Alzheimer's disease (AD) is characterized by significant accumulation of cerebral amyloid plaques and intraneuronal neurofibrillary tangles. Amyloid plaques are composed mainly of the β-amyloid peptide (Aβ). Aβ is a normal product of amyloid precursor protein (APP) metabolism. Several genes have been identified encoding enzymes that directly metabolize APP to generate Aβ; however, it is not fully understood how APP metabolism is regulated. Here we describe and validate a novel experimental approach for identifying genes encoding regulators of APP metabolism.
Aβ is generated by the successive proteolytic processing of APP, a process referred to as regulated intramembrane proteolysis (RIP) [1–3]. RIP occurs when a transmembrane protein is cleaved within the transmembrane domain, releasing a cytoplasmic fragment that can activate gene expression in the nucleus . RIP requires two cleavage events; the first, outside the membrane, often in response to ligand binding, can trigger the second, intramembraneous, cleavage. RIP liberates small, intracellular protein domains that are involved in nuclear signaling processes [1, 2]. Therefore, regulation of RIP is critical for controlling nuclear signaling. Identifying the regulatory mechanisms controlling these proteolytic steps is important for a fuller understanding of these processes.
APP is a type I transmembrane glycoprotein and is suggested to function in neuroprotection, synaptic transmission, signal transduction, and axonal transport [4, 5]. Upon being synthesized, APP undergoes maturation in the protein secretory pathway. APP is N-glycosylated in the ER and cis-Golgi followed by O-glycosylation in medial- and trans-Golgi. RIP of APP can occur via two alternative routes: amyloidogenic and non-amyloidogenic. In amyloidogenic processing, APP undergoes sequential cleavage by β-secretase (BACE) and γ-secretase to generate Aβ . BACE cleavage occurs in the APP extracellular domain to produce a soluble extracellular fragment called sAPPβ and a membrane associated, 99-residue C-terminal fragment called C99  The C99 fragment is a substrate for subsequent cleavage by the γ-secretase complex [8, 9]. The active γ-secretase complex is composed of the amino- and carboxy-terminal fragments of presenilin1 (PS1), a highly glycosylated form of nicastrin (NCSTN), Aph1α and Pen-2 [8, 9]. The amino- and carboxy-terminal fragments of PS1 (~27 and ~17 kDa respectively) are derived by endoproteolytic cleavage of the inactive, full length PS1 protein within the large hydrophilic loop that spans between transmembrane helices 6 and 7 and are thought to interact with each other . The products of γ-secretase cleavage are the cytoplasmic APP Intracellular Domain (AICD) fragment and Aβ peptides of varying length, mainly 40 and 42 residues long [11–13]. In non-amyloidogenic processing, the initial extracellular cleavage of APP is catalyzed by one of a group of proteases termed α-secretases. These enzymes include ADAM9, ADAM10, and ADAM17 (TACE). α-secretase cleavage produces a soluble extracellular fragment called sAPPα and a membrane associated, 83-residue C-terminal fragment called C83. This C83 fragment is then cleaved by the γ-secretase complex to produce AICD and a p3 peptide, which is not involved in amyloidogenesis .
A common feature of RIP processing is the liberation of an intracellular protein domain that initiates nuclear signaling [1, 2]. In the case of APP processing, nuclear signaling can be initiated by the production of the intracellular AICD fragment. Once generated by γ-secretase, the AICD fragment can be stabilized and transported to the nucleus by the cytoplasmic adaptor protein Fe65 [14, 15]. Upon entering the nucleus the AICD/Fe65 complex can form a tripartite, transcriptionally active complex with the histone acetyltransferase Tip60 [16, 17]. Consistent with this model, cells concomitantly over-expressing an APP-Gal4-DNA binding domain fusion protein and Fe65, and carrying a Gal4 UAS-driven reporter construct display a >2000 fold increase in reporter transcription compared to cells over-expressing just the Gal4 DNA binding domain and Fe65 . This increase in transactivation activity is dependent on Tip60 and can be abolished when the interaction between AICD and Fe65 is disrupted by mutagenesis of the AICD NPTY motif, the binding site for Fe65 . However, these data do not rule out a possible effect of full-length APP in inducing nuclear signaling. Indeed, APP nuclear signaling can occur in the absence of γ-secretase activity and therefore does not require the AICD fragment . The relative contribution of AICD-mediated versus holo-APP mediated nuclear signaling is not clear at this time [16–18].
The genomic targets of AICD- or APP-mediated nuclear signaling are not clearly defined. APP, BACE, Tip60, GSK-3β, Mn-SOD, KAI1, NEP and other genes have all been reported to be targets of APP mediated transcriptional activation [19–22]; however, there is a paucity of confirmatory reports . At this time, the biological role of AICD-mediated transactivation is not clear [20, 23, 24]. Despite this confusion, evidence suggests that defective APP signaling is involved in AD pathogenesis [25–29].
Given the centrality of APP in AD, it is crucial to identify regulators of APP metabolism, including, but not limited to, APP proteolysis. Regulation of APP metabolism can occur by numerous mechanisms, including regulation of APP transcription, APP translation, APP maturation, intracellular trafficking of full-length APP and APP cleavage products, APP proteolysis, and APP degradation. While Komano and colleagues have used a genetic screen to specifically identify regulators of γ-secretase activity , a screen that will identify APP metabolism regulators that act through multiple mechanisms is needed.
Here we describe a novel experimental approach to identify a variety of regulators of APP metabolism. We use an AICD-Gal4 mediated luciferase expression assay as a general reporter of APP metabolism in the human neuroblastoma cell lines, SH-SY5Y. To validate this assay, we utilized pharmacologic agents, as well as forward and reverse genetics, to modulate APP proteolysis, AICD trafficking and AICD transactivation. To determine if regulators of APP maturation and PS1 endoproteolysis also can be detected with this screening approach, we knocked-down Ubiquilin 1 and observed decreased AICD-Gal4 luciferase activity. Using Western blot analysis, we show that Ubiquilin 1 controls APP levels, the ratio of mature to immature APP, as well as presenilin1 endoproteolysis, confirming the previously reported role of Ubiquilin 1 in APP and presenilin1 metabolism in non-neuronal human cell lines [31–34]. Taken together, our results validate the use of the AICD-Gal4 mediated luciferase assay in combination with forward and reverse genetics as a screen to identify APP metabolism regulators.