, a concentration-dependent decrease in amyloid-beta levels was observed. However, no interaction between GSAP and the C-terminal fragment of amyloid precursor protein was evident in co-immunoprecipitation studies. In addition, subchronic administration of imatinib to rats had no effect on brain amyloid-beta levels. In summary, these findings suggest the roles of GSAP and imatinib in the regulation of gamma-secretase activity and amyloid-beta generation are uncertain.

that BIN1 is a genuine genetic determinant of AD. The Amphiphysins (Amph 1 and 2) are expressed in the brain and are involved in endocytosis processes. However, little is known about their potential implications in the pathological process. We aimed at characterizing how BIN1 may act in the AD process. Methods: mRNA levels of Amph 1 and BIN1 were measured in the frontal area of 64 AD brains and 64 controls using the Quantigene technology. The SKNSH-SY5Yand HEK cell lines, stably over-expressing APP695wt were transitorily transfected with a vector expressing BIN1. After 48 hours, supernatants were recovered and cells were lysed. Holo-APP and s-actin were measured by protein blotting. As1-40, As1-42, sAPPa and sAPPs were measured by sandwich ELISA. Finally, we assessed BIN1/Amph 1modulation of Tau and As -mediated neurotoxicity in D. melanogaster. using rough-eye and bristle phenotypes.Results:We observed an increase in expression of both Amph 1 and BIN1 genes in the brains of AD cases compared to controls. BIN1 over-expression in the two cell lines did not modify the APP metabolism whereas BIN1/Amph 1 knockdown suppressed Tau neurotoxicity in D. melanogaster. Conclusions: Our preliminary results indicated that BIN1 is over-expressed in AD brain and might play a role in AD process not by modulating APP metabolism but by controlling Tau proteinopathy-mediated neurotoxicity.