Zinc and copper effect mechanical cell adhesion properties of the amyloid precursor protein.

Alzheimer disease beta-amyloid (Abeta) peptides are generated via sequential proteolysis of amyloid precursor protein (APP) by BACE1 and gamma-secretase. A subset of BACE1 localizes to cholesterol-rich membrane microdomains, termed lipid rafts. BACE1 processing in raft microdomains of cultured cells and neurons was characterized in previous studies by disrupting the integrity of lipid rafts by cholesterol depletion. These studies found either inhibition or elevation of Abeta production depending on the extent of cholesterol depletion, generating controversy. The intricate interplay between cholesterol levels, APP trafficking, and BACE1 processing is not clearly understood because cholesterol depletion has pleiotropic effects on Golgi morphology, vesicular trafficking, and membrane bulk fluidity. In this study, we used an alternate strategy to explore the function of BACE1 in membrane microdomains without altering the cellular cholesterol level. We demonstrate that BACE1 undergoes S-palmitoylation at four Cys residues at the junction of transmembrane and cytosolic domains, and Ala substitution at these four residues is sufficient to displace BACE1 from lipid rafts. Analysis of wild type and mutant BACE1 expressed in BACE1 null fibroblasts and neuroblastoma cells revealed that S-palmitoylation neither contributes to protein stability nor subcellular localization of BACE1. Surprisingly, non-raft localization of palmitoylation-deficient BACE1 did not have discernible influence on BACE1 processing of APP or secretion of Abeta. These results indicate that post-translational S-palmitoylation of BACE1 is not required for APP processing, and that BACE1 can efficiently cleave APP in both raft and non-raft microdomains.

The amyloid precursor protein (APP) and its proteolytic product amyloid beta (Abeta) are associated with both familial and sporadic forms of Alzheimer disease (AD). Aberrant expression and function of microRNAs has been observed in AD. Here, we show that in rat hippocampal neurons cultured in vitro, the down-regulation of Argonaute-2, a key component of the RNA-induced silencing complex, produced an increase in APP levels. Using site-directed mutagenesis, a microRNA responsive element (RE) for miR-101 was identified in the 3'-untranslated region (UTR) of APP. The inhibition of endogenous miR-101 increased APP levels, whereas lentiviral-mediated miR-101 overexpression significantly reduced APP and Abeta load in hippocampal neurons. In addition, miR-101 contributed to the regulation of APP in response to the proinflammatory cytokine interleukin-1beta (IL-lbeta). Thus, miR-101 is a negative regulator of APP expression and affects the accumulation of Abeta, suggesting a possible role for miR-101 in neuropathological conditions.

Following the discovery that the amyloid precursor protein (APP) is the source of β-amyloid peptides (Aβ) that accumulate in Alzheimer’s disease (AD), structural analyses suggested that the holoprotein resembles a transmembrane receptor. Initial studies using reconstituted membranes demonstrated that APP can directly interact with the heterotrimeric G protein Gαo (but not other G proteins) via an evolutionarily G protein-binding motif in its cytoplasmic domain. Subsequent investigations in cell culture showed that antibodies against the extracellular domain of APP could stimulate Gαo activity, presumably mimicking endogenous APP ligands. In addition, chronically activating wild type APP or overexpressing mutant APP isoforms linked with familial AD could provoke Go-dependent neurotoxic responses, while biochemical assays using human brain samples suggested that the endogenous APP-Go interactions are perturbed in AD patients. More recently, several G protein-dependent pathways have been implicated in the physiological roles of APP, coupled with evidence that APP interacts both physically and functionally with Gαo in a variety of contexts. Work in insect models has demonstrated that the APP ortholog APPL directly interacts with Gαo in motile neurons, whereby APPL-Gαo signaling regulates the response of migratory neurons to ligands encountered in the developing nervous system. Concurrent studies using cultured mammalian neurons and organotypic hippocampal slice preparations have shown that APP signaling transduces the neuroprotective effects of soluble sAPPα fragments via modulation of the PI3K/Akt pathway, providing a mechanism for integrating the stress and survival responses regulated by APP. Notably, this effect was also inhibited by pertussis toxin, indicating an essential role for Gαo/i proteins. Unexpectedly, C-terminal fragments (CTFs) derived from APP have also been found to interact with Gαs, whereby CTF-Gαs signaling can promote neurite outgrowth via adenylyl cyclase/PKA-dependent pathways. These reports offer the intriguing perspective that G protein switching might modulate APP-dependent responses in a context-dependent manner. In this review, we provide an up-to-date perspective on the model that APP plays a variety of roles as an atypical G protein-coupled receptor in both the developing and adult nervous system, and we discuss the hypothesis that disruption of these normal functions might contribute to the progressive neuropathologies that typify AD.

The amyloid precursor protein (APP) is an integral membrane glycoprotein whose cleavage products, particularly amyloid-β, accumulate in Alzheimer disease (AD). APP is present at synapses and is thought to play a role in both the formation and plasticity of these critical neuronal structures. Despite the central role suggested for APP in AD pathogenesis, the mechanisms regulating APP in neurons and its processing into cleavage products remain incompletely understood. F-box only protein 2 (Fbxo2), a neuron-enriched ubiquitin ligase substrate adaptor that preferentially binds high-mannose glycans on glycoproteins, was previously implicated in APP processing by facilitating the degradation of the APP-cleaving β-secretase, β-site APP-cleaving enzyme. Here, we sought to determine whether Fbxo2 plays a similar role for other glycoproteins in the amyloid processing pathway. We present in vitro and in vivo evidence that APP is itself a substrate for Fbxo2. APP levels were decreased in the presence of Fbxo2 in non-neuronal cells, and increased in both cultured hippocampal neurons and brain tissue from Fbxo2 knock-out mice. The processing of APP into its cleavage products was also increased in hippocampi and cultured hippocampal neurons lacking Fbxo2. In hippocampal slices, this increase in cleavage products was accompanied by a significant reduction in APP at the cell surface. Taken together, these results suggest that Fbxo2 regulates APP levels and processing in the brain and may play a role in modulating AD pathogenesis.

The amyloid precursor protein (APP) and its homologs amyloid precursor-like protein1 (APLP1) and APLP2 have central physiological functions in transcellular adhesion that depend on copper and zinc mediated trans-directed dimerization of the extracellular domains E1 and E2. Copper binds to three distinct sites in APP, one in the copper binding (CuBD) and growthfactor-like (GFLD) domains each within E1, and one in the E2 domain. For APLP1 and APLP2, metal binding has so far only been shown for the E2 domain. Zinc binding has been reported for all APP family members to a unique site in the E2 domain and an additional site essential for APLP1 E2 domain trans-dimerization. Using isothermal titration calorimetry, co-immunoprecipitation, and invitro bead aggregation assays, we show that copper promotes cis- as well as trans-directed dimerization of APLP1 and APLP2, similar as reported previously for APP. Furthermore, we report a APP-specific zinc binding site with nanomolar affinity located in the E1 domain, whereas no binding of zinc to the individual subdomains GFLD or CuBD was detected. Zinc binding did not affect the cis- but trans-dimerization of APP and APLP1. Furthermore, zinc binding inhibited copper-induced trans-directed dimerization of APP. Together, we identified a high-affinity APP-specific zinc binding site in the E1 domain and revealed contrasting cis- and trans-directed dimerization properties of APP, APLP1, and APLP2 in dependence on zinc and copper ions. Consequently, changes in metal ion homeostasis, as reported in the context of synaptic activity and neurodegenerative diseases, appear as key modulators of homo- and heterotypic trans-cellular APP/APLPs complexes.

The amyloid β (Aβ) peptide, which is abundantly found in the brains of patients suffering from Alzheimer disease, is central in the pathogenesis of this disease. Therefore, to understand the processing of the amyloid precursor protein (APP) is of critical importance. Recently, we demonstrated that the metalloprotease meprin β cleaves APP and liberates soluble N-terminal APP (N-APP) fragments. In this work, we present evidence that meprin β can also process APP in a manner reminiscent of β-secretase. We identified cleavage sites of meprin β in the amyloid β sequence of the wild type and Swedish mutant of APP at positions p1 and p2, thereby generating Aβ variants starting at the first or second amino acid residue. We observed even higher kinetic values for meprin β than BACE1 for both the wild type and the Swedish mutant APP form. This enzymatic activity of meprin β on APP and Aβ generation was also observed in the absence of BACE1/2 activity using a β-secretase inhibitor and BACE knock-out cells, indicating that meprin β acts independently of β-secretase.

Alzheimer disease (AD) is characterized by senile plaques, which are mainly composed of beta amyloid (Abeta) peptides. Abeta is cleaved off from amyloid precursor protein (APP) with consecutive proteolytic processing: beta-secretase, followed by gamma-secretase. Here, we show that BRI3, a member of the BRI gene family that includes the familial British and Danish dementia gene BRI2, interacts with APP and serves as an endogenous negative regulator of Abeta production. BRI3 colocalizes with APP along neuritis in differentiated N2a cells; endogenous BRI3-APP complexes are readily detectable in mouse brain extract; reducing endogenous BRI3 levels by RNA interference results in increased Abeta secretion. BRI3 resembles BRI2, because BRI3 overexpression reduces both alpha- and beta-APP cleavage. We propose that BRI3 inhibits the various processing of APP by blocking the access of alpha- and beta-secretases to APP. However, unlike BRI2, the binding of BRI3 to the beta-secretase cleaved APP C-terminal fragment is negligible and BRI3 does not cause the massive accumulation of this APP fragment, suggesting that, unlike BRI2, BRI3 is a poor gamma-cleavage inhibitor. Competitive inhibition of APP processing by BRI3 may provide a new approach to AD therapy and prevention.

Sortilin-related receptor with A-type repeats (SORLA) is a sorting receptor that impairs processing of amyloid precursor protein (APP) to soluble (s) APP and to the amyloid beta-peptide in cultured neurons and is poorly expressed in patients with Alzheimer disease (AD). Here, we evaluated the consequences of Sorla gene defects on brain anatomy and function using mouse models of receptor deficiency. In line with a protective role for SORLA in APP metabolism, lack of the receptor results in increased amyloidogenic processing of endogenous APP and in aggravated plaque deposition when introduced into PDAPP mice expressing mutant human APP. Surprisingly, increased levels of sAPP caused by receptor deficiency correlate with pro-found stimulation of neuronal ERK signaling and with enhanced neurogenesis, providing in vivo support for neurotrophic functions of sAPP. Our data document a role for SORLA not only in control of plaque burden but also in APP-dependent neuronal signaling and suggest a molecular explanation for increased neurogenesis observed in some AD patients.

Elucidation of Abeta-lowering agents that inhibit processing of the wild-type (WT) beta-secretase amyloid precursor protein (APP) site, present in most Alzheimer disease (AD) patients, is a logical approach for improving memory deficit in AD. The cysteine protease inhibitors CA074Me and E64d were selected by inhibition of beta-secretase activity in regulated secretory vesicles that produce beta-amyloid (Abeta). The regulated secretory vesicle activity, represented by cathepsin B, selectively cleaves the WT beta-secretase site but not the rare Swedish mutant beta-secretase site. In vivo treatment of London APP mice, expressing the WT beta-secretase site, with these inhibitors resulted in substantial improvement in memory deficit assessed by the Morris water maze test. After inhibitor treatment, the improved memory function was accompanied by reduced amyloid plaque load, decreased Abeta40 and Abeta42, and reduced C-terminal beta-secretase fragment derived from APP by beta-secretase. However, the inhibitors had no effects on any of these parameters in mice expressing the Swedish mutant beta-secretase site of APP. The notable efficacy of these inhibitors to improve memory and reduce Abeta in an AD animal model expressing the WT beta-secretase APP site present in the majority of AD patients provides support for CA074Me and E64d inhibitors as potential AD therapeutic agents.

The analysis of potential sorting signals in amyloid precursor protein (APP) by site-directed mutagenesis and the disturbance of metabolic pathways by drugs is used here to define the parameters that determine polarized secretion of APP in Madin-Darby canine kidney cells. Endogenously produced APP751/770 and APP695 produced from transfected constructs are secreted almost exclusively into the basolateral compartment. The sorting mechanism is highly dependent on intracellular pH as demonstrated by its sensitivity to primary amines and inhibitors of the acidifying vacuolar protein ATPase. The role of potential basolateral sorting signals in the cytoplasmic, transmembrane, and beta A4 amyloid region of APP was investigated. Neither deletion of the endocytosis and putative basolateral sorting signal GY.NPTY nor complete deletion of the cytoplasmic domain causes apical secretion of soluble APP. Further deletion of the transmembrane domain and of the beta A4 amyloid region confirmed that the major basolateral sorting determinant resides in the extracellular domain of APP. Increased beta-secretase cleavage of APP after introduction of the "swedish" double mutation causes apical missorting of about 20% of beta-secretase-cleaved APP. The data underline the complexity of processing and sorting APP in polarized cells and suggest a possible problem of protein sorting in Alzheimer's Disease.

SorLA has been recognized as a novel sorting receptor that regulates trafficking and processing of the amyloid precursor protein (APP) and that represents a significant risk factor for sporadic Alzheimer disease. Here, we investigated the cellular mechanisms that control intracellular trafficking of sorLA and their relevance for APP processing. We demonstrate that sorLA acts as a retention factor for APP in trans-Golgi compartments/trans-Golgi network, preventing release of the precursor into regular processing pathways. Proper localization and activity of sorLA are dependent on functional interaction with GGA and PACS-1, adaptor proteins involved in protein transport to and from the trans-Golgi network. Aberrant targeting of sorLA to the recycling compartment or the plasma membrane causes faulty APP trafficking and imbalance in non-amyloidogenic and amyloidogenic processing fates. Thus, our findings identified altered routing of sorLA as a major cellular mechanism contributing to abnormal APP processing and enhanced amyloid beta-peptide formation.

Accumulation of the amyloid beta (Abeta) peptide derived from the proteolytic processing of amyloid precursor protein (APP) is the defining pathological hallmark of Alzheimer disease. We previously demonstrated that the C-terminal 37 amino acids of lipoprotein receptor-related protein (LRP) robustly promoted Abeta generation independent of FE65 and specifically interacted with Ran-binding protein 9 (RanBP9). In this study we found that RanBP9 strongly increased BACE1 cleavage of APP and Abeta generation. This pro-amyloidogenic activity of RanBP9 did not depend on the KPI domain or the Swedish APP mutation. In cells expressing wild type APP, RanBP9 reduced cell surface APP and accelerated APP internalization, consistent with enhanced beta-secretase processing in the endocytic pathway. The N-terminal half of RanBP9 containing SPRY-LisH domains not only interacted with LRP but also with APP and BACE1. Overexpression of RanBP9 resulted in the enhancement of APP interactions with LRP and BACE1 and increased lipid raft association of APP. Importantly, knockdown of endogenous RanBP9 significantly reduced Abeta generation in Chinese hamster ovary cells and in primary neurons, demonstrating its physiological role in BACE1 cleavage of APP. These findings not only implicate RanBP9 as a novel and potent regulator of APP processing but also as a potential therapeutic target for Alzheimer disease.

Identification and regulation of the high affinity binding site of the Alzheimer's disease amyloid protein precursor (APP) to glycosaminoglycans

The use of statins, 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors that block the synthesis of mevalonate (and downstream products such as cholesterol and nonsterol isoprenoids), as a therapy for Alzheimer disease is currently the subject of intense debate. It has been reported that statins reduce the risk of developing the disorder, and a link between cholesterol and Alzheimer disease pathophysiology has been proposed. Moreover, experimental studies focusing on the cholesterol-dependent effects of statins have demonstrated a close association between cellular cholesterol levels and amyloid production. However, evidence suggests that statins are pleiotropic, and the potential cholesterol-independent effects of statins on amyloid precursor protein (APP) metabolism and amyloid beta-peptide (A beta) genesis are unknown. In this study, we developed a novel in vitro system that enabled the discrete analysis of cholesterol-dependent and -independent (i.e. isoprenoid-dependent) statin effects on APP cleavage and A beta formation. Given the recent interest in the role that intracellular A beta may play in Alzheimer disease, we analyzed statin effects on both secreted and cell-associated A beta. As reported previously, low cellular cholesterol levels favored the alpha-secretase pathway and decreased A beta secretion presumably within the endocytic pathway. In contrast, low isoprenoid levels resulted in the accumulation of APP, amyloidogenic fragments, and A beta likely within biosynthetic compartments. Importantly, low cholesterol and low isoprenoid levels appeared to have completely independent effects on APP metabolism and A beta formation. Although the implications of these effects for Alzheimer disease pathophysiology have yet to be investigated, to our knowledge, these results provide the first evidence that isoprenylation is involved in determining levels of intracellular A beta.

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.