Abstract
γ-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 γ-secretase activating protein (GSAP) was identified that interacts with γ-secretase and the C-terminal fragment of amyloid precursor protein to selectively increase amyloid-β production. In this study we have further characterized the role of endogenous and exogenous GSAP in the regulation of γ-secretase activity and amyloid-β production in vitro. Knockdown of GSAP expression in N2a cells decreased amyloid-β levels. In contrast, overexpression of GSAP in HEK cells expressing amyloid precursor protein or in N2a cells had no overt effect on amyloid-β generation. Likewise, purified recombinant GSAP had no effect on amyloid-β generation in two distinct in vitro γ-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-β 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-β levels. In summary, these findings suggest the roles of GSAP and imatinib in the regulation of γ-secretase activity and amyloid-β generation are uncertain.
Highlights
␥-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
Mouse neuroblastoma N2a cells were selected for these studies as both GSAP mRNA expression and endogenous A levels were detectable in these cells and because this cell line was used by He et al [17] in their studies on GSAP. 48 h post-siRNA treatment, GSAP mRNA expression was reduced by 74% in N2a cells (Fig. 1b)
In siRNA knockdown studies we demonstrated that a reduction of endogenous GSAP expression significantly reduced A levels in N2a cells
Summary
␥-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. Subchronic administration of imatinib to rats had no effect on brain amyloid- levels These findings suggest the roles of GSAP and imatinib in the regulation of ␥-secretase activity and amyloid- generation are uncertain. ␥-Secretase is a large protein complex composed of four components, presenilin (PS), nicastrin, presenilin enhancer 2, and anterior pharynx-defective 1 (Aph-1) that are required for ␥-secretase activity [6] Additional ancillary proteins such as CD147 and TPM21 that can associate with the ␥-secretase complex and regulate A generation have been identified [7, 8]. Due to the fundamental role ␥-secretase plays in the intramembrane proteolysis of a number of other proteins [10], the development of these compounds was hinlular domain; PS, presenilin; CTF, carboxyl-terminal fragment; DMSO, dimethyl sulfoxide; GSAP, ␥-secretase activating protein; GSI, ␥-secretase inhibitor; NICD, Notch intracellular domain; ANOVA, analysis of variance; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; CHAPSO, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid. Drug discovery efforts have shifted to the development of ␥-secretase modulators that selectively lower A42 production, in the absence of any effect on Notch processing [15, 16] and should be safer and better tolerated than ␥-secretase inhibitors
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