Gamma-secretase activating protein is a therapeutic target for Alzheimer's disease

Abstract

Accumulation of neurotoxic amyloid-beta is a major hallmark of Alzheimer's disease. Formation of amyloid-beta is catalysed by gamma-secretase, a protease with numerous substrates. Little is known about the molecular mechanisms that confer substrate specificity on this potentially promiscuous enzyme. Knowledge of the mechanisms underlying its selectivity is critical for the development of clinically effective gamma-secretase inhibitors that can reduce amyloid-beta formation without impairing cleavage of other gamma-secretase substrates, especially Notch, which is essential for normal biological functions. Here we report the discovery of a novel gamma-secretase activating protein (GSAP) that drastically and selectively increases amyloid-beta production through a mechanism involving its interactions with both gamma-secretase and its substrate, the amyloid precursor protein carboxy-terminal fragment (APP-CTF). GSAP does not interact with Notch, nor does it affect its cleavage. Recombinant GSAP stimulates amyloid-beta production in vitro. Reducing GSAP concentrations in cell lines decreases amyloid-beta concentrations. Knockdown of GSAP in a mouse model of Alzheimer's disease reduces levels of amyloid-beta and plaque development. GSAP represents a type of gamma-secretase regulator that directs enzyme specificity by interacting with a specific substrate. We demonstrate that imatinib, an anticancer drug previously found to inhibit amyloid-beta formation without affecting Notch cleavage, achieves its amyloid-beta-lowering effect by preventing GSAP interaction with the gamma-secretase substrate, APP-CTF. Thus, GSAP can serve as an amyloid-beta-lowering therapeutic target without affecting other key functions of gamma-secretase.

Figure 1. Identification of gSAP as an imatinib target

a: A PS1-associated 16 kDa protein is labeled by a photoactivatable imatinib derivative. Left panel: photolysis of ¹²⁵I-G01 with membrane preparations. Middle panel: photolysis of ³H-G01 with intact HEK293 cells. Right panel: PS1-CTF migrated with a slower mobility than the labeled 16 kDa band and was not labeled by G01. Labeling specificity was confirmed by competition with unlabeled imatinib. b: Solubilized endogenous γ-secretase components from HEK293 cells were bound to immobilized biotin-imatinib (left panel). Among the proteins bound to biotin-imatinib, a ~ 16 kDa band was detected by silver staining and was identified as the C-terminal domain of gSAP (right panel, arrow and label “gSAP”). Biotin-coated beads and an inactive biotin-imatinib derivative (see supplementary Fig. 3) served as controls. c: Endogenous gSAP in N2a cells was synthesized as a full length 98 kDa-precursor protein and rapidly processed into a 16 kDa C-terminal fragment. Under steady-state conditions, the predominant cellular form of gSAP was 16 kDa. d: Endogenous gSAP-16K was specifically labeled by ³H-G01 in neuroblastoma cells. e: After gSAP siRNA knockdown in N2a cells, immobilized biotin-imatinib no longer captured PS1.

Figure 2. gSAP regulates Aβ production but does not influence Notch cleavage

a: siRNA-mediated knockdown of gSAP in N2a cells overexpressing APP695 lowered Aβ production. The Aβ-lowering effects of imatinib and of siRNA were not additive (mean ±s.d.; **p < 0.01; n ≥3). b: Transfection of N2a cells overexpressing APP695 with gSAP siRNA reduced the levels of Aβ38, Aβ40 and Aβ42 (mean ±s.d.; **p < 0.01; n ≥3). c: Recombinant gSAP-16K from E.coli stimulated Aβ production in an in vitro γ-secretase assay, inhibited AICD production and had no effect on Notch cleavage. The γ-secretase inhibitor, L685,458 (1 μM) abolished NICD, AICD and Aβ production (mean ±s.d.; **p < 0.01; n ≥3). d: Neither gSAP knockdown (left panel) nor its overexpression (right panel), affected Notch processing in HEK293 cells overexpressing extracellular domain truncated Notch (NotchΔE, with C-terminal Myc tag). NICD was detected using a Myc antibody and a cleavage-product specific antibody (Notch1 Val-1744). The γ-secretase inhibitor, L685,458 (1 μM) served as a control.

Figure 3. gSAP interacts with γ-secretase and APP-CTF but not with Notch

a: Endogenous gSAP-16K in solubilized membrane preparations from N2a cells co-migrated with γ-secretase components during gel filtration (void volume: fraction 6). b: Immunoprecipitation of endogenous gSAP from N2a cells resulted in co-immunoprecipitation of γ-secretase components. c: Endogenous gSAP-16K and γ-secretase components are highly enriched by an immobilized γ-secretase transition state analogue (GSI beads). d: In HEK293 cells, gSAP-16K and APP-CTF, but not NotchΔE, co-immunoprecipitated. e: Imatinib treatment reduced the co-immunoprecipitation of APP-CTF and gSAP in a concentration-dependent manner. An inactive imatinib derivative (IC200001, see supplementary Fig. 3) served as a negative control. f: In HEK293 cells, APP-CTF without the cytoplasmic domain (APPε-CTF) did not co-immunoprecipitate with gSAP-16K (upper panel); γ-cleavage of APPε-CTF was not stimulated by gSAP-16K in an in vitro assay (lower panel).

Figure 4. Knockdown of gSAP reduces Aβ production and plaque development in an AD mouse model

a: gSAP RNAi-AD mice were generated by crossing double transgenic AD mice with doxycycline-inducible gSAP RNAi mice. Six months after inducing gSAP shRNA expression, Aβ40 and Aβ42 levels in the crossed mouse brains were decreased by 42 ± 13% and 40 ± 7%, respectively (mean ±s.e.m.; **: p< 0.01. n= 4). Doxycycline treatment alone did not change Aβ levels in AD mice. b: Six months after inducing gSAP shRNA expression, amyloid plaque development was reduced in the crossed mouse brains by 38 ± 9%(mean ±s.e.m.; **: p< 0.01. n= 4). Doxycycline treatment alone did not change plaques in AD mice. Amyloid plaques were revealed by 6E10 immunostaining.