Structure of the intracellular domain of the amyloid precursor protein in complex with Fe65-PTB2

Cleavage of the amyloid precursor protein (APP) is a crucial event in Alzheimer disease pathogenesis that creates the amyloid-beta peptide (Abeta) and liberates the carboxy-terminal APP intracellular domain (AICD) into the cytosol. The interaction of the APP C terminus with the adaptor protein Fe65 mediates APP trafficking and signalling, and is thought to regulate APP processing and Abeta generation. We determined the crystal structure of the AICD in complex with the C-terminal phosphotyrosine-binding (PTB) domain of Fe65. The unique interface involves the NPxY PTB-binding motif and two alpha helices. The amino-terminal helix of the AICD is capped by threonine T(668), an Alzheimer disease-relevant phosphorylation site involved in Fe65-binding regulation. The structure together with mutational studies, isothermal titration calorimetry and nuclear magnetic resonance experiments sets the stage for understanding T(668) phosphorylation-dependent complex regulation at a molecular level. A molecular switch model is proposed.     

FKBP12 regulates the localization and processing of amyloid precursor protein in human cell lines

One of the pathological hallmarks of Alzheimer’s disease is the presence of insoluble extracellular amyloid plaques. These plaques are mainly constituted of amyloid beta peptide (Aβ), a proteolytic product of amyloid precursor protein (APP). APP processing also generates the APP intracellular domain (AICD). We have previously demonstrated that AICD interacts with FKBP12, a peptidyl-prolyl cis-trans isomerase (PPIase) ubiquitous in nerve systems. This interaction was interfered by FK506, a clinically used immunosuppressant that has recently been reported to be neuroprotective. To elucidate the roles of FKBP12 in the pathogenesis of Alzheimer’s disease, the effect of FKBP12 overexpression on APP processing was evaluated. Our results revealed that APP processing was shifted towards the amyloidogenic pathway, accompanied by a change in the subcellular localization of APP, upon FKBP12 overexpression. This FKBP12-overexpression-induced effect was reverted by FK506. These findings support our hypothesis that FKBP12 may participate in the regulation of APP processing. FKBP12 overexpression may lead to the stabilization of a certain isomer (presumably the cis form) of the Thr668-Pro669 peptide bond in AICD, therefore change its affinity to flotillin-1 or other raft-associated proteins, and eventually change the localization pattern and cause a shift in the proteolytic processing of APP.     

Ligand-Dependent Activation of EphA4 Signaling Regulates the Proteolysis of Amyloid Precursor Protein Through a Lyn-Mediated Pathway

Alzheimer's disease is the most common dementia afflicting the elderly in modern society. This disease arises from the neurotoxicity elicited by abnormal aggregates of amyloid-β (Aβ) protein. Such aggregates form through the cleavage of amyloid precursor protein (APP) by β-secretase and the subsequent proteolysis of the APP C-terminal fragment (APP-βCTF or C99) by γ-secretase to yield Aβ and APP intracellular domain (AICD). Recent evidence suggests that C99 and AICD may exert harmful effects on cells, suggesting that the proteolytic products of APP, including Aβ, C99, and AICD, could play a pivotal role in neuronal viability. Here, we demonstrate that ligand-activated EphA4 signaling governs the proteostasis of C99, AICD, and Aβ, without significantly affecting γ-secretase activity. EphA4 induced accumulation of C99 and AICD through a Lyn-dependent pathway; activation of this pathway triggered phosphorylation of EphA4, resulting in positive feedback of C99 and AICD proteostasis. Inhibition of EphA4 by dasatinib, a receptor tyrosine kinase inhibitor, effectively suppressed C99 and AICD accumulation. Furthermore, EphA4 signaling controlled C99 and AICD proteolysis through the ubiquitin–proteasome system. In conclusion, we have identified an EphA4–Lyn pathway that is essential for the metabolism of APP and its proteolytic derivatives, thereby providing novel pharmacological targets for the development of anti-Aβ therapeutics for AD.     

Phosphorylation of amyloid precursor protein (APP) at Tyr687 regulates APP processing by alpha- and gamma-secretase

Abnormal proteolytic processing of amyloid precursor protein (APP) is a pathologic feature of Alzheimer’s disease. Recent studies have demonstrated that serine/threonine phosphorylation specifically at amino-acid residue Thr668 (APP695 numbering) regulates APP processing. In this study, we investigated the possibility that tyrosine phosphorylation of APP regulates APP processing. A tyrosine kinase inhibitor decreased expression of the C83 fragment which is a cleaved product of APP by α-secretase. By overexpressing APP mutant proteins, Tyr687 was found to be the major tyrosine kinase phosphorylation site. Expression of the C83 fragment was decreased in APPY687A-expressing cells relative to APP wild-type (APPWT)-expressing cells, which likely reflects the different cellular localization patterns of these two proteins. Expression of APP intracellular domain (AICD) which is a cleaved product of the C83 fragment by γ-secretase was decreased in C83Y687A-expressing cells. These results suggest that phosphorylation of APP at Tyr687 regulates APP processing by α- and γ-secretases, determining the expression level of AICD.     

Apoptotic cell death influences the signaling activity of the amyloid precursor protein through ShcA and Grb2 adaptor proteins in neuroblastoma SH-SY5Y cells

The amyloid precursor protein (APP) is an ubiquitous receptor-like molecule involved in the pathogenesis of Alzheimer's disease (AD). APP and some of its C-terminal proteolytic fragments (CTFs) have been shown to be phosphorylated and to interact with cytosolic phosphotyrosine binding (PTB) domain containing proteins involved in cell signaling and vesicular transport. Among others, the interaction between tyrosine-phosphorylated CTFs and ShcA-Grb2 adaptors is highly enhanced in AD brain. Here we have identified in SH-SY5Y neuroblastoma cells an interaction between APP holoprotein and the adaptor Grb2. Upon activation of apoptotic cell death this interaction is rapidly degraded, APP is partially cleaved and the complex APP/Grb2 is replaced by a new complex between CTFs and ShcA that still involves Grb2. The formation of these complexes is regulated by beta-site APP-cleaving enzyme 1 and influences the phosphorylation of mitogen-activated protein kinase p44/42 extracellular signal-regulated kinase as well as the level of apoptotic death of the cells. These data suggest a dual role in cell signaling for APP and its CTFs in neuroblastoma cells, in a manner similar to that previously reported for other tyrosine kinase receptor, through a tightly regulated coupling with alternative intracellular adaptors to control the signaling of the cell.     

Regulated intramembrane proteolysis of amyloid precursor protein and regulation of expression of putative target genes

gamma-Secretase-dependent regulated intramembrane proteolysis of amyloid precursor protein (APP) releases the APP intracellular domain (AICD). The question of whether this domain, like the Notch intracellular domain, is involved in nuclear signalling is highly controversial. Although some reports suggest that AICD regulates the expression of KAI1, glycogen synthase kinase-3beta, Neprilysin and APP, we found no consistent effects of gamma-secretase inhibitors or of genetic deficiencies in the gamma-secretase complex or the APP family on the expression levels of these genes in cells and tissues. Finally, we demonstrate that Fe65, an important AICD-binding protein, transactivates a wide variety of different promoters, including the viral simian virus 40 promoter, independent of AICD coexpression. Overall, the four currently proposed target genes are at best indirectly and weakly influenced by APP processing. Therefore, inhibition of APP processing to decrease Abeta generation in Alzheimer's disease will not interfere significantly with the function of these genes.     

A TAG1-APP signalling pathway through Fe65 negatively modulates neurogenesis

The release of amyloid precursor protein (APP) intracellular domain (AICD) may be triggered by extracellular cues through gamma-secretase-dependent cleavage. AICD binds to Fe65, which may have a role in AICD-dependent signalling; however, the functional ligand has not been characterized. In this study, we have identified TAG1 as a functional ligand of APP. We found that, through an extracellular interaction with APP, TAG1 increased AICD release and triggered Fe65-dependent activity in a gamma-secretase-dependent manner. TAG1, APP and Fe65 colocalized in the neural stem cell niche of the fetal ventricular zone. Neural precursor cells from TAG1-/-, APP-/- and TAG1-/-;APP-/- mice had aberrantly enhanced neurogenesis, which was significantly reversed in TAG1-/- mice by TAG1 or AICD but not by AICD mutated at the Fe65 binding site. Notably, TAG1 reduced normal neurogenesis in Fe65+/+ mice. Abnormally enhanced neurogenesis also occurred in Fe65-/- mice but could not be reversed by TAG1. These results describe a TAG1-APP signalling pathway that negatively modulates neurogenesis through Fe65.     

A TAG on to the neurogenic functions of APP

The proteolytic processing of amyloid β precursor protein (APP) has long been studied because of its association with the pathology of Alzheimer's disease (AD). The ectodomain of APP is shed by α- or β-secretase cleavage. The remaining membrane bound stub can then undergo regulated intramembrane proteolysis (RIP) by γ-secretase. This cleavage can release amyloid β (Aβ) from the stub left by β-secretase cleavage but also releases the APP intracellular domain (AICD) after α- or β-secretase cleavage. The physiological functions of this proteolytic processing are not well understood. We compare the proteolytic processing of APP to the ligand-dependent RIP of Notch. In this review, we discuss recent evidence suggesting that TAG1 is a functional ligand for APP. The interaction between TAG1 and APP triggers γ-secretase-dependent release of AICD. TAG1, APP and Fe65 colocalise in the neurogenic ventricular zone and in fetal neural progenitor cells in vitro. Experiments in TAG1, APP and Fe65 null mice as well as TAG1 and APP double-null mice demonstrate that TAG1 induces a γ-secretase- and Fe65-dependent suppression of neurogenesis.     

Fe65 stimulates proteolytic liberation of the beta-amyloid precursor protein intracellular domain

The beta-amyloid precursor protein (APP)-binding protein Fe65 is involved in APP nuclear signaling and several steps in APP proteolytic processing. In this study, we show that Fe65 stimulates gamma-secretase-mediated liberation of the APP intracellular domain (AICD). The mechanism of Fe65-mediated stimulation of AICD formation appears to be through enhanced production of the carboxyl-terminal fragment substrates of gamma-secretase and direct stimulation of processing by gamma-secretase. The stimulatory capacity of Fe65 is isoform-dependent, as the non-neuronal and a2 isoforms promote APP processing more effectively than the exon 9 inclusive neuronal form of Fe65. Intriguingly, Fe65 stimulation of AICD production appears to be inversely related to pathogenic beta-amyloid production as the Fe65 isoforms profoundly stimulate AICD production and simultaneously decrease Abeta42 production. Despite the capacity of Fe65 to stimulate gamma-secretase-mediated APP proteolysis, it does not rescue the loss of proteolytic function associated with the presenilin-1 familial Alzheimer disease mutations. These data suggest that Fe65 regulation of APP proteolysis may be integrally associated with its nuclear signaling function, as all antecedent proteolytic steps prior to release of Fe65 from the membrane are fostered by the APP-Fe65 interaction.     

The phosphotyrosine interaction domains of X11 and FE65 bind to distinct sites on the YENPTY motif of amyloid precursor protein.

The phosphotyrosine interaction (PI) domains (also known as the PTB, or phosphotyrosine binding, domains) of Shc and IRS-1 are recently described domains that bind peptides phosphorylated on tyrosine residues. The PI/PTB domains differ from Src homology 2 (SH2) domains in that their binding specificity is determined by residues that lie amino terminal and not carboxy terminal to the phosphotyrosine. Recently, it has been appreciated that other cytoplasmic proteins also contain PI domains. We now show that the PI domain of X11 and one of the PI domains of FE65, two neuronal proteins, bind to the cytoplasmic domain of the amyloid precursor protein ((beta)APP). (beta)APP is an integral transmembrane glycoprotein whose cellular function is unknown. One of the processing pathways of (beta)APP leads to the secretion of A(beta), the major constituent of the amyloid deposited in the brain parenchyma and vessel walls of Alzheimer's disease patients. We have found that the X11 PI domain binds a YENPTY motif in the intracellular domain of (beta)APP that is strikingly similar to the NPXY motifs that bind the Shc and IRS-1 PI/PTB domains. However, unlike the case for binding of the Shc PI/PTB domain, tyrosine phosphorylation of the YENPTY motif is not required for the binding of (beta)APP to X11 or FE65. The binding site of the FE65 PI domain appears to be different from that of X11, as mutations within the YENPTY motif differentially affect the binding of X11 and FE65. Using site-directed mutagenesis, we have identified a crucial residue within the PI domain involved in X11 and FE65 binding to (beta)APP. The binding of X11 or FE65 PI domains to residues of the YENPTY motif of (beta)APP identifies PI domains as general protein interaction domains and may have important implications for the processing of (beta)APP.     

NGF controls APP cleavage by downregulating APP phosphorylation at Thr668: relevance for Alzheimer's disease

NGF has been implicated in forebrain neuroprotection from amyloidogenesis and Alzheimer's disease (AD). However, the underlying molecular mechanisms are still poorly understood. Here, we investigated the role of NGF signalling in the metabolism of amyloid precursor protein (APP) in forebrain neurons using primary cultures of septal neurons and acute septo‐hippocampal brain slices. In this study, we show that NGF controls the basal level of APP phosphorylation at Thr668 (T668) by downregulating the activity of the Ser/Thr kinase JNK(p54) through the Tyr kinase signalling adaptor SH2‐containing sequence C (ShcC). We also found that the specific NGF receptor, Tyr kinase A (TrkA), which is known to bind to APP, fails to interact with the fraction of APP molecules phosphorylated at T668 (APP^pT668). Accordingly, the amount of TrkA bound to APP is significantly reduced in the hippocampus of ShcC KO mice and of patients with AD in which elevated APP^pT668 levels are detected. NGF promotes TrkA binding to APP and APP trafficking to the Golgi, where APP–BACE interaction is hindered, finally resulting in reduced generation of sAPPβ, CTFβ and amyloid‐beta (1‐42). These results demonstrate that NGF signalling directly controls basal APP phosphorylation, subcellular localization and BACE cleavage, and pave the way for novel approaches specifically targeting ShcC signalling and/or the APP–TrkA interaction in AD therapy.     

A TAG on to the neurogenic functions of APP

The proteolytic processing of amyloid β precursor protein (APP) has long been studied because of its association with the pathology of Alzheimer''s disease (AD). The ectodomain of APP is shed by α- or β-secretase cleavage. The remaining membrane bound stub can then undergo regulated intramembrane proteolysis (RIP) by γ-secretase. This cleavage can release amyloid β (Aβ) from the stub left by β-secretase cleavage but also releases the APP intracellular domain (AICD) after α- or β-secretase cleavage. The physiological functions of this proteolytic processing are not well understood. We compare the proteolytic processing of APP to the ligand-dependent RIP of Notch. In this review, we discuss recent evidence suggesting that TAG1 is a functional ligand for APP. The interaction between TAG1 and APP triggers γ-secretase-dependent release of AICD. TAG1, APP and Fe65 colocalise in the neurogenic ventricular zone and in fetal neural progenitor cells in vitro. Experiments in TAG1, APP and Fe65 null mice as well as TAG1 and APP double-null mice demonstrate that TAG1 induces a γ-secretase- and Fe65-dependent suppression of neurogenesis.Key words: Amyloid β precursor protein, APP, TAG1, AICD, Fe65, neurogenesis, Alzheimer''s disease     

The neuronal adaptor protein X11beta reduces amyloid beta-protein levels and amyloid plaque formation in the brains of transgenic mice

Accumulation of cerebral amyloid beta-protein (Abeta) is believed to be part of the pathogenic process in Alzheimer's disease. Abeta is derived by proteolytic cleavage from a precursor protein, the amyloid precursor protein (APP). APP is a type-1 membrane-spanning protein, and its carboxyl-terminal intracellular domain binds to X11beta, a neuronal adaptor protein. X11beta has been shown to inhibit the production of Abeta in transfected non-neuronal cells in culture. However, whether this is also the case in vivo in the brain and whether X11beta can also inhibit the deposition of Abeta as amyloid plaques is not known. Here we show that transgenic overexpression of X11beta in neurons leads to a decrease in cerebral Abeta levels in transgenic APPswe Tg2576 mice that are a model of the amyloid pathology of Alzheimer's disease. Moreover, overexpression of X11beta retards amyloid plaque formation in these APPswe mice. Our findings suggest that modulation of X11beta function may represent a novel therapeutic approach for preventing the amyloid pathology of Alzheimer's disease.