hFE65L Influences Amyloid Precursor Protein Maturation and Secretion

The amyloid precursor protein (APP) is processed in the secretory and endocytic pathways, where both the neuroprotective alpha-secretase-derived secreted APP (APPs alpha) and the Alzheimer's disease-associated beta-amyloid peptide are generated. All three members of the FE65 protein family bind the cytoplasmic domain of APP, which contains two sorting signals, YTS and YENPTY. We show here that binding of APP to the C-terminal phosphotyrosine interaction domain of hFE65L requires an intact YENPTY clathrin-coated pit internalization sequence. To study the effects of the hFE65L/APP interaction on APP trafficking and processing, we performed pulse/chase experiments and examined APP maturation and secretion in an H4 neuroglioma cell line inducible for expression of the hFE65L protein. Pulse/chase analysis of endogenous APP in these cells showed that the ratio of mature to total cellular APP increased after the induction of hFE65L. We also observed a three-fold increase in the amount of APPs alpha recovered from conditioned media of cells overexpressing hFE65L compared with uninduced controls. The effect of hFE65L on the levels of APPs alpha secreted is due neither to a simple increase in the steady-state levels of APP nor to activation of the protein kinase C-regulated APP secretion pathway. We conclude that the effect of hFE65L on APP processing is due to altered trafficking of APP as it transits through the secretory pathway.     

Expression of human FE65 in amyloid precursor protein transgenic mice is associated with a reduction in beta-amyloid load

FE65 is an adaptor protein that interacts with the cytoplasmic tail of the amyloid precursor protein (APP). In cultured non-neuronal cells, the formation of the FE65-APP complex is a key element for the modulation of APP processing, signalling and beta-amyloid (Abeta) production. The functions of FE65 in vivo, including its role in the metabolism of neuronal APP, remain to be investigated. In this study, transgenic mice expressing human FE65 were generated and crossbred with APP transgenic mice, known to develop Abeta deposits at 6 months of age. Compared with APP mice, APP/FE65 double transgenic mice exhibited a lower Abeta accumulation in the cerebral cortex as demonstrated by immunohistochemistry and immunoassay, and a lower level of APP-CTFs. The reduced accumulation of Abeta in APP/FE65 double transgenics, compared with APP mice, could be linked to the low Abeta42 level observed at 4 months of age and to the lower APP-CTFs levels. The present work provides evidence that FE65 plays a role in the regulation of APP processing in an in vivo model.     

Production of intracellular amyloid-containing fragments in hippocampal neurons expressing human amyloid precursor protein and protection against amyloidogenesis by subtle amino acid substitutions in the rodent sequence.

A distinguishing feature of Alzheimer's disease (AD) is the deposition of amyloid plaques in brain parenchyma. These plaques arise by the abnormal accumulation of beta A4, a proteolytic fragment of amyloid precursor protein (APP). Despite the fact that neurons are dramatically affected in the course of the disease, little is known about the neuronal processing of APP. To address this question we have expressed in fully mature, synaptically active rat hippocampal neurons, the neuronal form of human APP (APP695), two mutant forms of human APP associated with AD, and the mouse form of APP (a species known not to develop amyloid plaques). Protein expression was achieved via the Semliki Forest Virus system. Expression of wild type human APP695 resulted in the secretion of beta A4-amyloid peptide and the intracellular accumulation of potential amyloidogenic and non-amyloidogenic fragments. The relative amount of amyloid-containing fragments increased dramatically during expression of the clinical mutants, while it decreased strongly when the mouse form of APP was expressed. 'Humanizing' the rodent APP sequence by introducing three mutations in the beta A4-region also led to increased production of amyloid peptide to levels similar to those obtained with human APP. The single Gly601 to Arg substitution alone was sufficient to triple the ratio of beta A4-peptide to non-amyloidogenic p3-peptide. Due to the capacity of these cells to secrete and accumulate intracellular amyloid fragments, we hypothesize that in the pathogenesis of AD there is a positive feed-back loop where neurons are both producers and victims of amyloid, leading to neuronal degeneration and dementia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neuronal protein trafficking associated with Alzheimer disease

Aberrant and/or cumulative amyloid-beta (Aβ) production, resulting from proteolytic processing of the amyloid precursor protein (APP) by β and γ-secretases, have been postulated to be a main etiological basis of Alzheimer disease (AD). A number of proteins influence the subcellular trafficking itinerary of APP and the b-site APP-cleaving enzyme (BACE1) between the cell surface, endosomes and the trans-Golgi network (TGN). Available evidence suggests that co-residence of APP and BACE1 in the endosomal compartments promotes amyloidogenesis. Retrograde transport of APP out of the endosome to the TGN reduces Aβ production, while APP routed to and kept at the cell surface enhances its non-amyloidogenic, α-secretase-mediated processing. Changes in post-Golgi membrane trafficking in aging neurons that may influence APP processing is particularly relevant to late-onset, idiopathic AD. Dystrophic axons are key features of AD pathology, and impaired axonal transport could play crucial roles in the pathogenesis of idiopathic AD. Recent evidence has also indicated that Aβ-induced synaptic defects and memory impairment could be explained by a loss of both AMPA and NMDA receptors through endocytosis. Detail understanding of factors that influence these neuronal trafficking processes will open up novel therapeutic avenues for preventing or delaying the onset of symptomatic AD.     

Aging impact on amyloid precursor protein neuronal trafficking

Neurons live a lifetime. Neuronal aging may increase the risk of Alzheimer's disease. How does neuronal membrane trafficking maintain synapse function during aging? In the normal aged brain, intraneuronal beta-amyloid (Aβ) accumulates without Alzheimer's disease mutations or risk variants. However, do changes with neuronal aging potentiate Aβ accumulation? We reviewed the membrane trafficking of the amyloid precursor protein in neurons and highlighted its importance in Aβ production. Importantly, we reviewed the evidence supporting the impact of aging on neuronal membrane trafficking, APP processing, and consequently Aβ production. Dissecting the molecular regulators of APP trafficking during neuronal aging is required to identify strategies to delay synaptic decline and protect from Alzheimer's disease.     

Huntingtin associated protein 1 regulates trafficking of the amyloid precursor protein and modulates amyloid beta levels in neurons

J. Neurochem. (2012) 122, 1010-1022. ABSTRACT: Amyloid precursor protein (APP) is involved in the pathogenesis of Alzheimer's disease. It is axonally transported, endocytosed and sorted to different cellular compartments where amyloid beta (Aβ) is produced. However, the mechanism of APP trafficking remains unclear. We present evidence that huntingtin associated protein 1 (HAP1) may reduce Aβ production by regulating APP trafficking to the non-amyloidogenic pathway. HAP1 and APP are highly colocalized in a number of brain regions, with similar distribution patterns in both mouse and human brains. They are associated with each other, the interacting site is the 371-599 of HAP1. APP is more retained in cis-Golgi, trans-Golgi complex, early endosome and ER-Golgi intermediate compartment in HAP1-/- neurons. HAP1 deletion significantly alters APP endocytosis and reduces the re-insertion of APP into the cytoplasmic membrane. Amyloid precursor protein-YFP(APP-YFP) vesicles in HAP1-/- neurons reveal a decreased trafficking rate and an increased number of motionless vesicles. Knock-down of HAP1 protein in cultured cortical neurons of Alzheimer's disease mouse model increases Aβ levels. Our data suggest that HAP1 regulates APP subcellular trafficking to the non-amyloidogenic pathway and may negatively regulate Aβ production in neurons.     

Rivastigmine lowers Aβ and increases sAPPα levels, which parallel elevated synaptic markers and metabolic activity in degenerating primary rat neurons

Overproduction of amyloid-β (Aβ) protein in the brain has been hypothesized as the primary toxic insult that, via numerous mechanisms, produces cognitive deficits in Alzheimer's disease (AD). Cholinesterase inhibition is a primary strategy for treatment of AD, and specific compounds of this class have previously been demonstrated to influence Aβ precursor protein (APP) processing and Aβ production. However, little information is available on the effects of rivastigmine, a dual acetylcholinesterase and butyrylcholinesterase inhibitor, on APP processing. As this drug is currently used to treat AD, characterization of its various activities is important to optimize its clinical utility. We have previously shown that rivastigmine can preserve or enhance neuronal and synaptic terminal markers in degenerating primary embryonic cerebrocortical cultures. Given previous reports on the effects of APP and Aβ on synapses, regulation of APP processing represents a plausible mechanism for the synaptic effects of rivastigmine. To test this hypothesis, we treated degenerating primary cultures with rivastigmine and measured secreted APP (sAPP) and Aβ. Rivastigmine treatment increased metabolic activity in these cultured cells, and elevated APP secretion. Analysis of the two major forms of APP secreted by these cultures, attributed to neurons or glia based on molecular weight showed that rivastigmine treatment significantly increased neuronal relative to glial secreted APP. Furthermore, rivastigmine treatment increased α-secretase cleaved sAPPα and decreased Aβ secretion, suggesting a therapeutic mechanism wherein rivastigmine alters the relative activities of the secretase pathways. Assessment of sAPP levels in rodent CSF following once daily rivastigmine administration for 21 days confirmed that elevated levels of APP in cell culture translated in vivo. Taken together, rivastigmine treatment enhances neuronal sAPP and shifts APP processing toward the α-secretase pathway in degenerating neuronal cultures, which mirrors the trend of synaptic proteins, and metabolic activity.     

AMPA Receptor Activation Promotes Non-Amyloidogenic Amyloid Precursor Protein Processing and Suppresses Neuronal Amyloid-β Production

Soluble oligomeric amyloid β peptide (Aβ) generated from processing of the amyloid precursor protein (APP) plays a central role in the pathogenesis of Alzheimer''s Disease (AD) and through actions at glutamatergic synapses affects excitability and plasticity. The physiological control of APP processing is not fully understood but stimulation of synaptic NMDA receptors (NMDAR) can suppress Aβ levels through an ERK-dependent increase in α-secretase activity. AMPA-type glutamate receptors (AMPAR) couple to ERK phosphorylation independently of NMDAR activation raising the possibility that stimulation of AMPAR might similarly promote non-amyloidogenic APP processing. We have tested this hypothesis by investigating whether AMPAR directly regulate APP processing in cultured mouse cortical neurons, by analyzing APP C-terminal fragments (CTFs), soluble APP (sAPP), Aβ levels, and cleavage of an APP-GAL4 reporter protein. We report that direct stimulation of AMPAR increases non-amyloidogenic α-secretase-mediated APP processing and inhibits Aβ production. Processing was blocked by the matrix metalloproteinase inhibitor TAPI-1 but was only partially dependent on Ca²⁺ influx and ERK activity. AMPAR can therefore, be added to the repertoire of receptors that couple to non-amyloidogenic APP processing at glutamatergic synapses and thus pharmacological targeting of AMPAR could potentially influence the development and progression of Aβ pathology in AD.     

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.     

Hormonal control of cerebral amyloidogenesis in Alzheimer's diseases

Estrogen reduces the risk of Alzheimer disease (AD) in postmenopausal women, β‐amyloid (Aβ) burden in animal models of AD, and secretion of Aβ from neuronal cultures. The biological basis for these effects remains unknown. Aβ is proteolytically derived from the β‐amyloid precursor protein (βAPP) within the secretory pathway by distinct enzymatic activities known as β‐ and gamma‐secretase. Aggregated Aβ peptides are found predominantly within extraneuronal space and are believed to initiate toxic and inflammatory cascades leading to neuronal death. The major population of secreted Aβ peptides is generated within the trans‐Golgi‐network (TGN), also the major site of βAPP residence in neurons at steady state. Utilizing cell‐free systems derived from both neuroblastoma cells and primary neurons, we demonstrate that 17β‐estradiol (17β‐E2) stimulates formation of vesicles containing βAPP, from the TGN. Accelerated βAPP trafficking precludes maximal Aβ generation within the TGN. 17β‐E2 appears to modulate TGN phospholipid levels, particularly those of phosphatidylinositol, and recruit soluble trafficking factors, such as Rab11, to the TGN. Together, these results suggest that estrogen may exert its anti‐Aβ effects by regulating βAPP trafficking within the late secretory pathway. These results suggest a novel mechanism through which 17β‐E2 may act in estrogen‐responsive tissues and illustrate how altering the kinetics of a protein's transport can influence its metabolic fate.     

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.     

Large quantities of Abeta peptide are constitutively released during amyloid precursor protein metabolism in vivo and in vitro

The metabolism of the amyloid precursor protein (APP) has been extensively investigated because its processing generates the amyloid-β-peptide (Aβ), which is a likely cause of Alzheimer disease. Much prior research has focused on APP processing using transgenic constructs and heterologous cell lines. Work to date in native neuronal cultures suggests that Aβ is produced in very large amounts. We sought to investigate APP metabolism and Aβ production simultaneously under more physiological conditions in vivo and in vitro using cultured rat cortical neurons and live pigs. We found in cultured neurons that both APP and Aβ are secreted rapidly and at extremely high rates into the extracellular space (2-4 molecules/neuron/s for Aβ). Little APP is degraded outside of the pathway that leads to extracellular release. Two metabolic pools of APP are identified, one that is metabolized extremely rapidly (t1/2;) = 2.2 h), and another, surface pool, composed of both synaptic and extrasynaptic elements, that turns over very slowly. Aβ release and accumulation in the extracellular medium can be accounted for stoichiometrically by the extracellular release of β-cleaved forms of the APP ectodomain. Two α-cleavages of APP occur for every β-cleavage. Consistent with the results seen in cultured neurons, an extremely high rate of Aβ production and secretion from the brain was seen in juvenile pigs. In summary, our experiments show an enormous and rapid production and extracellular release of Aβ and the soluble APP ectodomain. A small, slowly metabolized, surface pool of full-length APP is also identified.     

Coordinated metabolism of Alcadein and amyloid beta-protein precursor regulates FE65-dependent gene transactivation

The Alcadeins (Alcs)/calsyntenins and the amyloid beta-protein precursor (APP) associate with each other in the brain by binding via their cytoplasmic domains to X11L (the X11-like protein). We previously reported that the formation of this APP-X11L-Alc tripartite complex suppresses the metabolic cleavages of APP. We show here that the metabolism of the Alcs markedly resembles that of APP. The Alcs are subjected to a primary cleavage event that releases their extracellular domain. Alcs then undergo a secondary presenilin-dependent gamma-cleavage that leads to the secretion of the amyloid beta-protein-like peptide and the liberation of an intracellular domain fragment (AlcICD). However, when Alc is in the tripartite complex, it escapes from these cleavages, as does APP. We also found that AlcICD suppressed the FE65-dependent gene transactivation activity of the APP intracellular domain fragment, probably because AlcICD competes with the APP intracellular domain fragment for binding to FE65. We propose that the Alcs and APP are coordinately metabolized in neurons and that their cleaved cytoplasmic fragments are reciprocally involved in the regulation of FE65-dependent gene transactivation. Any imbalance in the metabolism of Alcs and APP may influence the FE65-dependent gene transactivation, which together with increased secretion of amyloid beta-protein may contribute to neural disorders.     

Mint Proteins Are Required for Synaptic Activity-dependent Amyloid Precursor Protein (APP) Trafficking and Amyloid β Generation

Aberrant amyloid β (Aβ) production plays a causal role in Alzheimer disease pathogenesis. A major cellular pathway for Aβ generation is the activity-dependent endocytosis and proteolytic cleavage of the amyloid precursor protein (APP). However, the molecules controlling activity-dependent APP trafficking in neurons are less defined. Mints are adaptor proteins that directly interact with the endocytic sorting motif of APP and are functionally important in regulating APP endocytosis and Aβ production. We analyzed neuronal cultures from control and Mint knockout neurons that were treated with either glutamate or tetrodotoxin to stimulate an increase or decrease in neuronal activity, respectively. We found that neuronal activation by glutamate increased APP endocytosis, followed by elevated APP insertion into the cell surface, stabilizing APP at the plasma membrane. Conversely, suppression of neuronal activity by tetrodotoxin decreased APP endocytosis and insertion. Interestingly, we found that activity-dependent APP trafficking and Aβ generation were blocked in Mint knockout neurons. We showed that wild-type Mint1 can rescue APP internalization and insertion in Mint knockout neurons. In addition, we found that Mint overexpression increased excitatory synaptic activity and that APP was internalized predominantly to endosomes associated with APP processing. We demonstrated that presenilin 1 (PS1) endocytosis requires interaction with the PDZ domains of Mint1 and that this interaction facilitates activity-dependent colocalization of APP and PS1. These findings demonstrate that Mints are necessary for activity-induced APP and PS1 trafficking and provide insight into the cellular fate of APP in endocytic pathways essential for Aβ production.     

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.     

The role of membrane trafficking in the processing of amyloid precursor protein and production of amyloid peptides in Alzheimer's disease

Alzheimer's disease (AD) is characterized by progressive accumulation of misfolded proteins, which form senile plaques and neurofibrillary tangles, and the release of inflammatory mediators by innate immune responses. β-Amyloid peptide (Aβ) is derived from sequential processing of the amyloid precursor protein (APP) by membrane-bound proteases, namely the β-secretase, BACE1, and γ-secretase. Membrane trafficking plays a key role in the regulation of APP processing as both APP and the processing secretases traffic along distinct pathways. Genome wide sequencing studies have identified several AD susceptibility genes which regulate membrane trafficking events. To understand the pathogenesis of AD it is critical that the cell biology of APP and Aβ production in neurons is well defined. This review discusses recent advances in unravelling the membrane trafficking events associated with the production of Aβ, and how AD susceptible alleles may perturb the sorting and transport of APP and BACE1. Mechanisms whereby inflammation may influence APP processing are also considered.