The polarity protein Par3 regulates APP trafficking and processing through the endocytic adaptor protein Numb.
ABSTRACT: The processing of amyloid precursor protein (APP) into ?-amyloid peptide (A?) is a key step in the pathogenesis of Alzheimer's disease (AD), and trafficking dysregulations of APP and its secretases contribute significantly to altered APP processing. Here we show that the cell polarity protein Par3 plays an important role in APP processing and trafficking. We found that the expression of full length Par3 is significantly decreased in AD patients. Overexpression of Par3 promotes non-amyloidogenic APP processing, while depletion of Par3 induces intracellular accumulation of A?. We further show that Par3 functions by regulating APP trafficking. Loss of Par3 decreases surface expression of APP by targeting APP to the late endosome/lysosome pathway. Finally, we show that the effects of Par3 are mediated through the endocytic adaptor protein Numb, and Par3 functions by interfering with the interaction between Numb and APP. Together, our studies show a novel role for Par3 in regulating APP processing and trafficking.
Project description:The cleavage of amyloid precursor protein (APP) by ?-site APP cleaving enzyme 1 (BACE1) is the rate-limiting step in beta amyloid generation during Alzheimer's disease (AD) pathogenesis. In AD brains, BACE1 is abnormally accumulated in endocytic compartments, where the acidic pH is optimal for its activity. However, mechanisms regulating the endosome-to-trans-Golgi network (TGN) retrieval of BACE1 remain unclear. Here, we show that partitioning defective 3 (Par3) facilitates BACE1 retrograde trafficking from endosomes to the TGN. Par3 functions through aPKC-mediated phosphorylation of BACE1 on Ser498, which in turn promotes the interaction between BACE1 and phosphofurin acidic cluster sorting protein 1 and facilitates the retrograde trafficking of BACE1 to the TGN. In human AD brains, there is a significant decrease in Ser498 phosphorylation of BACE1 suggesting that defective phosphorylation-dependent retrograde transport of BACE1 is important in AD pathogenesis. Together, our studies provide mechanistic insight into a novel role for Par3 and aPKC in regulating the retrograde endosome-to-TGN trafficking of BACE1 and shed light on the mechanisms of AD pathogenesis.
Project description:The convergence between amyloid precursor protein (APP) and its ?-secretase ?-site APP cleaving enzyme 1 (BACE1) is a prerequisite for the generation of ?-amyloid peptide, a key pathogenic agent for Alzheimer's disease. Yet the underlying molecular mechanisms regulating their convergence remain unclear. Here, we show that the polarity protein partitioning-defective 3 (Par3) regulates the polarized convergence between APP and BACE1 in hippocampal neurons. Par3 forms a complex with BACE1 through its first PDZ domain, which is important for regulating BACE1 endosome-to-TGN trafficking. In the absence of Par3, there is an increase in the convergence between internalized APP and BACE1. In hippocampal neurons, loss of Par3 leads to increased APP and BACE1 convergence in axons but not in dendrites. This polarized convergence mainly occurs in retrograde or stalled axonal late endocytic organelles and is likely due to compartment-specific regulation of APP trafficking by Par3. Together, our data show a novel function for Par3 in regulating polarized convergence between APP and BACE1 in hippocampal neurons.
Project description:Central to the pathogenesis of Alzheimer disease is the aberrant processing of the amyloid precursor protein (APP) to generate amyloid beta-peptide (Abeta), the principle component of amyloid plaques. The cell fate determinant Numb is a phosphotyrosine binding domain (PTB)-containing endocytic adapter protein that interacts with the carboxyl-terminal domain of APP. The physiological relevance of this interaction is unknown. Mammals produce four alternatively spliced variants of Numb that differ in the length of their PTB and proline-rich region. In the current study, we determined the influence of the four human Numb isoforms on the intracellular trafficking and processing of APP. Stable expression of Numb isoforms that differ in the PTB but not in the proline-rich region results in marked differences in the sorting of APP to the recycling and degradative pathways. Neural cells expressing Numb isoforms that lack the insert in the PTB (short PTB (SPTB)) exhibited marked accumulation of APP in Rab5A-labeled early endosomal and recycling compartments, whereas those expressing isoforms with the insertion in the PTB (long PTB (LPTB)) exhibited reduced amounts of cellular APP and its proteolytic derivatives relative to parental control cells. Neither the activities of the beta- and gamma-secretases nor the expression of APP mRNA were significantly different in the stably transfected cells, suggesting that the differential effects of the Numb proteins on APP metabolism is likely to be secondary to altered APP trafficking. In addition, the expression of SPTB-Numb increases at the expense of LPTB-Numb in neuronal cultures subjected to stress, suggesting a role for Numb in stress-induced Abeta production. Taken together, these results suggest distinct roles for the human Numb isoforms in APP metabolism and may provide a novel potential link between altered Numb isoform expression and increased Abeta generation.
Project description:Epithelial-mesenchymal transition (EMT), which can be caused by aberrant tyrosine kinase signalling, marks epithelial tumour progression and metastasis, yet the underlying molecular mechanism is not fully understood. Here, we report that Numb interacts with E-cadherin (E-cad) through its phosphotyrosine-binding domain (PTB) and thereby regulates the localization of E-cad to the lateral domain of epithelial cell-cell junction. Moreover, Numb engages the polarity complex Par3-aPKC-Par6 by binding to Par3 in polarized Madin-Darby canine kidney cells. Intriguingly, after Src activation or hepatocyte growth factor (HGF) treatment, Numb decouples from E-cad and Par3 and associates preferably with aPKC-Par6. Binding of Numb to aPKC is necessary for sequestering the latter in the cytosol during HGF-induced EMT. Knockdown of Numb by small hairpin RNA caused a basolateral-to-apicolateral translocation of E-cad and beta-catenin accompanied by elevated actin polymerization, accumulation of Par3 and aPKC in the nucleus, an enhanced sensitivity to HGF-induced cell scattering, a decrease in cell-cell adhesion, and an increase in cell migration. Our work identifies Numb as an important regulator of epithelial polarity and cell-cell adhesion and a sensor of HGF signalling or Src activity during EMT.
Project description:Alzheimer's disease (AD) is the most common cause of dementia and is likely caused by defective amyloid precursor protein (APP) trafficking and processing in neurons leading to amyloid plaques containing the amyloid-β (Aβ) APP peptide byproducts. Understanding how APP is targeted to selected destinations inside neurons and identifying the mechanisms responsible for the generation of Aβ are thus the keys for the advancement of new therapies. We previously developed a mouse model with a mutation at tyrosine (Tyr) 682 in the C-terminus of APP. This residue is needed for APP to bind to the coating protein Clathrin and to the Clathrin adaptor protein AP2 as well as for the correct APP trafficking and sorting in neurons. By extending these findings to humans, we found that APP binding to Clathrin is decreased in neural stem cells from AD sufferers. Increased APP Tyr phosphorylation alters APP trafficking in AD neurons and it is associated to Fyn Tyr kinase activation. We show that compounds affecting Tyr kinase activity and counteracting defects in AD neurons can control APP location and compartmentalization. APP Tyr phosphorylation is thus a potential therapeutic target for AD.
Project description:Amyloid precursor protein (APP) is cleaved by ?-site amyloid precursor protein-cleaving enzyme 1 (BACE1) to produce ?-amyloid (A?), a critical pathogenic peptide in Alzheimer's disease (AD). A? generation can be affected by the intracellular trafficking of APP or its related secretases, which is thus important to understanding its pathological alterations. Although sorting nexin (SNX) family proteins regulate this trafficking, the relevance and role of sorting nexin-4 (SNX4) regarding AD has not been studied yet.In this study, human brain tissue and APP/PS1 mouse brain tissue were used to check the disease relevance of SNX4. To investigate the role of SNX4 in AD pathogenesis, several experiments were done, such as coimmunoprecipitation, Western blotting, immunohistochemistry, and gradient fractionation.We found that SNX4 protein levels changed in the brains of patients with AD and of AD model mice. Overexpression of SNX4 significantly increased the levels of BACE1 and A?. Downregulation of SNX4 had the opposite effect. SNX4 interacts with BACE1 and prevents BACE1 trafficking to the lysosomal degradation system, resulting in an increased half-life of BACE1 and increased production of A?.We show that SNX4 regulates BACE1 trafficking. Our findings suggest novel therapeutic implications of modulating SNX4 to regulate BACE1-mediated ?-processing of APP and subsequent A? generation.
2017-01-01 | S-EPMC5251330 | BioStudies
Project description:Cleavage of amyloid precursor protein (APP) by BACE-1 (?-site APP cleaving enzyme-1) is the rate-limiting step in amyloid-? (A?) production and a neuropathological hallmark of Alzheimer's disease (AD). Despite decades of research, mechanisms of amyloidogenic APP processing remain highly controversial. Here, we show that in neurons APP processing and A? production are controlled by the protein complex-2 (AP-2), an endocytic adaptor known to be required for APP endocytosis. Now we find that AP-2 prevents amyloidogenesis by additionally functioning downstream of BACE1 endocytosis, regulating BACE1 endosomal trafficking and its delivery to lysosomes. AP-2 is decreased in iPSC-derived neurons from patients with late-onset AD, while conditional AP-2 knockout (KO) mice exhibit increased A? production, resulting from accumulation of BACE1 within late endosomes and autophagosomes. Deletion of BACE1 decreases amyloidogenesis and mitigates synapse loss in neurons lacking AP-2. Taken together, these data suggest a mechanism for BACE1 intracellular trafficking and degradation via an endocytosis-independent function of AP-2 and reveal a novel role for endocytic proteins in AD
Project description:Cleavage of amyloid precursor protein (APP) by BACE-1 (?-site APP cleaving enzyme 1) is the rate-limiting step in amyloid-? (A?) production and a neuropathological hallmark of Alzheimer's disease (AD). Despite decades of research, mechanisms of amyloidogenic APP processing remain highly controversial. Here, we show that in neurons, APP processing and A? production are controlled by the protein complex-2 (AP-2), an endocytic adaptor known to be required for APP endocytosis. Now, we find that AP-2 prevents amyloidogenesis by additionally functioning downstream of BACE1 endocytosis, regulating BACE1 endosomal trafficking and its delivery to lysosomes. AP-2 is decreased in iPSC-derived neurons from patients with late-onset AD, while conditional AP-2 knockout (KO) mice exhibit increased A? production, resulting from accumulation of BACE1 within late endosomes and autophagosomes. Deletion of BACE1 decreases amyloidogenesis and mitigates synapse loss in neurons lacking AP-2. Taken together, these data suggest a mechanism for BACE1 intracellular trafficking and degradation via an endocytosis-independent function of AP-2 and reveal a novel role for endocytic proteins in AD.
Project description:In Drosophila, the partition defective (Par) complex containing Par3, Par6 and atypical protein kinase C (aPKC) directs the polarized distribution and unequal segregation of the cell fate determinant Numb during asymmetric cell divisions. Unequal segregation of mammalian Numb has also been observed, but the factors involved are unknown. Here, we identify in vivo phosphorylation sites of mammalian Numb and show that both mammalian and Drosophila Numb interact with, and are substrates for aPKC in vitro. A form of mammalian Numb lacking two protein kinase C (PKC) phosphorylation sites (Numb2A) accumulates at the cell membrane and is refractory to PKC activation. In epithelial cells, mammalian Numb localizes to the basolateral membrane and is excluded from the apical domain, which accumulates aPKC. In contrast, Numb2A is distributed uniformly around the cell cortex. Mutational analysis of conserved aPKC phosphorylation sites in Drosophila Numb suggests that phosphorylation contributes to asymmetric localization of Numb, opposite to aPKC in dividing sensory organ precursor cells. These results suggest a model in which phosphorylation of Numb by aPKC regulates its polarized distribution in epithelial cells as well as during asymmetric cell divisions.
Project description:The scaffold protein PAR3 and the kinase PAR1 are essential proteins that control cell polarity. Their precise opposite localisations define plasma membrane domains with specific functions. PAR3 and PAR1 are mutually inhibited by direct or indirect phosphorylations, but their fates once phosphorylated are poorly known. Through precise spatiotemporal quantification of PAR3 localisation in the Drosophila oocyte, we identify several mechanisms responsible for its anterior cortex accumulation and its posterior exclusion. We show that PAR3 posterior plasma membrane exclusion depends on PAR1 and an endocytic mechanism relying on RAB5 and PI(4,5)P2. In a second phase, microtubules and the dynein motor, in connection with vesicular trafficking involving RAB11 and IKK-related kinase, IKK?, are required for PAR3 transport towards the anterior cortex. Altogether, our results point to a connection between membrane trafficking and dynein-mediated transport to sustain PAR3 asymmetry.