FE65 proteins regulate NMDA receptor activation-induced amyloid precursor protein processing

Amyloid precursor protein (APP) family members and their proteolytic products are implicated in normal nervous system function and Alzheimer's disease pathogenesis. APP processing and Aβ secretion are regulated by neuronal activity. Various data suggest that NMDA receptor (NMDAR) activity plays a role in both non-amyloidogenic and amyloidogenic APP processing depending on whether synaptic or extrasynaptic NMDARs are activated, respectively. The APP-interacting FE65 proteins modulate APP trafficking and processing in cell lines, but little is known about their contribution to APP trafficking and processing in neurons, either in vivo or in vitro. In this study, we examined the contribution of the FE65 protein family to APP trafficking and processing in WT and FE65/FE65L1 double knockout neurons under basal conditions and following NMDAR activation. We report that FE65 proteins facilitate neuronal Aβ secretion without affecting APP fast axonal transport to pre-synaptic terminals. In addition, FE65 proteins facilitate an NMDAR-dependent non-amyloidogenic APP processing pathway. Generation of high-molecular weight (HMW) species bearing an APP C-terminal epitope was also observed following NMDAR activation. These HMW species require proteasomal and calpain activities for their accumulation. Recovery of APP polypeptide fragments from electroeluted HMW species having molecular weights consistent with calpain I cleavage of APP suggests that HMW species are complexes formed from APP metabolic products. Our results indicate that the FE65 proteins contribute to physiological APP processing and accumulation of APP metabolic products resulting from NMDAR activation.     

The Alzheimer amyloid precursor protein (APP) and FE65, an APP-binding protein, regulate cell movement.

FE65 binds to the Alzheimer amyloid precursor protein (APP), but the function of this interaction has not been identified. Here, we report that APP and FE65 are involved in regulation of cell movement. APP and FE65 colocalize with actin and Mena, an Abl-associated signaling protein thought to regulate actin dynamics, in lamellipodia. APP and FE65 specifically concentrate with beta 1-integrin in dynamic adhesion sites known as focal complexes, but not in more static adhesion sites known as focal adhesions. Overexpression of APP accelerates cell migration in an MDCK cell wound--healing assay. Coexpression of APP and FE65 dramatically enhances the effect of APP on cell movement, probably by regulating the amount of APP at the cell surface. These data are consistent with a role for FE65 and APP, possibly in a Mena-containing macromolecular complex, in regulation of actin-based motility.     

gamma-Secretase cleavage and binding to FE65 regulate the nuclear translocation of the intracellular C-terminal domain (ICD) of the APP family of proteins

Regulated intramembrane proteolysis (RIP) of the amyloid precursor protein (APP) produces amyloid beta-protein (Abeta), the probable causative agent of Alzheimer's disease (AD), and is therefore an important target for therapeutic intervention. However, there is a burgeoning consensus that gamma-secretase, one of the proteases that generates Abeta, is also critical for the signal transduction of APP and a growing list of other receptors. APP is a member of a gene family that includes two amyloid precursor-like proteins, APLP1 and APLP2. Although APP and the APLPs undergo similar proteolytic processing, there is little information about the role of their gamma-secretase-generated intracellular domains (ICDs). Here, we show that APLP1 and 2 undergo presenilin-dependent RIP similar to APP, resulting in the release of a approximately 6 kDa ICD for each protein. Each of the ICDs are degraded by an insulin degrading enzyme-like activity, but they can be stabilized by members of the FE65 family and translocate to the nucleus. Given that modulation of APP processing is a therapeutic target and that the APLPs are processed in a manner similar to APP, any strategy aimed at altering APP proteolysis will have to take into account possible effects on signaling by APLP 1 and 2.     

Membrane palmitoylated proteins regulate trafficking and processing of nectins

Nectins are cell-cell adhesion molecules involved in the formation of various intercellular junctions and the establishment of apical-basal polarity at cell-cell adhesion sites. To have a better understanding of the roles of nectins in the formation of cell-cell junctions, we searched for new cytoplasmic binding partners for nectin. We report that nectin-1α associates with membrane palmitoylated protein 3 (MPP3), one of the human homologues of a Drosophila tumor suppressor gene, Disc large. Two major forms of MPP3 at 66 and 98 kDa were detected, in conjunction with nectin-1α, suggesting that an association between the two may occur in various cell types. Nectin-1α recruits MPP3 to cell-cell contact sites, mediated by a PDZ-binding motif at the carboxyl terminus of nectin-1α. Association with MPP3 increases cell surface expression of nectin-1α and enhances nectin-1α ectodomain shedding, indicating that MPP3 regulates trafficking and processing of nectin-1α. Further study showed that MPP3 interacts with nectin-3α, but not with nectin-2α, showing that the association of nectins with MPP3 is isoform-specific. MPP5, another MPP family member, interacts with nectins with varying affinity and facilitates surface expression of nectin-1α, nectin-2α, and nectin-3α. These data suggest that wide interactions between nectins and MPP family members may occur in various cell-cell junctions and that these associations may regulate trafficking and processing of nectins.     

Rab35 and glucocorticoids regulate APP and BACE1 trafficking to modulate Aβ production

Chronic stress and elevated glucocorticoids (GCs), the major stress hormones, are risk factors for Alzheimer’s disease (AD) and promote AD pathomechanisms, including overproduction of toxic amyloid-β (Aβ) peptides and intraneuronal accumulation of hyperphosphorylated Tau protein. The latter is linked to downregulation of the small GTPase Rab35, which mediates Tau degradation via the endolysosomal pathway. Whether Rab35 is also involved in Aβ overproduction remains an open question. Here, we find that hippocampal Rab35 levels are decreased not only by stress/GC but also by aging, another AD risk factor. Moreover, we show that Rab35 negatively regulates Aβ production by sorting amyloid precursor protein (APP) and β-secretase (BACE1) out of the endosomal network, where they interact to produce Aβ. Interestingly, Rab35 coordinates distinct intracellular trafficking steps for BACE1 and APP, mediated by its effectors OCRL and ACAP2, respectively. Finally, we demonstrate that Rab35 overexpression prevents the amyloidogenic trafficking of APP and BACE1 induced by high GC levels. These studies identify Rab35 as a key regulator of APP processing and suggest that its downregulation may contribute to stress-related and AD-related amyloidogenesis.Subject terms: Cellular neuroscience, Alzheimer''s disease     

Dab1 binds to Fe65 and diminishes the effect of Fe65 or LRP1 on APP processing

Fe65 and Dab1 are adaptor proteins that interact with the cytoplasmic domain of amyloid precursor protein (APP) via phosphotyrosine‐binding (PTB) domain and that affect APP processing and Aβ production. Co‐expression of Dab1 with Fe65 and APP resumed nuclear translocation of Fe65 despite of its cytoplasmic anchor, APP. The decreased amount of Fe65 bound to APP was shown in co‐immunoprecipitation assay from the cells with Dab1 which also displayed the effect on APP processing. These data suggested that Fe65 and Dab1 compete for binding to APP. Surprisingly, we found that Fe65 interacts with Dab1 via C‐terminal region of Dab1 and unphosphorylated Dab1 is capable of binding Fe65. Dab1 interacts with the low‐density lipoprotein receptor‐related protein (LRP) as well as APP through its PTB domain. Dab1 significantly decreased the amount of APP bound to LRP and the level of secreted APP and APP‐CTF in LRP expressing cells, unlike Fe65. It implies that overexpression of Dab1 diminish LRP–APP complex formation, resulting in altered APP processing. The competition for overlapped binding site among adaptor proteins may be related to the regulation mechanism of APP metabolism in various conditions. J. Cell. Biochem. 111: 508–519, 2010. © 2010 Wiley‐Liss, Inc.     

Structural features of the KPI domain control APP dimerization, trafficking, and processing

The two major isoforms of human APP, APP695 and APP751, differ by the presence of a Kunitz-type protease inhibitor (KPI) domain in the extracellular region. APP processing and function is thought to be regulated by homodimerization. We used bimolecular fluorescence complementation (BiFC) to study dimerization of different APP isoforms and mutants. APP751 was found to form significantly more homodimers than APP695. Mutation of dimerization motifs in the TM domain did not affect fluorescence complementation, but native folding of KPI is critical for APP751 homodimerization. APP751 and APP695 dimers were mostly localized at steady state in the Golgi region, suggesting that most of the APP751 and 695 dimers are in the secretory pathway. Mutation of the KPI led to the retention of the APP homodimers in the endoplasmic reticulum. We finally showed that APP751 is more efficiently processed through the nonamyloidogenic pathway than APP695. These findings provide new insight on the particular role of KPI domain in APP dimerization. The correlation observed between dimerization, subcellular localization, and processing suggests that dimerization acts as an efficient regulator of APP trafficking in the secretory compartments that has major consequences on its processing.     

Epac signaling pathway involves STEF, a guanine nucleotide exchange factor for Rac, to regulate APP processing

The amyloid precursor protein (APP) is a key protein involved in the development of Alzheimer's disease. We previously identified a signal transduction secretory pathway in which the small G protein Rac sets downstream of the cAMP/Epac/Rap1 signalling cascade regulating the alpha cleavage of APP [Maillet, M. et al. (2003) Crosstalk between Rap and Rac regulates secretion of sAPP alpha. Nat. Cell Biol. 5, 633-639]. We now report that Rap1 can physically and specifically associate with the guanine nucleotide exchange factor (GEF) STEF through its TSS region. A deleted TSS domain of STEF cells fails to activate Rac1 and dramatically decreases secretion of the non-amyloidogenic soluble form of APP (sAPP alpha) induced by the cAMP-binding protein Epac. Altogether, our data show that upon Epac activation, Rap1 recruits STEF through its TSS region and activates Rac1, which mediates APP processing.     

Lipoprotein receptors and cholesterol in APP trafficking and proteolytic processing,implications for Alzheimer's disease

Amyloid-β (Aβ) peptide accumulation in the brain is central to the pathogenesis of Alzheimer's disease (AD). Aβ is produced through proteolytic processing of a transmembrane protein, β-amyloid precursor protein (APP), by β- and γ-secretases. Mounting evidence has demonstrated that alterations in APP cellular trafficking and localization directly impact its processing to Aβ. Members of the low-density lipoprotein receptor family, including LRP, LRP1B, SorLA/LR11, and apoER2, interact with APP and regulate its endocytic trafficking. Additionally, APP trafficking and processing are greatly affected by cellular cholesterol content. In this review, we summarize the current understanding of the roles of lipoprotein receptors and cholesterol in APP trafficking and processing and their implication for AD pathogenesis and therapy.     

Syk-dependent actin dynamics regulate endocytic trafficking and processing of antigens internalized through the B-cell receptor

Antigen binding to the B-cell receptor (BCR) induces multiple signaling cascades that ultimately lead to B lymphocyte activation. In addition, the BCR regulates the key trafficking events that allow the antigen to reach endocytic compartments devoted to antigen processing, i.e., that are enriched for major histocompatibility factor class II (MHC II) and accessory molecules such as H2-DM. Here, we analyze the role in antigen processing and presentation of the tyrosine kinase Syk, which is activated upon BCR engagement. We show that convergence of MHC II- and H2-DM-containing compartments with the vesicles that transport BCR-uptaken antigens is impaired in cells lacking Syk activity. This defect in endocytic trafficking compromises the ability of Syk-deficient cells to form MHC II-peptide complexes from BCR-internalized antigens. Altered endocytic trafficking is associated to a failure of Syk-deficient cells to properly reorganize their actin cytoskeleton in response to BCR engagement. We propose that, by modulating the actin dynamics induced upon BCR stimulation, Syk regulates the positioning and transport of the vesicles that carry the molecules required for antigen processing and presentation.     

Immunity as a link between obesity and insulin resistance

Obesity is a major public health problem in the United States and worldwide. Further, obesity is causally linked to the pathogenesis of insulin resistance, metabolic syndrome and type-2 diabetes (T2D). A chronic low-grade inflammation occurring in adipose tissue is at least in part responsible for the obesity-induced insulin resistance. This adipose tissue inflammation is characterized by changes in immune cell populations giving rise to altered adipo/cytokine profiles, which in turn induces skeletal muscle and hepatic insulin resistance. Detailed molecular mechanisms of insulin resistance, adipose tissue inflammation and the implications of these findings on therapeutic strategies are discussed in this review.     

A novel cyclin provides a link between dopamine and RNA processing.

Regulation of gene expression by dopamine may play an important role in learning, reward, and addiction. Hyman and colleagues now report the characterization of ania-6, a novel cyclin that associates with RNA polymerase II and is induced by dopamine or cocaine in the neostriatum. Ania-6 may thus provide a link between dopamine and gene expression at the level of mRNA processing.     

Employing a superior BACE1 cleavage sequence to probe cellular APP processing

The involvement of beta-secretase (BACE1; beta-site APP-cleaving enzyme) in producing the beta-amyloid component of plaques found in the brains of Alzheimer's patients, has fueled a major research effort to characterize this protease. Here, we describe work toward understanding the substrate specificity of BACE1 that began by considering the natural APP substrate and its Swedish mutant, APPSw, and proceeded on to include oxidized insulin B chain and ubiquitin substrates. From these findings, and the study of additional synthetic peptides, we determined that a decapeptide derived from APP in which the P3-P2' sequence, ...VKM--DA..., was replaced by ...ISY--EV... (-- = beta site of cleavage), yielded a substrate that was cleaved by BACE1 seven times faster than the corresponding APPSw peptide, SEVNL--DAEFR. The expanded peptide, GLTNIKTEEISEISY--EVEFRWKK, was cleaved an additional seven times faster than its decapeptide counterpart (boldface), and provides a substrate allowing assay of BACE1 at picomolar concentrations. Several APP mutants reflecting these beta-site amino acid changes were prepared as the basis for cellular assays. The APPISYEV mutant proved to be a cellular substrate that was superior to APPSw. The assay based on APPISYEV is highly specific for measuring BACE1 activity in cells; its homolog, BACE2, barely cleaved APPISYEV at the beta-site. Insertion of the optimized ISY--EV motif at either the beta-site (Asp1) or beta'-site (Glu11) directs the rate of cellular processing of APP at these two accessible sites. Thus, we have identified optimal BACE1 substrates that will be useful to elucidate the cellular enzymatic actions of BACE1, and for design of inhibitors that might be of therapeutic benefit in Alzheimer's disease.     

Interactions between GIPC-APPL and GIPC-TRP1 regulate melanosomal protein trafficking and melanogenesis in human melanocytes

By virtue of the presence of multiple protein–protein interaction and signaling domains, PDZ proteins play important roles in assembling protein complexes that participate in diverse cell biological processes. GIPC is a versatile PDZ protein that binds a variety of target proteins in different cell types. In previous studies we showed that, in epidermal melanocytes, GIPC interacts with newly synthesized melanosomal protein TRP1 in the Golgi region and proposed that this interaction may facilitate intracellular trafficking of TRP1. However, since GIPC contains a single PDZ domain and no other known protein interaction motifs, it is not known how GIPC–TRP1 interaction affects melanosome biogenesis and/or melanin pigmentation. Here, we show that in human primary melanocytes GIPC interacts with AKT-binding protein APPL (adaptor protein containing pleckstrin homology, leucine zipper and phosphotyrosine binding domains), which readily co-precipitates with newly synthesized TRP1. Knockdown of either GIPC or APPL inhibits melanogenesis by decreasing tyrosinase protein levels and enzyme activity. In melanocytes, APPL exists in a complex with GIPC and phospho-AKT. Inhibition of AKT phosphorylation using a PI3-kinase inhibitor abolishes this interaction and results in retardation TRP1 in the Golgi. These data suggest that interactions between TRP1–GIPC and GIPC–APPL–AKT provide a potential link between melanogenesis and PI3 kinase signaling.     

Exploiting human anatomical variability as a link between genome and cognome

Although talents and disabilities appear to run in families, direct links between genes and cognitive ability are difficult to establish. Investigators are currently searching for intermediate phenotypes with plausible links to both genome and cognome (the cognitive phenotype). Cortical anatomy could provide one such intermediate phenotype. Variation in cortical size, asymmetry and sulcal pattern is influenced by genetic variation in neurotrophic factors and can predict variation in verbal and mathematical talent. Anecdotal evidence suggests that individuals with a rare morphological variant of Sylvian fissure sometimes have superior visualization ability combined with verbal deficits. Documentation of such 'cognitive cortical syndromes' might prove as genetically informative as the identification of dysmorphic syndromes associated with mental retardation. A necessary prerequisite for the establishment of such syndromes is a reliable technique for the identification of cortical patterns. Recent technical advances in software for automatically labeling and measuring cortical sulci now provide the possibility of establishing standard measures for their shape, size and location. Such measures are a prerequisite for genetic studies of cortical patterns that could illuminate the neurodevelopmental pathways by which genes affect cognitive ability.     

Exosomes as a messager to regulate the crosstalk between macrophages and cardiomyocytes under hypoxia conditions

Recent studies have confirmed that cardiomyocyte‐derived exosomes have many pivotal biological functions, like influencing the progress of coronary artery disease via modulating macrophage phenotypes. However, the mechanisms underlying the crosstalk between cardiomyocytes and macrophages have not been fully characterized. Hence, this study aimed to observe the interaction between cardiomyocytes under hypoxia and macrophages through exosome communication and further evaluate the ability of exosomes derived from cardiomyocytes cultured under hypoxic conditions (Hypo‐Exo) to polarize macrophages, and the effect of alternatively activated macrophages (M2) on hypoxic cardiomyocytes. Our results revealed that hypoxia facilitated the production of transforming growth factor‐beta (TGF‐β) in H9c2 cell‐derived exosomes. Moreover, exosomes derived from cardiomyocytes cultured under normal conditions (Nor‐Exo) and Hypo‐Exo could induce RAW264.7 cells into classically activated macrophages (M1) and M2 macrophages respectively. Likewise, macrophage activation was induced by circulating exosomes isolated from normal human controls (hNor‐Exo) or patients with acute myocardial infarction (hAMI‐Exo). Thus, our findings support that the profiles of hAMI‐Exo have been changed, which could regulate the polarization of macrophages and subsequently the polarized M2 macrophages reduced the apoptosis of cardiomyocytes in return. Based on our findings, we speculate that exosomes have emerged as important inflammatory response modulators regulating cardiac oxidative stress injury.     

The insulin-degrading enzyme as a link between insulin and neuropeptides metabolism

We have applied a recently developed HPLC-MS enzymatic assay to investigate the cryptic peptides generated by the action of the insulin-degrading enzyme (IDE) on some neuropeptides (NPs) involved in the development of tolerance and dependence to opioids. Particularly, the tested NPs are generated from the NPFF precursor (pro-NPFF (A)): NPFF (FLFQPQRF) and NPAF (AGEGLSSPFWSLAAPQRF). The results show that IDE is able to cleave NPFF and NPAF, generating specific cryptic peptides. As IDE is also responsible for the processing of many other peptides in the brain (amyloid beta protein among the others), we have also performed competitive degradation assays using mixtures of insulin and the above mentioned NPs. Data show that insulin is able to slow down the degradation of both NPs tested, whereas, surprisingly, NPAF is able to accelerate insulin degradation, hinting IDE as the possible link responsible of the mutual influence between insulin and NPs metabolism.     

APP transgenic mice and their application to drug discovery

The development of transgenic mice expressing mutated forms of the human amyloid precursor protein (APP) and presenilin-1 (PS1), proteins associated with familial forms of Alzheimer's disease (AD), has provided a backbone for translational studies of potential novel drug therapies. Such mice model some aspects of AD pathology in that they develop senile plaque-like deposits of the amyloid beta-protein (Aβ) together with inflammatory pathology and some degree of neurodegeneration. Aβ deposition is considered to be a potentially pathogenic feature of AD and drug discovery programmes utilising such mice and associated with drugs now reaching the clinic have been largely directed towards decreasing the deposition. This goal has been achieved in the mouse models, although the agents developed have not, to date, shown evidence of efficacy in AD sufferers and, in some cases, have worsened the clinical state. Nevertheless, reducing the pathological features of the disease continues to be the objective of pharmacological intervention and ongoing programmes continue to use transgenic mice expressing mutated APP and PS1 transgenes in attempts to overcome issues and difficulties arising from the initial clinical trials and to explore new approaches to AD treatment.