Inhibition of APP trafficking by tau protein does not increase the generation of amyloid-beta peptides

Amyloid-beta, a peptide derived from the precursor protein APP, accumulates in the brain and contributes to the neuropathology of Alzheimer's disease. Increased generation of amyloid-beta might be caused by axonal transport inhibition, via increased dwell time of APP vesicles and thereby higher probability of APP cleavage by secretase enzymes residing on the same vesicles. We tested this hypothesis using a neuronal cell culture model of inhibited axonal transport and by imaging vesicular transport of fluorescently tagged APP and beta-secretase (BACE1). Microtubule-associated tau protein blocks vesicle traffic by inhibiting the access of motor proteins to the microtubule tracks. In neurons co-transfected with CFP-tau, APP-YFP traffic into distal neurites was strongly reduced. However, this did not increase amyloid-beta levels. In singly transfected axons, APP-YFP was transported in large tubules and vesicles moving very fast (on average 3 microm/s) and with high fluxes in the anterograde direction (on average 8.4 vesicles/min). By contrast, BACE1-CFP movement was in smaller tubules and vesicles that were almost 2x slower (on average 1.6 microm/s) with approximately 18x lower fluxes (on average 0.5 vesicles/min). Two-colour microscopy of co-transfected axons confirmed that the two proteins were sorted into distinct carriers. The results do not support the above hypothesis. Instead, they indicate that APP is transported on vesicles distinct from the secretase components and that amyloid-beta is not generated in transit when transport is blocked by tau.     

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.     

Suppression of APP-containing vesicle trafficking and production of β-amyloid by AID/DHHC-12 protein

The metabolism of amyloid β-protein precursor (APP) is regulated by various cytoplasmic and/or membrane-associated proteins, some of which are involved in the regulation of intracellular membrane trafficking. We found that a protein containing Asp–His–His–Cys (DHHC) domain, alcadein and APP interacting DHHC protein (AID)/DHHC-12, strongly inhibited APP metabolism, including amyloid β-protein (Aβ) generation. In cells expressing AID/DHHC-12, APP was tethered in the Golgi, and APP-containing vesicles disappeared from the cytoplasm. Although DHHC domain-containing proteins are involved in protein palmitoylation, a AID/DHHC-12 mutant of which the enzyme activity was impaired by replacing the DHHC sequence with Ala–Ala–His–Ser (AAHS) made no detectable difference in the generation and trafficking of APP-containing vesicles in the cytoplasm or the metabolism of APP. Furthermore, the mutant AID/DHHC-12 significantly increased non-amyloidogenic α-cleavage of APP along with activation of a disintegrin and metalloproteinase 17, a major α-secretase, suggesting that protein palmitoylation involved in the regulation of α-secretase activity. AID/DHHC-12 can modify APP metabolism, including Aβ generation in multiple ways by regulating the generation and/or trafficking of APP-containing vesicles from the Golgi and their entry into the late secretary pathway in an enzymatic activity-independent manner, and the α-cleavage of APP in the enzymatic activity-dependent manner.     

Trafficking of Alzheimer's disease-related membrane proteins and its participation in disease pathogenesis

Alzheimer's disease (AD) is a common neurodegenerative disorder that causes senile dementia. The pathological characteristics are the appearance of neurofibrillary tangles comprising abnormally phosphorylated tau and senile plaques composed of amyloid beta-protein depositions. Amyloid beta-protein precursor (APP) and presenilin (PS) are known to be causative genes of familial AD. Recent analyses have documented that APP functions in the axonal transport of vesicles and PS regulates intracellular protein trafficking. Dystrophic neurites, in which APP and Alcadein accumulate in swollen axons, are also observed in AD brain. These pathological characteristics and the features of AD-related proteins suggest that AD is a disease of the vesicular transport system. Here we review recent progress of research on AD pathogenesis from the viewpoint of membrane trafficking.     

Enhanced generation of Alzheimer's amyloid-β following chronic exposure to phorbol ester correlates with differential effects on alpha and epsilon isozymes of protein kinase C

Alzheimer's amyloid precursor protein (APP) sorting and processing are modulated through signal transduction mechanisms regulated by protein phosphorylation. Notably, protein kinase C (PKC) appears to be an important component in signaling pathways that control APP metabolism. PKCs exist in at least 11 conventional and unconventional isoforms, and PKCα and PKCε isoforms have been specifically implicated in controlling the generation of soluble APP and amyloid-β (Aβ) fragments of APP, although identification of the PKC substrate phospho-state-sensitive effector proteins remains challenging. In the current study, we present evidence that chronic application of phorbol esters to cultured cells in serum-free medium is associated with several phenomena, namely: (i) PKCα down-regulation; (ii) PKCε up-regulation; (iii) accumulation of APP and/or APP carboxyl-terminal fragments in the trans Golgi network; (iv) disappearance of fluorescence from cytoplasmic vesicles bearing a green fluorescent protein tagged form of APP; (v) insensitivity of soluble APP release following acute additional phorbol application; and (vi) elevated cellular APP mRNA levels and holoprotein, and secreted Aβ. These data indicate that, unlike acute phorbol ester application, which is accompanied by lowered Aβ generation, chronic phorbol ester treatment causes differential regulation of PKC isozymes and increased Aβ generation. These data have implications for the design of amyloid-lowering strategies based on modulating PKC activity.     

SorCS1 variants and amyloid precursor protein (APP) are co‐transported in neurons but only SorCS1c modulates anterograde APP transport

Processing of amyloid precursor protein (APP) into amyloid‐β peptide (Aβ) is crucial for the development of Alzheimer's disease (AD). Because this processing is highly dependent on its intracellular itinerary, altered subcellular targeting of APP is thought to directly affect the degree to which Aβ is generated. The sorting receptor SorCS1 has been genetically linked to AD, but the underlying molecular mechanisms are poorly understood. We analyze two SorCS1 variants; one, SorCS1c, conveys internalization of surface‐bound ligands whereas the other, SorCS1b, does not. In agreement with previous studies, we demonstrate co‐immunoprecipitation and co‐localization of both SorCS1 variants with APP. Our results suggest that SorCS1c and APP are internalized independently, although they mostly share a common post‐endocytic pathway. We introduce functional Venus‐tagged constructs to study SorCS1b and SorCS1c in living cells. Both variants are transported by fast anterograde axonal transport machinery and about 30% of anterograde APP‐positive transport vesicles contain SorCS1. Co‐expression of SorCS1b caused no change of APP transport kinetics, but SorCS1c reduced the anterograde transport rate of APP and increased the number of APP‐positive stationary vesicles. These data suggest that SorCS1 and APP share trafficking pathways and that SorCS1c can retain APP from insertion into anterograde transport vesicles.

     

Expression of tau reduces secretion of Abeta without altering the amyloid precursor protein content in CHOsw cells

Insoluble deposits of tau and amyloid precursor protein (APP) peptides Abeta characterize Alzheimer's disease. We studied the role of tau in the metabolism of APP in cells stably expressing APP Swedish mutation (CHOsw). Transient expression of tau in CHOsw cells caused morphological changes, bundling of microtubules and perinuclear aggregation of Golgi-derived vesicles. It also reduced the secretion of Abeta(1-40) and Abeta(1-42) without altering the APP steady state levels. This was accompanied by a reduction in the gamma-secretase and an increase in the insulin degrading enzyme activities. Our results suggest that tau may play an inhibitory role in the amyloidogenic activity of APP.     

Dicumarol, an inhibitor of ADP-ribosylation of CtBP3/BARS, fragments golgi non-compact tubular zones and inhibits intra-golgi transport

Dicumarol (3,3'-methylenebis[4-hydroxycoumarin]) is an inhibitor of brefeldin-A-dependent ADP-ribosylation that antagonises brefeldin-A-dependent Golgi tubulation and redistribution to the endoplasmic reticulum. We have investigated whether dicumarol can directly affect the morphology of the Golgi apparatus. Here we show that dicumarol induces the breakdown of the tubular reticular networks that interconnect adjacent Golgi stacks and that contain either soluble or membrane-associated cargo proteins. This results in the formation of 65-120-nm vesicles that are sometimes invaginated. In contrast, smaller vesicles (45-65 nm in diameter, a size consistent with that of coat-protein-I-dependent vesicles) that excluded cargo proteins from their lumen are not affected by dicumarol. All other endomembranes are largely unaffected by dicumarol, including Golgi stacks, the ER, multivesicular bodies and the trans-Golgi network. In permeabilized cells, dicumarol activity depends on the function of CtBP3/BARS protein and pre-ADP-ribosylation of cytosol inhibits the breakdown of Golgi tubules by dicumarol. In functional experiments, dicumarol markedly slows down intra-Golgi traffic of VSV-G transport from the endoplasmic reticulum to the medial Golgi, and inhibits the diffusional mobility of both galactosyl transferase and VSV-G tagged with green fluorescent protein. However, it does not affect: transport from the trans-Golgi network to the cell surface; Golgi-to-endoplasmic reticulum traffic of ERGIC58; coat-protein-I-dependent Golgi vesiculation by AlF4 or ADP-ribosylation factor; or ADP-ribosylation factor and beta-coat protein binding to Golgi membranes. Thus the ADP-ribosylation inhibitor dicumarol induces the selective breakdown of the tubular components of the Golgi complex and inhibition of intra-Golgi transport. This suggests that lateral diffusion between adjacent stacks has a role in protein transport through the Golgi complex.     

The hemicholinium-3 sensitive high affinity choline transporter is internalized by clathrin-mediated endocytosis and is present in endosomes and synaptic vesicles

Synthesis of acetylcholine depends on the plasma membrane uptake of choline by a high affinity choline transporter (CHT1). Choline uptake is regulated by nerve impulses and trafficking of an intracellular pool of CHT1 to the plasma membrane may be important for this regulation. We have generated a hemagglutinin (HA) epitope tagged CHT1 to investigate the organelles involved with intracellular trafficking of this protein. Expression of CHT1-HA in HEK 293 cells establishes Na+-dependent, hemicholinium-3 sensitive high-affinity choline transport activity. Confocal microscopy reveals that CHT1-HA is found predominantly in intracellular organelles in three different cell lines. Importantly, CHT1-HA seems to be continuously cycling between the plasma membrane and endocytic organelles via a constitutive clathrin-mediated endocytic pathway. In a neuronal cell line, CHT1-HA colocalizes with the early endocytic marker green fluorescent protein (GFP)-Rab 5 and with two markers of synaptic-like vesicles, VAMP-myc and GFP-VAChT, suggesting that in cultured cells CHT1 is present mainly in organelles of endocytic origin. Subcellular fractionation and immunoisolation of organelles from rat brain indicate that CHT1 is present in synaptic vesicles. We propose that intracellular CHT1 can be recruited during stimulation to increase choline uptake in nerve terminals.     

AtPEX2 and AtPEX10 are targeted to peroxisomes independently of known endoplasmic reticulum trafficking routes

Controversy exists in the literature over the involvement of the endoplasmic reticulum (ER) in the delivery of membrane proteins to peroxisomes. In this study, the involvement of the ER in the trafficking of two Arabidopsis (Arabidopsis thaliana) peroxisomal membrane proteins was investigated using confocal laser scanning microscopy of living cells expressing fusions between enhanced yellow fluorescent protein (eYFP) and AtPEX2 and AtPEX10. The fusion proteins were always detected in peroxisomes and cytosol irrespective of the location of the eYFP tag or the level of expression. The cytosolic fluorescence was not due to cleavage of the eYFP reporter from the C-terminal fusion proteins. Blocking known ER transport routes using the fungal metabolite Brefeldin A or expressing dominant negative mutants of Sar1 or RabD2a had no effect on the trafficking of AtPEX2 and AtPEX10 to peroxisomes. We conclude that AtPEX2 and AtPEX10 are inserted into peroxisome membranes directly from the cytosol.     

Localization and function of soluble N-ethylmaleimide-sensitive factor attachment protein-25 and vesicle-associated membrane protein-2 in functioning gastric parietal cells

The soluble N-ethylmaleimide-sensitive factor attachment protein of 25 kDa (SNAP-25) plays an important role in vesicle trafficking. Together with vesicle-associated membrane protein-2 (VAMP-2) and syntaxin, SNAP-25 forms a ternary complex implicated in docking and fusion of secretory vesicles with the plasma membrane during exocytosis. These so-called SNARE proteins are believed to regulate tubulovesicle trafficking and fusion during the secretory cycle of the gastric parietal cell. Here we examined the cellular localization and functional importance of SNAP-25 in parietal cell cultures. Adenoviral constructs were used to express SNAP-25 tagged with cyan fluorescent protein, VAMP-2 tagged with yellow fluorescent protein, and SNAP-25 in which the C-terminal 25 amino acids were deleted (SNAP-25 Delta181-206). Membrane fractionation experiments and fluorescent imaging showed that SNAP-25 is localized to the apical plasma membrane. The expression of the mutant SNAP-25 Delta181-226 inhibited the acid secretory response of parietal cells. Also, SNAP Delta181-226 bound poorly in vitro with recombinant syntaxin-1 compared with wild type SNAP-25, indicating that pairing between syntaxin-1 and SNAP-25 is required for parietal cell activation. Dual expression of SNAP-25 tagged with cyan fluorescent protein and VAMP-2 tagged with yellow fluorescent protein revealed a dynamic change in distribution associated with acid secretion. In resting cells, SNAP-25 is at the apical plasma membrane and VAMP-2 is associated with cytoplasmic H,K-ATPase-rich tubulovesicles. After stimulation, the two proteins co-localize on the apical plasma membrane. These data demonstrate the functional significance of SNAP-25 as a SNARE protein in the parietal cell and show the dynamic stimulation-associated redistribution of VAMP-2 from H,K-ATPase-rich tubulovesicles to co-localize with SNAP-25 on the apical plasma membrane.     

MARK/PAR1 kinase is a regulator of microtubule-dependent transport in axons

Microtubule-dependent transport of vesicles and organelles appears saltatory because particles switch between periods of rest, random Brownian motion, and active transport. The transport can be regulated through motor proteins, cargo adaptors, or microtubule tracks. We report here a mechanism whereby microtubule associated proteins (MAPs) represent obstacles to motors which can be regulated by microtubule affinity regulating kinase (MARK)/Par-1, a family of kinases that is known for its involvement in establishing cell polarity and in phosphorylating tau protein during Alzheimer neurodegeneration. Expression of MARK causes the phosphorylation of MAPs at their KXGS motifs, thereby detaching MAPs from the microtubules and thus facilitating the transport of particles. This occurs without impairing the intrinsic activity of motors because the velocity during active movement remains unchanged. In primary retinal ganglion cells, transfection with tau leads to the inhibition of axonal transport of mitochondria, APP vesicles, and other cell components which leads to starvation of axons and vulnerability against stress. This transport inhibition can be rescued by phosphorylating tau with MARK.     

Increased Expression of β-Amyloid Protein Precursor and Microtubule-Associated Protein τ During the Differentiation of Murine Embryonal Carcinoma Cells

Expression of the genes encoding the beta/A4 amyloid protein precursor (APP) and microtubule-associated protein tau was studied in an embryonal carcinoma cell line (P19) that differentiates in vitro into cholinergic neurons after treatment with retinoic acid. Expression of APP increased 34- (mRNA) and 50-fold (protein) during neuronal differentiation; APP-695 accounted for most of this increase. These remarkable increases in APP expression coincided with a proliferation of neuronal processes and with an increase in content of tau mRNA. Moreover, subsequent decreases in the levels of APP and tau mRNA coincided with the onset of the degeneration of the neuronal processes. Immunocytochemical staining suggested that greater than 85% of the P19-derived neurons are cholinergic and that APP is present in the neuronal processes and cell bodies. These results suggest that APP may play an important role in construction of neuronal networks and neuronal differentiation and also indicate that this embryonal carcinoma cell line provides an ideal model system to investigate biological functions of APP and the roles of APP and tau protein in development of Alzheimer's disease in cholinergic neurons.     

Uncoupled packaging of amyloid precursor protein and presenilin 1 into coat protein complex II vesicles

Mutant forms of presenilin (PS) 1 and 2 and amyloid precursor protein (APP) lead to familial Alzheimer's disease. Several reports indicate that PS may modulate APP export from the endoplasmic reticulum (ER). To develop a test of this possibility, we reconstituted the capture of APP and PS1 in COPII (coat protein complex II) vesicles formed from ER membranes in permeabilized cultured cells. The recombinant forms of mammalian COPII proteins were active in a reaction that measures coat subunit assembly and coated vesicle budding on chemically defined synthetic liposomes. However, the recombinant COPII proteins were not active in cargo capture and vesicle budding from microsomal membranes. In contrast, rat liver cytosol was active in stimulating the sorting and packaging of APP, PS1, and p58 (an itinerant ER to Golgi marker protein) into transport vesicles from donor ER membranes. Budding was stimulated in dilute cytosol by the addition of recombinant COPII proteins. Fractionation of the cytosol suggested one or more additional proteins other than the COPII subunits may be essential for cargo selection or vesicle formation from the mammalian ER membrane. The recombinant Sec24C specifically recognized the APP C-terminal region for packaging. Titration of Sarla distinguished the packaging requirements of APP and PS1. Furthermore, APP packaging was not affected by deletion of PS1 or PS1 and 2, suggesting APP and PS1 trafficking from the ER are normally uncoupled.     

The spatial organization of lipid synthesis in the yeast Saccharomyces cerevisiae derived from large scale green fluorescent protein tagging and high resolution microscopy

The localization pattern of proteins involved in lipid metabolism in the yeast Saccharomyces cerevisiae was determined using C-terminal green fluorescent protein tagging and high resolution confocal laser scanning microscopy. A list of 493 candidate proteins ( approximately 9% of the yeast proteome) was assembled based on proteins of known function in lipid metabolism, their interacting proteins, proteins defined by genetic interactions, and regulatory factors acting on selected genes or proteins. Overall 400 (81%) transformants yielded a positive green fluorescent protein signal, and of these, 248 (62% of the 400) displayed a localization pattern that was not cytosolic. Observations for many proteins with known localization patterns were consistent with published data derived from cell fractionation or large scale localization approaches. However, in many cases, high resolution microscopy provided additional information that indicated that proteins distributed to multiple subcellular locations. The majority of tagged enzymes localized to the endoplasmic reticulum (91), but others localized to mitochondria (27), peroxisomes (17), lipid droplets (23), and vesicles (53). We assembled enzyme localization patterns for phospholipid, sterol, and sphingolipid biosynthetic pathways and propose a model, based on enzyme localization, for concerted regulation of sterol and sphingolipid metabolism that involves shuttling of key enzymes between endoplasmic reticulum, lipid droplets, vesicles, and Golgi.     

Transport of microinjected alcohol oxidase from Pichia pastoris into vesicles in mammalian cells: involvement of the peroxisomal targeting signal

This report describes the microinjection of a purified peroxisomal protein, alcohol oxidase, from Pichia pastoris into mammalian tissue culture cells and the subsequent transport of this protein into vesicular structures. Transport was into membrane-enclosed vesicles as judged by digitonin-permeabilization experiments. The transport was time and temperature dependent. Vesicles containing alcohol oxidase could be detected as long as 6 d after injection. Coinjection of synthetic peptides containing a consensus carboxyterminal tripeptide peroxisomal targeting signal resulted in abolition of alcohol oxidase transport into vesicles in all cell lines examined. Double-label experiments indicated that, although some of the alcohol oxidase was transported into vesicles that contained other peroxisomal proteins, the bulk of the alcohol oxidase did not appear to be transported to preexisting peroxisomes. While the inhibition of transport of alcohol oxidase by peptides containing the peroxisomal targeting signal suggests a competition for some limiting component of the machinery involved in the sorting of proteins into peroxisomes, the organelles into which the majority of the protein is targeted appear to be unusual and distinct from endogenous peroxisomes by several criteria. Microinjected alcohol oxidase was transported into vesicles in normal fibroblasts and also in cell lines derived from patients with Zellweger syndrome, which are unable to transport proteins containing the ser-lys-leu-COOH peroxisomal targeting signal into peroxisomes (Walton et al., 1992). The implications of this result for the mechanism of peroxisomal protein transport are discussed.     

Visualization of APP dimerization and APP-Notch2 heterodimerization in living cells using bimolecular fluorescence complementation

We previously demonstrated that the amyloid precursor protein (APP) interacts with Notch receptors. Here, we confirmed the APP/Notch1 endogenous interaction in embryonic day 17 rat brain tissue, suggesting the interaction was not as a result of over-expression artifacts. To investigate potential homodimeric and heterodimeric interactions of APP and Notch2 (N2), we have visualized the subcellular localization of the APP/N2 complexes formed in living cells using bimolecular fluorescence complementation (BiFC) analysis. BiFC was accomplished by fusing the N-terminal fragment or the C-terminal fragment of yellow fluorescent protein (YFP) to APP, N2, and a C-terminally truncated form of N2. When expressed in COS-7 cells, these tagged proteins alone did not produce a fluorescent signal. The tagged APP homodimer produced a weak fluorescent signal, while neither full-length N2, nor a truncated N2 alone, produced a visible signal, suggesting that N2 receptors do not form homodimers. The strongest fluorescent signal was obtained with co-expression of the C-terminal fragment of YFP fused to APP and the N-terminal fragment of YFP fused to the truncated form of N2. This heterodimer localized to plasma membrane, endoplasmic reticulum (ER), Golgi and other compartments. The results were confirmed and quantified by flow cytometry. The BiFC method of specifically visualizing APP/Notch interactions can be applied to study APP and Notch signaling during development, aging and neurodegeneration.     

The role of selective transport in neuronal protein sorting

To assess whether selective microtubule-based vesicle transport underlies the polarized distribution of neuronal proteins, we expressed green fluorescent protein- (GFP-) tagged chimeras of representative axonal and dendritic membrane proteins in cultured hippocampal neurons and visualized the transport of carrier vesicles containing these proteins in living cells. Vesicles containing a dendritic protein, transferrin receptor (TfR), were preferentially transported into dendrites and excluded from axons. In contrast, vesicles containing the axonal protein NgCAM (neuron-glia cell adhesion molecule) were transported into both dendrites and axons. These data demonstrate that neurons utilize two distinct mechanisms for the targeting of polarized membrane proteins, one (for dendritic proteins) based on selective transport, the other (for axonal proteins) based on a selectivity "filter" that occurs downstream of transport.