We have reported that imatinib (STI571, Gleevec®) decreases production of all Aβ species by inhibiting γ-cleavage of APP-CTF5
. To identify the direct target responsible for imatinib's selective Aβlowering activity, we synthesized a photoactivatable azido imatinib derivative, G01 (supplementary Methods and supplementary Fig. 2
). When 125
I-G01 was incubated with a membrane preparation followed by photolysis, none of the four components of γ-secretase were labeled. Rather, 125
I-G01 labeled a ~ 16 kDa protein () which co-immunoprecipitated with the more slowly migrating 18 kDa presenilin-1-CTF (). This result was confirmed by intact cell photolabeling using cell permeable 3
H-G01: the 3
H-imatinib derivative did not bind to any of the four γ-secretase components, but did label a band of ~16 kDa that co-immunoprecipitated with PS1 ().
Identification of gSAP as an imatinib target
To purify the potential target protein, we synthesized a biotinylated derivative of imatinib, “biotin-imatinib” (supplementary Methods and supplementary Fig. 3
). Solubilized γ-secretase components, including presenilin-1, Pen-2, and nicastrin, were specifically captured by the immobilized biotin-imatinib (). A ~16 kDa band was observed by silver staining () after biotin-imatinib bound proteins were separated by SDS-PAGE, concurring with the photolabeling results. Peptide fragments, derived from this band after trypsin digestion, and analyzed by tandem mass spectrometry, corresponded to the C-terminal region of an uncharacterized protein, pigeon homologue protein (PION
) (human accession number: NP_059135). The identification was made based on two unique tryptic peptides (766
) covering approximately 20% of the 16 kDa fragment. Its sequence, especially the C-terminal region, is highly conserved among multiple species from chicken to human (supplementary Fig. 4
). Expression pattern analysis indicates that this gene is expressed in diverse tissues, including brain (supplementary Fig. 5
). In this report, we characterize PION as a g
Based on its predicted sequence, the full opening reading frame of human gSAP encodes a protein of 854 amino acids (~98 kDa). To determine whether the 16 kDa fragment was derived from a high molecular weight precursor, the metabolism of endogenous gSAP in cells was monitored by pulse-chase analysis. The results showed that gSAP is synthesized as a holo-protein (~98 kDa) and is rapidly processed into a ~ 16 kDa C-terminal fragment (gSAP-16K) (). In the steady state, the 16 kDa fragment is the predominant form ().
Incubation of cells with 3H-G01, followed by photolysis and immunoprecipitation with anti-gSAP antibody, confirmed that imatinib directly binds gSAP-16K (). When gSAP levels were reduced using siRNA, the amount of γ-secretase () associated with biotin-imatinib dramatically decreased. This indicates that the affinity of imatinib for the γ-secretase complex depends on gSAP.
The effect of gSAP on Aβ generation is shown in . When siRNA was used to reduce gSAP level (by 72 ± 15%) in N2a cells overexpressing APP695, the level of Aβ decreased about 50±7 % (); imatinib had little or no additional effect on Aβ levels. This result indicates that gSAP is the molecule through which imatinib lowers Aβ. gSAP knockdown resulted in decreased levels of all major Aβ species; Aβ38 by 43±%, Aβ40 by 53±13%, and Aβ42 by 48±7%, respectively (). gSAP showed no detectable effect on α- and β-cleavages (supplementary Fig. 6
). To further investigate whether gSAP can modulate γ-secretase activity, the effect of purified gSAP on Aβ production was examined in an in vitro
γ-secretase assay. When recombinant gSAP-16K (aa 733-854 of full length human gSAP), isolated after expression in E.coli
, was added to membrane preparations from HEK cells containing overexpressed APP-β-CTF, Aβ level was increased and AICD level was reduced ().
gSAP regulates Aβ production but does not influence Notch cleavage
APP-CTF is cleaved by γ-secretase in the middle of its transmembrane domain to generate Aβ (γ-cleavage) and near its cytosolic membrane boundary to generate APP intracellular domain (AICD) (ε-cleavage). The effect of gSAP on AICD production was examined in N2a cells overexpressing APP695. Both gSAP knockdown and imatinib treatment increased levels of AICD (supplementary Fig. 7a
). gSAP overexpression in HEK293 cells reduced AICD production (supplementary Fig. 7b
). These results indicate that gSAP differentially regulates γ- andε-cleavage of APP-CTF to form Aβ and AICD respectively.
One distinctive feature of imatinib is its selective inhibition of Aβ production while sparing Notch cleavage5
. The effect of gSAP on Notch cleavage was evaluated using cells expressing Notch ΔE (Notch without its extracellular domain), the Notch substrate for γ-secretase. As shown in , the level of theγ-secretase cleavage product, the Notch intracellular domain (NICD), was not changed either by reducing gSAP levels using shRNA (left panel) or by overexpressing gSAP (right panel). In addition, gSAP had no effect on Notch cleavage in an in vitro
γ-secretase assay (). Thus, gSAP modulates the γ-secretase cleavage of APP, but not of Notch.
Additional evidence that endogenous gSAP forms a complex with γ-secretase was provided by examining the distribution of the proteins in subcellular fractions and in co-immunoprecipitation studies. Using a sucrose gradient, endogenous gSAP co-fractionated with a trans-Golgi network (TGN) marker, and with PS1-CTF (Supplementary Figure 8
) and other γ-secretase components (not shown). Using gel filtration to separate membrane proteins from neuroblastoma cells solubilized in 1% CHAPSO, endogenous gSAP-16K and γ-secretase co-migrated as a high molecular weight complex (). Further, endogenous gSAP co-immunoprecipitated with γ-secretase components, providing additional evidence that these proteins exist in a complex (). Endogenous γ-secretase was isolated using an immobilized biotinylated derivative of the transition-state analogue L-685,4586
. Endogenous gSAP-16K co-isolated with the enzyme–inhibitor complex, strongly suggesting that gSAP-16K is a co-factor for γ-secretase ().
gSAP interacts with γ-secretase and APP-CTF but not with Notch
A number of proteases with broad substrate recognition can achieve specificity through auxiliary factors that couple the core enzyme to selective substrates7,8
. To explore the mechanism by which gSAP might confer such specificity, we analyzed its association with specific substrates. gSAP-16K coimmunoprecipitated with APP-CTF but not with Notch δE (); the interaction was reduced by imatinib in a concentration-dependent manner (). Disruption of this interaction by imatinib likely explains its Aβ-lowering activity. Domain mapping studies demonstrated that the juxtamembrane region of APP-CTF interacts with gSAP (supplementary Fig. 9
). A truncated form of APP-CTF lacking the cytoplasmic domain (APPεCTF)9
did not interact with gSAP and its γ-cleavage was no longer stimulated by gSAP-16K in an in vitro
To further determine the structural basis for the selective interaction of gSAP with APP-CTF, chimeric proteins were constructed by exchanging the AICD fragment in APP-CTF with the NICD fragment in NotchδE (supplementary Fig. 10a
). gSAP selectively interacted with AICD, but not NICD in chimeric proteins (supplementary Fig. 10b
). gSAP knockdown selectively increased AICD production, but had no influence on NICD production from the chimeric proteins (Supplementary Fig 10c
). These results further demonstrated that the selective effect of gSAP on APP-CTF cleavage by γ-secretase involves gSAP binding to the cytoplasmic domain of the substrate.
To determine whether our findings are relevant to AD pathology, the effects of gSAP on Aβ levels and plaque development were examined in vivo
. A conditional gSAP RNAi mouse line was generated by integration of a tetracycline-inducible gSAP shRNA vector into the mouse genomic locus. gSAP RNAi mice were then crossed with an AD mouse model (APPswe and PS1δE9 mutations; AD 2 X Tg-mice)10
. To examine the long term effect of gSAP knockdown on Aβ levels and plaque development, the crossed gSAP RNAi- AD 2 X mice were continuously induced for 6 months. After induction, gSAP mRNA levels in these hybrid mouse brains were reduced by 85 ± 12% and similar decreases were achieved in other tissues; after six months induction, Aβ40 and Aβ42 levels in the crossed mouse brains were lowered by 42 ± 13% and 40 ± 7%, respectively (). Amyloid plaque load in crossed mouse brains with gSAP knockdown was reduced by 38 ± 9%, compared to the same line of mouse brains without induction (). Doxycycline did not have an effect on either Aβ or plaque levels in AD 2 X mice. The Aβ-lowering effects of gSAP knockdown are similar to those caused by the γ-secretase inhibitor, dibenzazepine (DBZ)11
, administered at 10 μmol/kg for 5 days (Supplementary Fig. 11a
). In contrast, gSAP knockdown did not cause the intestinal mucosal cell metaplasia seen with DBZ (supplementary Fig. 11b
): this latter effect is mediated by impaired Notch processing4,11
. Furthermore, gSAP knockdown did not cause any pathological changes in spleen (data not shown), contrary to the severe marginal zone lymphoid depletion caused by DBZ administration12
. These results indicate that gSAP knockdown reduces Aβ levels and plaque formation without affecting Notch-dependent pathways.
Knockdown of gSAP reduces Aβ production and plaque development in an AD mouse model
γ-Secretase processes diverse substrates with low homologies at their cleavage sites13
. The various roles of γ-secretase during development and in tissue homeostasis require that its activity be tightly regulated. TMP2114
, orphan G-protein-coupled receptor 315
and different Aph-1 isoforms16
have been reported to modulate Aβ production through γ-secretase but to spare Notch cleavage. However, the underlying molecular mechanisms by which they impart their specificities were not elucidated in those studies. Nevertheless, those important studies demonstrated that it is possible to selectively regulate substrate specificity of this vitally important and potentially promiscuous enzyme. gSAP appears to confer substrate specificity on γ-secretase by forming a ternary complex with γ-secretase and the substrate APP-CTF. The present results support the concept that appropriate cofactors impart substrate specificity on the γ-secretase core enzyme complex, as they do on a number of other proteases7,8
The literature on the relationship between γ-cleavage and ε-cleavage of APP-CTF is controversial. For instance, there is some evidence supporting sequential cleavage of APP-CTF9,17
. There is also extensive evidence reported in the literature that these two types of cleavage can occur independently18,19,20
. Our data support the latter proposal. We hypothesize that removal of gSAP from the gSAP/γ-secretase/APP-CTF ternary complex alters the structural relationship between γ-secretase and APP-CTF facilitating ε-cleavage at the expense of γ-cleavage (supplementary Fig. 1
). To elucidate the detailed mechanism by which gSAP modulates the cleavage of APP-CTF, it will be important to compare the stoichiometry of the various γ-secretase cleavage products in the presence and absence of gSAP.
Anti-amyloid therapy remains a rationale approach to the treatment of Alzheimer's disease. One promising anti-amyloid compound failed in limited clinical trials, owning to lack of accumulation in the brain21
. Similarly, imatinib is actively excluded from the brain by a highly potent P-glycoprotein pump, a component of the blood-brain barrier22
. The development of compounds which accumulate in the brain and target gSAP represents a valid approach for development of potential therapies against Alzheimer's disease.