The γ-secretase complex represents an evolutionarily conserved family of transmembrane aspartyl proteases that cleave numerous type-I membrane proteins, including the β-amyloid precursor protein (APP) and the receptor Notch. All known rare mutations in APP and the γ-secretase catalytic component, presenilin, which lead to increased amyloid βpeptide production, are responsible for early-onset familial Alzheimer’s disease. β-amyloid protein precursor-like (APPL) is the
The γ-secretase complex, which consists of at least four proteins-Presenilin (PS), Nicastrin, Aph-1, and Pen-2, serves as a transmembrane aspartyl protease that plays a critical role in Alzheimer’s disease (AD) and Notch signaling pathway (Bai et al., 2015; De Strooper, 2003; Fortini, 2009). Amyloid βpeptides (Aβs), which are the main constituents of senile plaques present in the brain affected with AD, are generated by sequential cleavages of the β-amyloid precursor protein (APP) by β-secretase (BACE) and γ-secretase (Esler and Wolfe, 2001; Goedert, 2015). Another well-known substrate for γ-secretase is Notch, whose signaling controls a large number of cell fate decisions during development (Fortini, 2009; Louvi and Artavanis-Tsakonas, 2006). The cleavage of both APP and Notch within their single-pass transmembrane domains by γ-secretase releases cytosolic fragments and allows them to enter the nucleus, thereby regulating gene transcription (Brown et al., 2000; Cao and Südhof, 2001; Louvi and Artavanis-Tsakonas, 2006; Wang et al., 2014; Figs. 1A and 1B). This γ-secretase-dependent regulated intramembrane proteolysis (RIP) is tightly controlled (Brown et al., 2000), and thus alterations in the RIP of APP and Notch result in developmental defects and diseases (Esler and Wolfe, 2001; Louvi and Artavanis-Tsakonas, 2006).
AD is the most common form of senile dementia characterized by the deposition of aggregation-prone Aβs and neurofibrillary tangles in the brain (Selkoe, 1998). Interestingly, early-onset familial Alzheimer’s disease (EOAD) is caused by rare mutations in
Canonical Notch signaling, which is highly conserved in the animal kingdom, is initiated by the binding of ligands including Delta, Serrate, and LAG-2 (Fortini, 2009; Louvi and Artavanis-Tsakonas, 2006). Ligand binding induces sequential cleavages of the Notch receptor by ADAM family metalloproteases and γ-secretase, releasing the Notch intracellular domain (NICD) (Fortini, 2009; Louvi and Artavanis-Tsakonas, 2006). NICD then enters the nucleus and forms an active transcriptional complex with the DNA-binding protein Suppressor of Hairless (Su(H)) and Mastermind (MAM), triggering expression of Notch target genes (Fortini, 2009; Louvi and Artavanis-Tsakonas, 2006; Fig. 1B). In the absence of Notch signaling, Su(H) proteins associate with various core-pressors to actively repress the transcription of Notch target genes (Fortini, 2009; Louvi and Artavanis-Tsakonas, 2006; Fig. 1B). These findings demonstrate that Su(H) is the key effector of Notch signaling.
Here, we have created a
The UAS-N™-SV and UAS-APPL-SV constructs were generated by PCR and/or restriction enzyme-based strategies and subcloned into the pUAST vector (Brand and Perrimon, 1993). For the Litmus 28-Su(H)-VP16 (SV), a NcoI-PstI fragment from pGEX-Su(H) (Bailey and Posakony, 1995), which contains the DNA-binding domain (amino acids 109–457; GenBank: AAF53434.1) of Su(H), and a PstI-XbaI fragment from pAct-GAL4-VP16 (Han and Manley, 1993), which contains the transcriptional activation domain (amino acids 2–79; GenBank: AIZ65950.1) of VP16, were subcloned into the Litmus 28 vector (New England Biolabs, Inc.). For the Litmus 28-N™-SV, a SalI-NcoI fragment from a
Expression patterns of transgenes were visualized using anti-VP16 monoclonal antibody (14-5, Santa Cruz Biotech.) and 3,3′-diaminobenzidine reaction, as described previously (Jeong et al., 2012; Kim et al., 1995).
Using two pairs of forceps, the wings of female and male adult flies (younger than 3 days) were carefully cut off and subsequently were arranged in the same orientation on a glass slide. A coverslip was applied and each corner of the coverslip was sealed with a regular nail polish. In wild-type wing, the average L4 to L3 vein length ratio was 0.97 and the average L5 to L3 vein length ratio was 0.58 (see
All quantitative data are presented as mean ± standard error of the mean (S.E.M.). Statistical significance was determined using unpaired
We decided to take advantage of the Notch signaling pathway to generate transgenic reporters for
To monitor the proteolytic cleavage of Notch and APPL by γ-secretase within their transmembrane domains, we explored a sensitive reporter system, in which the chimeric transcriptional activator Su(H)-VP16 (SV) is fused to either a subfragment of Notch (N™) or full-length APPL (
To determine whether both the N™-SV and APPL-SV chimeric reporter proteins transduce Notch signaling activity, we ectopically overexpressed these transgenes using
To further address the difference in phenotypic severity between N™-SV and APPL-SV (Figs. 3D and 3G), we obtained a total of 5 independent transgenic lines for N™-SV and a total of 4 lines for APPL-SV. On average, ectopic expression in 5 different lines for N™-SV caused 43.1% L4 vein truncations and 43.8% L5 vein truncations (n = 84), while expression in 4 different lines for APPL-SV resulted in 90.4% L4 vein truncations and 96.8% L5 vein truncations (n = 62) (Fig. 3I). An almost 2-fold difference in the percentage of both vein truncations between N™-SV and APPL-SV (
Based on wing vein phenotypes, we deduced that the N™-SV and APPL-SV reporter proteins transduce Notch activity (Fig. 3). To further address this issue, we investigated genetic interactions among
We next analyzed genetic interactions among
Since Notch plays a role in apoptosis during
To examine whether APPL-SV-induced vein phenotypes result from apoptosis, we coexpressed
One of the hallmarks of AD is senile plaques largely consisting of Aβs, which are produced by sequential cleavages of APP by β-secretase (BACE) and γ-secretase (Esler and Wolfe, 2001; Goedert, 2015). Given that all known mutations responsible for early-onset familial Alzheimer’s disease (EOAD) are localized in
Several γ-secretase associated proteins (GSAPs), in addition to the four main components of the γ-secretase complex, have been discovered (Chen et al., 2006; He et al., 2010; Teranishi et al., 2015; Wakabayashi et al., 2009; Zhou et al., 2005). These GSAP proteins, including TMP21, pigeon homologue protein, and proton myo-inositol cotransporter, regulate substrate selectivity and Aβ production. Therefore, these GSAPs can serve as therapeutic targets for the treatment of AD. These observations might indicate the existence of unknown GSAP proteins regulating Aβ production without affecting proteolytic cleavage of other γ-secretase substrates including Notch. Future work using the