Apolipoprotein E modulates gamma-secretase cleavage of the amyloid precursor protein

Polymorphisms in the apolipoprotein E (APOE) gene affect the risk of Alzheimer disease and the amount of amyloid beta-protein (Abeta) deposited in the brain. The apoE protein reduces Abeta levels in conditioned media from cells in culture, possibly through Abeta clearance mechanisms. To explore this effect, we treated multiple neural and non-neural cell lines for 24 h with apoE at concentrations similar to those found in the cerebrospinal fluid (1-5 microg/mL). The apoE treatment reduced Abeta40 by 60-80% and Abeta42 to a lesser extent (20-30%) in the conditioned media. Surprisingly, apoE treatment resulted in an accumulation of amyloid precursor protein (APP)-C-terminal fragments in cell extracts and a marked reduction of APP intracellular domain-mediated signaling, consistent with diminished gamma-secretase processing of APP. All three isoforms of apoE, E2, E3 and E4, had similar effects on Abeta and APP-C-terminal fragments, and the effects were independent of the low-density lipoprotein receptor family. Apolipoprotein E had minimal effects on Notch cleavage and signaling in cell-based assays. These data suggest that apoE reduces gamma-secretase cleavage of APP, lowering secreted Abeta levels, with stronger effects on Abeta40. The apoE modulation of Abeta production and APP signaling is a potential mechanism affecting Alzheimer disease risk.     

Environment- and mutation-dependent aggregation behavior of Alzheimer amyloid beta-protein

The deposition of amyloid beta-protein in the brain is a fundamental process in the development of Alzheimerís disease; however, the mechanism underlying aggregation of amyloid beta-protein remains to be determined. Here, we report that a membrane-mimicking environment, generated in the presence of detergents or a ganglioside, is sufficient per se for amyloid fibril formation from soluble amyloid beta-protein. Furthermore, hereditary variants of amyloid beta-protein, which are caused by amyloid precursor protein gene mutations, including the Dutch (E693Q), Flemish (A692G) and Arctic (E693G) types, show mutually different aggregation behavior in these environments. Notably, the Arctic-type amyloid beta-protein, in contrast to the wild-type and other variant forms, shows a markedly rapid and higher level of amyloid fibril formation in the presence of sodium dodecyl sulfate or GM1 ganglioside. These results suggest that there are favorable local environments for fibrillogenesis of amyloid beta-protein.     

Inhibition of amyloid beta-peptide production by blockage of beta-secretase cleavage site of amyloid precursor protein

Amyloid beta-peptide (Abeta) is implicated as the major causative agent in Alzheimer's disease (AD). Abeta is produced by the processing of the amyloid precursor protein (APP) by BACE1 (beta-secretase) and gamma-secretase. Many inhibitors have been developed for the secretases. However, the inhibitors will interfere with the processing of not only APP but also of other secretase substrates. In this study, we describe the development of inhibitors that prevent production of Abeta by specific binding to the beta-cleavage site of APP. We used the hydropathic complementarity (HC) approach for the design of short peptide inhibitors. Some of the HC peptides were bound to the substrate peptide (Sub W) corresponding to the beta-cleavage site of APP and blocked its cleavage by recombinant human BACE1 (rhBACE1) in vitro. In addition, HC peptides specifically inhibited the cleavage of Sub W, and not affecting other BACE1 substrates. Chemical modification allowed an HC peptide (CIQIHF) to inhibit the processing of APP as well as the production of Abeta in the treated cells. Such novel APP-specific inhibitors will provide opportunity for the development of drugs that can be used for the prevention and treatment of AD with minimal side effects.     

Caspase cleavage of members of the amyloid precursor family of proteins

The synapse loss and neuronal cell death characteristic of Alzheimer's disease (AD) are believed to result in large part from the neurotoxic effects of beta-amyloid peptide (Abeta), a 40-42 amino acid peptide(s) derived proteolytically from beta-amyloid precursor protein (APP). However, APP is also cleaved intracellularly to generate a second cytotoxic peptide, C31, and this cleavage event occurs in vivo as well as in vitro and preferentially in the brains of AD patients (Lu et al. 2000). Here we show that APPC31 is toxic to neurons in primary culture, and that like APP, the APP family members APLP1 and possibly APLP2 are cleaved by caspases at their C-termini. The carboxy-terminal peptide derived from caspase cleavage of APLP1 shows a degree of neurotoxicity comparable to APPC31. Our results suggest that even though APLP1 and APLP2 cannot generate Abeta, they may potentially contribute to the pathology of AD by generating peptide fragments whose toxicity is comparable to that of APPC31.     

Frame-shifted amyloid precursor protein found in Alzheimer's disease and Down's syndrome increases levels of secreted amyloid beta40

Frame-shifted amyloid precursor protein (APP(+1)), which has a truncated out-of-frame C-terminus, accumulates in the neuropathological hallmarks of patients with Alzheimer's disease pathology. To study a possible involvement of APP(+1) in the pathogenesis of Alzheimer's disease, we expressed APP695 and APP(+1) in the HEK293 cell-line and studied whether the processing of APP695 was affected. APP(+1) is a secretory protein, but high expression of APP695 and APP(+1) results in the formation of intracellular aggregate-like structures containing both proteins and Fe65, an adaptor protein that interacts with APP695. APP(+1) is shown to interact with APP695, suggesting that these structures consist of functional protein complexes. Such an interaction can also be anticipated in post-mortem brains of young Down's syndrome patients without any sign of neuropathology. Here we observed APP(+1) immunoreactivity in beaded fibres. Additional support for functional consequences on the processing of APP695 comes from a 1.4-fold increase in levels of secreted amyloid beta40 in cells co-expressing APP695 and APP(+1), although APP(+1) itself does not contain the amyloid beta sequence. Taken together, these data show that co-expression of APP695 and APP(+1) affects the processing of APP695 in a pro-amyloidogenic way and this could gradually contribute to Alzheimer's disease pathology, as has been implicated in Down's syndrome patients.     

Amyloid precursor protein cross-linking stimulates beta amyloid production and pro-inflammatory cytokine release in monocytic lineage cells

Beta amyloid peptide-containing neuritic plaques are a defining feature of Alzheimer's disease pathology. Beta amyloid are 38-43 residue peptides derived by proteolytic cleavage of amyloid precursor protein. Although much attention has focused on the proteolytic events leading to beta amyloid generation, the function of amyloid precursor protein remains poorly described. Previously, we reported that amyloid precursor protein functions as a pro-inflammatory receptor on monocytic lineage cells and defined a role for amyloid precursor protein in adhesion by demonstrating that beta(1) integrin-mediated pro-inflammatory activation of monocytes is amyloid precursor protein dependent. We demonstrated that antibody-induced cross-linking of amyloid precursor protein in human THP-1 monocytes and primary mouse microglia stimulates a tyrosine kinase-based pro-inflammatory signaling response leading to acquisition of a reactive phenotype. Here, we have identified pro-inflammatory mediators released upon amyloid precursor protein-dependent activation of monocytes and microglia. We show that amyloid precursor protein cross-linking stimulated tyrosine kinase-dependent increases in pro-inflammatory cytokine release and a tyrosine kinase-independent increase in beta amyloid 1-42 generation. These data provide much needed insight into the function of amyloid precursor protein and provide potential therapeutic targets to limit inflammatory changes associated with the progression of Alzheimer's disease.     

Effects of human apolipoprotein E isoforms on the amyloid beta-protein concentration and lipid composition in brain low-density membrane domains

Apolipoprotein E4 (apoE4) encoded by epsilon 4 allele is a strong genetic risk factor for Alzheimer's disease (AD). ApoE4 carriers have accelerated amyloid beta-protein (A beta) deposition in their brains, which may account for their unusual susceptibility to AD. We hypothesized that the accelerated A beta deposition in the brain of apoE4 carriers is mediated through cholesterol-enriched low-density membrane (LDM) domains. Thus, the concentrations of A beta and various lipids in LDM domains were quantified in the brains of homozygous apoE3 and apoE4 knock-in (KI) mice, and in the brains of those mice bred with beta-amyloid precursor protein (APP) transgenic mice (Tg2576). The A beta 40 and A beta 42 concentrations and the A beta 42 proportions in LDM domains did not differ between apoE3 and apoE4 KI mice up to 18 months of age. The A beta 40 concentration in the LDM domains was slightly, but significantly higher in apoE3/APP mice than in apoE4/APP mice. The lipid composition of LDM domains was modulated in an apoE isoform-specific manner, but its significance for A beta deposition remains unknown. These data show that the apoE isoform-specific effects on the A beta concentration in LDM domains do not occur in KI mouse models.     

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.     

Cross-seeding of wild-type and hereditary variant-type amyloid beta-proteins in the presence of gangliosides

We investigated the molecular mechanism underlying the ganglioside-induced initiation of the assembly of wild and hereditary variant-type amyloid beta-proteins, including Arctic-, Dutch-, and Flemish-type amyloid beta-proteins. We monitored the assembly of amyloid beta-protein by thioflavin-T assay, western blotting and electron microscopy. We also examined how externally added amyloid beta-protein assembles in a cell culture. The assembly of wild-, Arctic-, Dutch-, and Flemish-type amyloid beta-proteins were accelerated in the presence of GM1, GM1, GM3 and GD3 gangliosides. Notably, all of these amyloid beta-proteins accelerated the assembly of different type of amyloid beta-protein, following prior binding to a specific ganglioside. A specific-ganglioside-bound form of variant-type amyloid beta-protein was recognized by the antibody (4396C) specific to the GM1-ganglioside-induced altered conformation of wild-type amyloid beta-protein. Moreover, the assembly of these amyloid beta-proteins in the presence of a specific ganglioside was markedly suppressed by coincubation with 4396C. This study suggests that cross-seeding can occur between wild and hereditary variant-type amyloid beta-proteins despite differences in their amino acid sequences.     

Oral administration of a potent and selective non-peptidic BACE-1 inhibitor decreases beta-cleavage of amyloid precursor protein and amyloid-beta production in vivo

Generation and deposition of the amyloid beta (Abeta) peptide following proteolytic processing of the amyloid precursor protein (APP) by BACE-1 and gamma-secretase is central to the aetiology of Alzheimer's disease. Consequently, inhibition of BACE-1, a rate-limiting enzyme in the production of Abeta, is an attractive therapeutic approach for the treatment of Alzheimer's disease. We have designed a selective non-peptidic BACE-1 inhibitor, GSK188909, that potently inhibits beta-cleavage of APP and reduces levels of secreted and intracellular Abeta in SHSY5Y cells expressing APP. In addition, we demonstrate that this compound can effectively lower brain Abeta in vivo. In APP transgenic mice, acute oral administration of GSK188909 in the presence of a p-glycoprotein inhibitor to markedly enhance the exposure of GSK188909 in the brain decreases beta-cleavage of APP and results in a significant reduction in the level of Abeta40 and Abeta42 in the brain. Encouragingly, subchronic dosing of GSK188909 in the absence of a p-glycoprotein inhibitor also lowers brain Abeta. This pivotal first report of central Abeta lowering, following oral administration of a BACE-1 inhibitor, supports the development of BACE-1 inhibitors for the treatment of Alzheimer's disease.     

ELISA analysis of beta-secretase cleavage of the Swedish amyloid precursor protein in the secretory and endocytic pathways

Limiting beta amyloid (Abeta) production could become an important therapeutic target in Alzheimer's disease (AD). Abeta is derived by the sequential cleavage of amyloid precursor protein (APP) by beta- and gamma-secretases. A double missense mutation in APP found in a Swedish pedigree (APPsw) elevates Abeta40 and Abeta42 production. Abeta production and, therefore, beta-secretase cleavage of APPsw reportedly occur in the endoplasmic reticulum (ER), Golgi and endocytic compartments. However, the relative contribution of beta-secretase cleavage occurring in each compartment has not been determined. Experiments described here use a novel ELISA to measure the beta-cleaved product, APPswbeta. Using this ELISA, we provide new information regarding the relative amount of beta-secretase cleavage of APPsw that occurs in secretory and endocytic pathways. Using a dilysine retrieval motif to retain APPsw in the ER, we discovered that less than 15% of the beta-secretase cleavage was still detected. Experiments utilizing endocytosis-impaired mutants of APPsw revealed that little or no beta-secretase cleavage of APPsw appears to take place through endocytosis. Surprisingly, deletion of the entire cytoplasmic tail increased both APPswbeta and Abeta secretion, suggesting that protein interactions with this region normally impede beta-secretase cleavage. These results suggest that APPsw is cleaved by beta-secretase late in the secretory pathway.     

A role for 12/15 lipoxygenase in the amyloid beta precursor protein metabolism

12/15 Lipoxygenase (12/15LO) protein levels and activity are increased in pathologically affected regions of Alzheimer's disease (AD) brains, compared with controls. Its metabolic products are elevated in cerebrospinal fluid of patients with AD and individuals with mild cognitive impairment, suggesting that this enzyme may be involved early in AD pathogenesis. Herein, we investigate the effect of pharmacologic inhibition of 12/15LO on the amyloid beta precursor protein (APP) metabolism. To this end, we used CHO and N2A cells stably expressing human APP with the Swedish mutant, and two structurally distinct and selective 12/15LO inhibitors, PD146176 and CDC. Our results demonstrated that both drugs dose-dependently reduced Abeta formation without affecting total APP levels. Interestingly, in the same cells we observed a significant reduction in secreted (s)APPbeta and beta-secretase (BACE), but not sAPPalpha and ADAM10 protein levels. Together, these data show for the first time that this enzymatic pathway influences Abeta formation whereby modulating the BACE proteolytic cascade. We conclude that specific pharmacologic inhibition of 12/15LO could represent a novel therapeutic target for treating or preventing AD pathology in humans.     

Characterization of amyloid beta peptides from brain extracts of transgenic mice overexpressing the London mutant of human amyloid precursor protein

Alzheimer's disease (AD) is marked by the presence of neurofibrillary tangles and amyloid plaques in the brain of patients. To study plaque formation, we report on further quantitative and qualitative analysis of human and mouse amyloid beta peptides (Abeta) from brain extracts of transgenic mice overexpressing the London mutant of human amyloid precursor protein (APP). Using enzyme-linked immunosorbant assays (ELISAs) specific for either human or rodent Abeta, we found that the peptides from both species aggregated to form plaques. The ratios of deposited Abeta1-42/1-40 were in the order of 2-3 for human and 8-9 for mouse peptides, indicating preferential deposition of Abeta42. We also determined the identity and relative levels of other Abeta variants present in protein extracts from soluble and insoluble brain fractions. This was done by combined immunoprecipitation and mass spectrometry (IP/MS). The most prominent peptides truncated either at the carboxyl- or the amino-terminus were Abeta1-38 and Abeta11-42, respectively, and the latter was strongly enriched in the extracts of deposited peptides. Taken together, our data indicate that plaques of APP-London transgenic mice consist of aggregates of multiple human and mouse Abeta variants, and the human variants that we identified were previously detected in brain extracts of AD patients.     

Late endocytic dysfunction as a putative cause of amyloid fibril formation in Alzheimer's disease

The assembly of amyloid β-protein to amyloid fibrils is a critical event in Alzheimer's disease. Evidence exists that endocytic pathway abnormalities, including the enlargement of early endosomes, precede the extraneuronal amyloid fibril deposition in the brain. We determined whether endocytic dysfunction potently promotes the assembly of amyloid β-protein on the surface of cultured cells. Blocking the early endocytic pathway by clathrin suppression, inactivation of small GTPases, removal of membrane cholesterol, and Rab5 knockdown did not result in amyloid fibril formation on the cell surface from exogenously added soluble amyloid β-protein. In contrast, blocking the late endocytic pathway by Rab7 suppression markedly induced the amyloid fibril formation in addition to the enlargement of early endosomes. Notably, a monoclonal antibody specific to GM1-ganglioside-bound amyloid β-protein, an endogenous seed for Alzheimer amyloid, completely blocks the amyloid fibril formation. Our results suggest that late but not early endocytic dysfunction contributes to the amyloid fibril formation by facilitating the generation of amyloid seed in the Alzheimer's brain.     

Beta-secretase-cleaved amyloid precursor protein in Alzheimer brain: a morphologic study

β-amyloid (Aβ) is the main constituent of senile plaques seen in Alzheimer's disease. Aβ is derived from the amyloid precursor protein (APP) via proteolytic cleavage by proteases β- and β-secretase. In this study, we examined content and localization of β-secretase-cleaved APP (β-sAPP) in brain tissue sections from the frontal, temporal and occipital lobe. Strong granular β-sAPP staining was found throughout the gray matter of all three areas, while white matter staining was considerably weaker. β-sAPP was found to be localized in astrocytes and in axons. We found the β-sAPP immunostaining to be stronger and more extensive in gray matter in Alzheimer disease (AD) cases than controls. The axonal β-sAPP staining was patchy and unevenly distributed for the AD cases, indicating impaired axonal transport. β-sAPP was also found surrounding senile plaques and cerebral blood vessels. The results presented here show altered β-sAPP staining in the AD brain, suggestive of abnormal processing and transport of APP.     

Asp664 cleavage of amyloid precursor protein induces tau phosphorylation by decreasing protein phosphatase 2A activity

Caspase cleavage of amyloid precursor protein (APP) has been reported to be important in amyloid beta protein (Aβ)‐mediated neurotoxicity. However, the underlying mechanisms are not clearly understood. In this study, we explored the effect of caspase cleavage of APP on tau phosphorylation in relation to Aβ. We found that Asp664 cleavage of APP increased tau phosphorylation at Thr212 and Ser262 in N2A cells and primary cultured hippocampal neurons. Compared with wild‐type APP, protein phosphatase 2A (PP2A) activity was significantly increased when Asp664 cleavage was blocked by the D664A point mutation. Furthermore, we found that over‐expression of C31 reduced PP2A activity. C31 binds directly to the PP2A catalytic subunit, through the asparagine, proline, threonine, tyrosine (NPTY) motif, which is essential for C31‐induced tau hyperphosphorylation. However, it appears that the other fragment produced by Asp664 cleavage, Jcasp, modulates neither PP2A activity nor tau hyperphosphorylation. Asp664 cleavage and accompanying tau hyperphosphorylation were remarkably diminished by blockage of Aβ production using a γ‐secretase inhibitor. Taken together, our results suggest that Asp664 cleavage of APP leads to tau hyperphosphorylation at specific epitopes by modulating PP2A activity as a downstream of Aβ. Direct binding of C31 to PP2A through the C31‐NPTY domain was identified as a mechanism underlying this effect.     

The Arctic Alzheimer mutation favors intracellular amyloid-beta production by making amyloid precursor protein less available to alpha-secretase

Mutations within the amyloid-beta (Abeta) domain of the amyloid precursor protein (APP) typically generate hemorrhagic strokes and vascular amyloid angiopathy. In contrast, the Arctic mutation (APP E693G) results in Alzheimer's disease. Little is known about the pathologic mechanisms that result from the Arctic mutation, although increased formation of Abeta protofibrils in vitro and intraneuronal Abeta aggregates in vivo suggest that early steps in the amyloidogenic pathway are facilitated. Here we show that the Arctic mutation favors proamyloidogenic APP processing by increased beta-secretase cleavage, as demonstrated by altered levels of N- and C-terminal APP fragments. Although the Arctic mutation is located close to the alpha-secretase site, APP harboring the Arctic mutation is not an inferior substrate to a disintegrin and metalloprotease-10, a major alpha-secretase. Instead, the localization of Arctic APP is altered, with reduced levels at the cell surface making Arctic APP less available for alpha-secretase cleavage. As a result, the extent and subcellular location of Abeta formation is changed, as revealed by increased Abeta levels, especially at intracellular locations. Our findings suggest that the unique clinical symptomatology and neuropathology associated with the Arctic mutation, but not with other intra-Abeta mutations, could relate to altered APP processing with increased steady-state levels of Arctic Abeta, particularly at intracellular locations.     

Enhancement of alpha-secretase cleavage of amyloid precursor protein by a metalloendopeptidase nardilysin

Amyloid-beta (Abeta) peptide, the principal component of senile plaques in the brains of patients with Alzheimer's disease, is derived from proteolytic cleavage of amyloid precursor protein (APP) by beta- and gamma-secretases. Alternative cleavage of APP by alpha-secretase occurs within the Abeta domain and precludes generation of Abeta peptide. Three members of the ADAM (a disintegrin and metalloprotease) family of proteases, ADAM9, 10 and 17, are the main candidates for alpha-secretases. However, the mechanism that regulates alpha-secretase activity remains unclear. We have recently demonstrated that nardilysin (EC 3.4.24.61, N-arginine dibasic convertase; NRDc) enhances ectodomain shedding of heparin-binding epidermal growth factor-like growth factor through activation of ADAM17. In this study, we show that NRDc enhances the alpha-secretase activity of ADAMs, which results in a decrease in the amount of Abeta generated. When expressed with ADAMs in cells, NRDc dramatically increased the secretion of alpha-secretase-cleaved soluble APP and reduced the amount of Abeta peptide generated. A peptide cleavage assay in vitro also showed that recombinant NRDc enhances ADAM17-induced cleavage of the peptide substrate corresponding to the alpha-secretase cleavage site of APP. A reduction of endogenous NRDc by RNA interference was accompanied by a decrease in the cleavage by alpha-secretase of APP and increase in the amount of Abeta generated. Notably, NRDc is clearly expressed in cortical neurons in human brain. Our results indicate that NRDc is involved in the metabolism of APP through regulation of the alpha-secretase activity of ADAMs, which may be a novel target for the treatment of Alzheimer's disease.     

Effects of familial Alzheimer's disease mutations on the folding nucleation of the amyloid beta-protein

The effect of single amino acid substitutions associated with the Italian (E22K), Arctic (E22G), Dutch (E22Q) and Iowa (D23N) familial forms of Alzheimer's disease and cerebral amyloid angiopathy on the structure of the 21-30 fragment of the Alzheimer amyloid β-protein (Aβ) is investigated by replica-exchange molecular dynamics simulations. The 21-30 segment has been shown in our earlier work to adopt a bend structure in solution that may serve as the folding nucleation site for Aβ. Our simulations reveal that the 24-28 bend motif is retained in all E22 mutants, suggesting that mutations involving residue E22 may not affect the structure of the folding nucleation site of Aβ. Enhanced aggregation in Aβ with familial Alzheimer's disease substitutions may result from the depletion of the E22-K28 salt bridge, which destabilizes the bend structure. Alternately, the E22 mutations may affect longer-range interactions outside the 21-30 segment that can impact the aggregation of Aβ. Substituting at residue D23, on the other hand, leads to the formation of a turn rather than a bend motif, implying that in contrast to E22 mutants, the D23N mutant may affect monomer Aβ folding and subsequent aggregation. Our simulations suggest that the mechanisms by which E22 and D23 mutations affect the folding and aggregation of Aβ are fundamentally different.     

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.