Cytoplasmic Domain Of Amyloid Precursor Protein Research Articles

Phosphorylation of the amyloid precursor protein (APP) at Ser-675 promotes APP processing involving meprin β

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by abnormal deposition of β-amyloid (Aβ) peptides. Aβ is a cleavage product of the amyloid precursor protein (APP), and aberrant posttranslational modifications of APP can alter APP processing and increase Aβ generation. In the AD brain, seven different residues, including Ser-675 (APP695 numbering) in the APP cytoplasmic domain has been found to be phosphorylated. Here, we show that expression of a phosphomimetic variant of Ser-675 in APP (APP-S675E), in human neuroblastoma SK-N-AS cells, reduces secretion of the soluble APP ectodomain (sAPPα), even though the total plasma membrane level of APP was unchanged compared with APP levels in cells expressing APPwt or APP-S675A. Moreover, the level of an alternative larger C-terminal fragment (CTF) increased in the APP-S675E cells, whereas the CTF form that was most abundant in cells expressing APPwt or APP-S675A decreased in the APP-S675E cells. Upon siRNA-mediated knockdown of the astacin metalloprotease meprin β, the levels of the alternative CTF decreased and the CTF ratio was restored back to APPwt levels. Our findings suggest that APP–Ser-675 phosphorylation alters the balance of APP processing, increasing meprin β–mediated and decreasing α-secretase–mediated processing of APP at the plasma membrane. As meprin β cleavage of APP has been shown to result in formation of highly aggregation-prone, truncated Aβ2–40/42 peptides, enhanced APP processing by this enzyme could contribute to AD pathology. We propose that it would be of interest to clarify in future studies how APP–Ser-675 phosphorylation promotes meprin β–mediated APP cleavage.

Adaptor Protein Sorting Nexin 17 Regulates Amyloid Precursor Protein Trafficking and Processing in the Early Endosomes

Accumulation of extracellular amyloid beta peptide (Abeta), generated from amyloid precursor protein (APP) processing by beta- and gamma-secretases, is toxic to neurons and is central to the pathogenesis of Alzheimer disease. Production of Abeta from APP is greatly affected by the subcellular localization and trafficking of APP. Here we have identified a novel intracellular adaptor protein, sorting nexin 17 (SNX17), that binds specifically to the APP cytoplasmic domain via the YXNPXY motif that has been shown previously to bind several cell surface adaptors, including Fe65 and X11. Overexpression of a dominant-negative mutant of SNX17 and RNA interference knockdown of endogenous SNX17 expression both reduced steady-state levels of APP with a concomitant increase in Abeta production. RNA interference knockdown of SNX17 also decreased APP half-life, which led to the decreased steady-state levels of APP. Immunofluorescence staining confirmed a colocalization of SNX17 and APP in the early endosomes. We also showed that a cell surface adaptor protein, Dab2, binds to the same YXNPXY motif and regulates APP endocytosis at the cell surface. Our results thus provide strong evidence that both cell surface and intracellular adaptor proteins regulate APP endocytic trafficking and processing to Abeta. The identification of SNX17 as a novel APP intracellular adaptor protein highly expressed in neurons should facilitate the understanding of the relationship between APP intracellular trafficking and processing to Abeta.

Reduction of mint-1, mint-2, and APP overexpression in okadaic acid-treated neurons

Treatment of neurons with okadaic acid, a protein phosphatase-2A inhibitor, has been used to induce tau phosphorylation and neuronal death, and to create a research model of Alzheimer's disease. Amyloid precursor protein (APP) is the precursor protein of the beta-amyloid peptide that accumulates in extracellular plaques in Alzheimer's disease. Several studies have shown that mint-1 (munc18-interacting protein 1) and mint-2 bind to the YENPTY motif in the cytoplasmic domain of APP and inhibit processing of APP to beta-amyloid peptide. Here, we report that, upon neurodegeneration with okadaic acid, mint-1 and mint-2 levels were reduced by proteolytic cleavage, and that these changes were followed by increases in APP levels. We also show that the mint-1 and mint-2 cleavage and APP overexpression were prevented by calpain inhibitor-I and inhibitor-II. These results indicate that mint cleavage might play a role in the pathophysiology of Alzheimer's disease.

Amyloid precursor protein cytoplasmic domain with phospho-Thr668 accumulates in Alzheimer’s disease and its transgenic models: a role to mediate interaction of Aβ and tau

Abnormal accumulation of Abeta and tau in senile plaques (SP) and neurofibrillary tangles (NFTs) is a key event in Alzheimer's disease (AD). Here, we show that T668-phosphorylated cytoplasmic domain of APP (pT668-ACD) accumulates Abeta and tau in AD and its transgenic models. Anti-pT668 immunostaining of AD brain sections with hydrated autoclave enhancement identified SP neurites and NFTs in which pT668-ACD colocalizes with tau. We produced and examined transgenic (Tg) mice that overexpress human APP695, harboring the double Swedish/London mutation, and develop age-dependently Abeta plaques in the brain. All Abeta plaques contain co-accumulations of pT668-ACD, but co-accumulation of tau appears in only a fraction of Abeta plaques in older animals. We also examined the established tau Tg mice that overexpress the smallest human brain tau isoform and develop neuronal accumulations of tau in older animals. Examination of the old tau Tg mice showed that neuronal cells affected by tau accumulation induce co-accumulation of pT668-ACD. We speculate that in AD brains, extracellular Abeta deposition is accompanied by intracellular accumulation of pT668-ACD, followed by tau accumulation in the SP with dystrophic neurites and that neuronal cells affected by tau accumulation induce co-accumulation of pT668-ACD in NFTs. Thus, pT668-ACD is likely to mediate pathological interaction between Abeta and tau.

Amyloid precursor protein cytoplasmic domain antagonizes reelin neurite outgrowth inhibition of hippocampal neurons

The function of the amyloid precursor protein (APP), a key molecule in Alzheimer's disease (AD) remains unknown. Among the proteins that interact with the APP cytoplasmic domain in vitro and in heterologous systems is Disabled-1, a signaling molecule of the reelin pathway. The physiological consequence of this interaction is unknown. Here we used an in vitro model of hippocampal neurons grown on a reelin substrate that inhibits neurite outgrowth. Our results show that an excess of APP cytoplasmic domain internalized by a cell permeable peptide, is able to antagonize the neurite outgrowth inhibition of reelin. The APP cytoplasmic domain binds Disabled-1 and retains it in the cytoplasm, preventing it from reaching the plasma membrane and sequesters tyrosine phosphorylated Disabled-1, both of which disrupt reelin signaling. In the context of AD, increased formation of APP cytoplasmic domain in the cytosol released after cleavage of the Aβ peptide, could then inhibit reelin signaling pathway in the hippocampus and thus influence synaptic plasticity.

Down-regulation of Endogenous Amyloid Precursor Protein Processing due to Cellular Aging

Processing of amyloid precursor protein (APP) is a well acknowledged central pathogenic mechanism in Alzheimer disease. However, influences of age-associated cellular alterations on the biochemistry of APP processing have not been studied in molecular detail so far. Here, we report that processing of endogenous APP is down-regulated during the aging of normal human fibroblasts (IMR-90). The generation of intracellular APP cleavage products C99, C83, and AICD gradually declines with increasing life span and is accompanied by a reduced secretion of soluble APP (sAPP) and sAPPalpha. Further, the maturation of APP was reduced in senescent cells, which has been shown to be directly mediated by age-associated increased cellular cholesterol levels. Of the APP processing secretases, protein levels of constituents of the gamma-secretase complex, presenilin-1 (PS1) and nicastrin, were progressively reduced during aging, resulting in a progressive decrease in gamma-secretase enzymatic activity. ADAM10 (a disintegrin and metalloprotease 10) and BACE (beta-site APP-cleaving enzyme) protein levels exhibited no age-associated regulation, but interestingly, BACE enzymatic activity was increased in aged cells. PS1 and BACE are located in detergent-resistant membranes (DRMs), well structured membrane microdomains exhibiting high levels of cholesterol, and caveolin-1. Although total levels of both structural components of DRMs were up-regulated in aged cells, their particular DRM association was decreased. This age-dependent membrane modification was associated with an altered distribution of PS1 and BACE between DRM and non-DRM fractions, very likely affecting their APP processing potential. In conclusion, we have found a significant modulation of endogenous APP processing and maturation in human fibroblasts caused by age-associated alterations in cellular biochemistry.

Notch 1 interacts with the amyloid precursor protein in a Numb‐independent manner

We hypothesized that the physical interaction between the amyloid precursor protein (APP) and Notch 1 (N1) may be mediating the reported cross-talk between the respective signaling pathways. Immunoprecipitation of mouse N1 (mN1) or extracellular domain truncated mN1 (mN1-TM, mimics TACE-produced membrane-bound C-terminal fragment) specifically coprecipitated APP(751). Conversely, immunoprecipitation of APP(751) specifically coprecipitated mN1, furin-generated membrane-bound mN1 C-terminal fragment (f.mN1-TM), or mN1-TM. The London mutation of APP did not affect the APP(751)/mN1 interaction. Coexpression of APP(751) and mN1 did not affect APP processing or production of mN1 intracellular domain (mNICD). The APP(751)/mN1 interaction was Numb-independent, insofar as it was observed in HEK293 cells that lack detectable levels of Numb and was unaffected by the expression of exogenous Numb or deletion of the APP cytoplasmic domain, including the Numb-binding YENPTY sequence. This interaction was unaffected even when the N-terminal 647 amino acids of APP were replaced by a sequence of secreted alkaline phosphatase. These data combined with data showing interaction between mN1-TM and APP(751) suggest that their transmebrane domains and short sequences around them are sufficient for the interaction and that APP(751) and mN1 interact in cis. Our results imply novel functions of APP and/or N1 that derive from their interaction.

Nicastrin Functions as a γ-Secretase-Substrate Receptor

gamma-secretase catalyzes the intramembrane cleavage of amyloid precursor protein (APP) and Notch after their extracellular domains are shed by site-specific proteolysis. Nicastrin is an essential glycoprotein component of the gamma-secretase complex but has no known function. We now show that the ectodomain of nicastrin binds the new amino terminus that is generated upon proteolysis of the extracellular APP and Notch domains, thereby recruiting the APP and Notch substrates into the gamma-secretase complex. Chemical- or antibody-mediated blocking of the free amino terminus, addition of purified nicastrin ectodomain, or mutations in the ectodomain markedly reduce the binding and cleavage of substrate by gamma-secretase. These results indicate that nicastrin is a receptor for the amino-terminal stubs that are generated by ectodomain shedding of type I transmembrane proteins. Our data are consistent with a model where nicastrin presents these substrates to gamma-secretase and thereby facilitates their cleavage via intramembrane proteolysis.

Differential Roles of JIP Scaffold Proteins in the Modulation of Amyloid Precursor Protein Metabolism

We previously found that the JNK-interacting proteins JIP1b and JIP2 associate with the cytoplasmic domain of the Alzheimer's amyloid precursor protein (APP) (Taru, H., Iijima, K., Hase, M., Kirino, Y., Yagi, Y., and Suzuki, T. (2002) J. Biol. Chem. 277, 20070-20078). This interaction involves the carboxyl-terminal phosphotyrosine interaction (PI) domain of JIP1b or JIP2 and the GYENPTY motif in the APP cytoplasmic domain. The expression of JIP1b stabilizes immature APP and suppresses the production of a secreted large extracellular amino-terminal domain of APP, the generation of a cleaved intracellular carboxyl-terminal fragment of APP, and the secretion of beta-amyloid 40 and 42. Deletion of the PI domain or alteration of PI amino acid residues prevents JIP1b from interacting with APP and affecting its metabolism, but deletion of the JNK-binding domain of JIP1b has no effect. JIP2, a weaker APP-binding protein, does not influence the processing of APP, although it is known that both JIP1b and JIP2 equally regulate the JNK signaling cascade. The present results suggest that JIP1b can directly modulate APP metabolism by interacting with the APP cytoplasmic domain, independent of its regulation of the JNK signaling cascade.

Demonstration by Fluorescence Resonance Energy Transfer of Two Sites of Interaction between the Low-Density Lipoprotein Receptor-Related Protein and the Amyloid Precursor Protein: Role of the Intracellular Adapter Protein Fe65

Amyloid-beta, the major constituent of senile plaques in Alzheimer's disease, is derived from the amyloid precursor protein (APP) by proteolysis. Kunitz protease inhibitor (KPI) containing forms of APP (APP751/770) interact with a multifunctional endocytic receptor, the low-density lipoprotein receptor-related protein (LRP), which modulates its proteolytic processing affecting production of amyloid-beta. We used fluorescence resonance energy transfer (FRET) using labeled LRP and APP in H4 cell line to examine the subcellular localization and the molecular domains involved in the APP-LRP interaction. KPI-containing forms of APP (APP770) demonstrated FRET with LRP that was sensitive to the LRP inhibitor receptor-associated protein (RAP), suggesting an interaction between the extracellular domains of APP770 and LRP. APP695 also interacts with LRP to lesser degree (as measured by extracellular domain probes), and this ectodomain interaction is not altered by RAP. By using C-terminally tagged LRP and APP, we demonstrate a second site of interaction between the C termini of both APP695 and APP770 and the C terminus of LRP, and that the interactions at these regions are not sensitive to RAP. We next examined the possibility that the C-termini APP-LRP interaction was mediated by Fe65, an adaptor protein that interacts with the cytoplasmic tails of LRP and APP. FRET studies confirmed a close proximity between the amino Fe65 phosphotyrosine binding (PTB) domain and LRP cytoplasmic domain and between the carboxyl Fe65 PTB domain and the APP cytoplasmic domain. These findings demonstrate that LRP interaction with APP occurs via both extracellular and intracellular protein interaction domains.

C-Jun N-Terminal Kinase (JNK)-Interacting Protein-1b/Islet-Brain-1 Scaffolds Alzheimer's Amyloid Precursor Protein with JNK

Using a yeast two-hybrid method, we searched for amyloid precursor protein (APP)-interacting molecules by screening mouse and human brain libraries. In addition to known interacting proteins containing a phosphotyrosine-interaction-domain (PID)-Fe65, Fe65L, Fe65L2, X11, and mDab1, we identified, as a novel APP-interacting molecule, a PID-containing isoform of mouse JNK-interacting protein-1 (JIP-1b) and its human homolog IB1, the established scaffold proteins for JNK. The APP amino acids Tyr(682), Asn(684), and Tyr(687) in the G(681)YENPTY(687) region were all essential for APP/JIP-1b interaction, but neither Tyr(653) nor Thr(668) was necessary. APP-interacting ability was specific for this additional isoform containing PID and was shared by both human and mouse homologs. JIP-1b expressed by mammalian cells was efficiently precipitated by the cytoplasmic domain of APP in the extreme Gly(681)-Asn(695) domain-dependent manner. Reciprocally, both full-length wild-type and familial Alzheimer's disease mutant APPs were precipitated by PID-containing JIP constructs. Antibodies raised against the N and C termini of JIP-1b coprecipitated JIP-1b and wild-type or mutant APP in non-neuronal and neuronal cells. Moreover, human JNK1beta1 formed a complex with APP in a JIP-1b-dependent manner. Confocal microscopic examination demonstrated that APP and JIP-1b share similar subcellular localization in transfected cells. These data indicate that JIP-1b/IB1 scaffolds APP with JNK, providing a novel insight into the role of the JNK scaffold protein as an interface of APP with intracellular functional molecules.

Proteolytic processing and cell biological functions of the amyloid precursor protein

Recent research has identified some key players involved in the proteolytic processing of amyloid precursor protein (APP) to amyloid beta-peptide, the principal component of the amyloid plaques in Alzheimer patients. Interesting parallels exists with the proteolysis of other proteins involved in cell differentiation, cholesterol homeostasis and stress responses. Since the cytoplasmic domain of APP is anchored to a complex protein network that might function in axonal elongation, dendritic arborisation and neuronal cell migration, the proteolysis of APP might be critically involved in intracellular signalling events.

The disabled 1 phosphotyrosine-binding domain binds to the internalization signals of transmembrane glycoproteins and to phospholipids.

Disabled gene products are important for nervous system development in drosophila and mammals. In mice, the Dab1 protein is thought to function downstream of the extracellular protein Reln during neuronal positioning. The structures of Dab proteins suggest that they mediate protein-protein or protein-membrane docking functions. Here we show that the amino-terminal phosphotyrosine-binding (PTB) domain of Dab1 binds to the transmembrane glycoproteins of the amyloid precursor protein (APP) and low-density lipoprotein receptor families and the cytoplasmic signaling protein Ship. Dab1 associates with the APP cytoplasmic domain in transfected cells and is coexpressed with APP in hippocampal neurons. Screening of a set of altered peptide sequences showed that the sequence GYXNPXY present in APP family members is an optimal binding sequence, with approximately 0.5 microM affinity. Unlike other PTB domains, the Dab1 PTB does not bind to tyrosine-phosphorylated peptide ligands. The PTB domain also binds specifically to phospholipid bilayers containing phosphatidylinositol 4P (PtdIns4P) or PtdIns4,5P2 in a manner that does not interfere with protein binding. We propose that the PTB domain permits Dab1 to bind specifically to transmembrane proteins containing an NPXY internalization signal.

Proteasome Inhibitors Induce the Association of Alzheimer's Amyloid Precursor Protein with Hsc73

Amyloid precursor protein (APP) is a secretory membrane-bound protein that undergoes restrictive proteolysis and degradation with a short life span in the constitutive secretory pathway or in the endosomal/lysosomal compartment. The degradation machinery, including cellular trafficking and the restrictive cleavage of APP, is poorly understood. To gain further insight into the intracellular degradation mechanism of APP, we searched for effector proteins that interact with APP. We found that a cytosolic molecular chaperon, Hsc73, effectively interacts with the cytoplasmic domain of APP in the presence of proteasome inhibitors. Hsc73 binds to the cytoplasmic domain near the post-transmembrane region of APP and not to the KFERQ-related sequence, KFFEQ, at the C-terminal tail that is assumed to be the selective targeting signal for lysosomal proteolysis. The amounts of Hsc73 that bind to several APP species such as those found in pathological Familial Alzheimer's disease (FAD), Swedish, or Dutch type mutation, are almost identical, suggesting that an abnormal conformation around the secretory cleavage site or a pathological imbalance in APP processing are not irrelevant to the efficiency of Hsc73 binding.

Insertion of Lysosomal Targeting Sequences to the Amyloid Precursor Protein Reduces Secretion of βA4

The processing of the amyloid precursor protein (APP) was investigated in cells stably expressing different APP hybrid proteins. The cytoplasmic domain of APP was either deleted or replaced by the corresponding domain of the membrane protein TGN38, lamp-1, or LIMPII. The cytosolic domain of TGN38 in the APP molecule did not alter the secretion of beta A4 when compared with the wild-type APP; however, APP associated with the cell surface and the nonamyloidogenic processing of APP were reduced. With the APP molecules carrying the lysosomal targeting signals of lamp-1 or LIMPII, a decrease in the secretion of beta A4 was observed. Cell surface association and nonamyloidogenic processing were also impaired. This suggests increased degradation of APP and thus efficient targeting to the lysosomal system. Cells expressing the Swedish APP variant generated intracellular beta A4 that accumulated after treatment with chloroquine. This effect was more dramatic with APP mutants carrying lysosomal targeting signals than with full-length APP. Our data suggest the existence of an intracellular site of beta A4 generation from where beta A4 is degraded rather than secreted.

The release of Alzheimer's disease beta amyloid peptide is reduced by phorbol treatment.

Amyloid precursor protein (APP) is cleaved predominantly within the beta amyloid peptide (BAP) domain to release a non-amyloidogenic amino-terminal PN2 fragment. Treatment of cells with phorbol dibutyrate, an agent which activates protein kinase C, has been shown to increase the release of an amino-terminal fragment. A panel of mutant APP reporter constructs was expressed in which each of the potential phosphorylation sites located within the cytoplasmic domain of APP was replaced with alanine residues. Phorbol response patterns were unchanged for each of these mutants, suggesting that induced cleavage occurs independently of APP substrate phosphorylation. We find that phorbol (a) increases the release of a PN2 fragment that is consistent with the normal secretase activity, (b) decreases the release of a shorter amino-terminal APP fragment that is cleaved near the amino terminus of BAP, and (c) decreases the release of BAP which was identified based on electrophoretic mobility, epitope mapping, and radio-sequencing. These data demonstrate that pharmacological treatment can reduce the formation of BAP and suggests that protein kinase C activators could be developed as therapeutic agents to block BAP formation.

Alzheimer beta/A4 amyloid precursor protein in human brain: aging-associated increases in holoprotein and in a proteolytic fragment.

Alzheimer beta/A4 amyloid precursor protein (APP) has been suggested to play a central role in the pathogenesis of Alzheimer disease. We have measured the content of different species of APP holoprotein and carboxyl-terminal fragments in human brains from young individuals, nondemented aged individuals, and aged individuals with Alzheimer disease. By using an antibody directed against the cytoplasmic domain of APP, five species were resolved. Three of these, of molecular masses 106, 113, and 133 kDa, represent presumptive immature and mature isoforms of APP holoprotein. Two smaller proteins, of molecular masses 15 and 19 kDa, represent presumptive proteolytic carboxyl-terminal fragments of APP. The 133-, 113-, 106-, and 15-kDa species were found in both grey and white matter, whereas the 19-kDa species was found only in grey matter. Total APP immunoreactivity (sum of all five species) and the levels of the 113-, 106-, and 15-kDa species were not significantly different in brain samples from young individuals, nondemented aged individuals, and aged individuals with Alzheimer disease. In contrast, the levels of the 133- and 19-kDa species increased 2- to 3-fold with age. A correlation was observed between the levels of the 133- and 19-kDa species, suggesting a possible precursor-product relationship. The size of the 19-kDa fragment indicated that it might have an intact beta/A4 domain and therefore be amyloidogenic. The age-dependent increase either in a mature APP isoform and/or in a putative amyloidogenic fragment could explain why Alzheimer disease is associated with advanced age.