Investigating novel interaction partners of amyloid precursor protein: the mechanistic target of rapamycin and pikyve complex

Previous Work Although the amyloid precursor protein (APP) is known to have a central role in Alzheimer's disease, its cellular function is poorly characterised. To better understand the cellular functions of APP, an interactome of APP’s intracellular domain (AICD) was generated using a proteo-lipososome based assay, which enabled interactions to be identified within a membrane context. In addition to proteins known to bind AICD, novel interactors were identified, including the mechanistic target of rapamycin complex 1 (mTORC1) and the phosphoinositide kinase PIKfyve complex. Binding of AICD to the two complexes was confirmed by Western blotting of treated AICDproteoliposomes and pulldowns of purified protein by AICD. Project Aims This project aimed to investigate the biological relevance of the APP/mTOR and APP/PIKfyve complex interactions. Results Investigation of the APP/mTOR interaction showed mTOR signalling increased in mammalian cells overexpressing APP/AICD, while loss of function studies determined C. elegans APP (APL-1) is involved in mTOR ortholog function. The APP/PIKfyve interaction was investigated with APP family knockdown, and TAT-AICD: a new molecular tool to allow acute AICD overexposure within the cell. Knockdown decreased PIKfyve function, while TAT-AICD exposure increased PIKfyve function in mammalian tissue culture. mTOR and PIKfyve are important to degradative pathway progression, and results suggested APP modulates the activity of these proteins. Protein degradation is important in human disease, including Alzheimer's disease. Experiments elaborating APP relevance in the lysosome demonstrated that APP degradation is dependent on sorting, endosomal acidification and the inhibition of mTOR. Further experiments linked PIKfyve inhibition to these degradative processes, in particular, to lower organelle acidification and altered late endosome morphology. Summary These results suggest an interdependence between APP, mTOR and PIKfyve, where APP appears to impact lysosomal function, while also being dependent upon it for down-regulation

A cell-permeable tool for analysing APP intracellular domain function and manipulation of PIKfyve activity

The mechanisms for regulating PIKfyve complex activity are currently emerging. The PIKfyve complex, consisting of the phosphoinositide kinase PIKfyve (also known as FAB1), VAC14 and FIG4, is required for the production of phosphatidylinositol-3,5-bisphosphate (PI(3,5)P2). PIKfyve function is required for homeostasis of the endo/lysosomal system and is crucially implicated in neuronal function and integrity, as loss of function mutations in the PIKfyve complex lead to neurodegeneration in mouse models and human patients. Our recent work has shown that the intracellular domain of the Amyloid Precursor Protein (APP), a molecule central to the aetiology of Alzheimer's disease binds to VAC14 and enhances PIKfyve function. Here we utilise this recent advance to create an easy-to-use tool for increasing PIKfyve activity in cells. We fused APP's intracellular domain (AICD) to the HIV TAT domain, a cell permeable peptide allowing proteins to penetrate cells. The resultant TAT-AICD fusion protein is cell permeable and triggers an increase of PI(3,5)P2. Using the PI(3,5)P2 specific GFP-ML1Nx2 probe we show that cell-permeable AICD alters PI(3,5)P2 dynamics. TAT-AICD also provides partial protection from pharmacological inhibition of PIKfyve. All three lines of evidence show that the APP intracellular domain activates the PIKfyve complex in cells, a finding that is important for our understanding of the mechanism of neurodegeneration in Alzheimer's disease

The amyloid precursor protein controls PIKfyve function

While the Amyloid Precursor Protein (APP) plays a central role in Alzheimer's disease, its cellular function still remains largely unclear. It was our goal to establish APP function which will provide insights into APP's implication in Alzheimer's disease. Using our recently developed proteo-liposome assay we established the interactome of APP's intracellular domain (known as AICD), thereby identifying novel APP interactors that provide mechanistic insights into APP function. By combining biochemical, cell biological and genetic approaches we validated the functional significance of one of these novel interactors. Here we show that APP binds the PIKfyve complex, an essential kinase for the synthesis of the endosomal phosphoinositide phosphatidylinositol-3,5-bisphosphate. This signalling lipid plays a crucial role in endosomal homeostasis and receptor sorting. Loss of PIKfyve function by mutation causes profound neurodegeneration in mammals. Using C. elegans genetics we demonstrate that APP functionally cooperates with PIKfyve in vivo. This regulation is required for maintaining endosomal and neuronal function. Our findings establish an unexpected role for APP in the regulation of endosomal phosphoinositide metabolism with dramatic consequences for endosomal biology and important implications for our understanding of Alzheimer's disease

APP controls the formation of PI(3,5)P2 vesicles through its binding of the PIKfyve complex

Phosphoinositides are signalling lipids that are crucial for major signalling events as well as established regulators of membrane trafficking. Control of endosomal sorting and endosomal homeostasis requires phosphatidylinositol-3-phosphate (PI(3)P) and phosphatidylinositol-3,5-bisphosphate (PI(3,5)P2), the latter a lipid of low abundance but significant physiological relevance. PI(3,5)P2 is formed by phosphorylation of PI(3)P by the PIKfyve complex which is crucial for maintaining endosomal homeostasis. Interestingly, loss of PIKfyve function results in dramatic neurodegeneration. Despite the significance of PIKfyve, its regulation is still poorly understood. Here we show that the Amyloid Precursor Protein (APP), a central molecule in Alzheimer’s disease, associates with the PIKfyve complex (consisting of Vac14, PIKfyve and Fig4) and that the APP intracellular domain directly binds purified Vac14. We also show that the closely related APP paralogues, APLP1 and 2 associate with the PIKfyve complex. Whether APP family proteins can additionally form direct protein–protein interaction with PIKfyve or Fig4 remains to be explored. We show that APP binding to the PIKfyve complex drives formation of PI(3,5)P2 positive vesicles and that APP gene family members are required for supporting PIKfyve function. Interestingly, the PIKfyve complex is required for APP trafficking, suggesting a feedback loop in which APP, by binding to and stimulating PI(3,5)P2 vesicle formation may control its own trafficking. These data suggest that altered APP processing, as observed in Alzheimer’s disease, may disrupt PI(3,5)P2 metabolism, endosomal sorting and homeostasis with important implications for our understanding of the mechanism of neurodegeneration in Alzheimer’s disease

APL-1 overexpression, while able to rescue partial loss of PPK-3 function, failed to rescue the ppk-3(mc46) null allele.

<p>(A) Expression of APL-1::GFP failed to rescue lethality of ppk-3 null animals and failed to rescue vacuolation in apl-1::GFP; ppk-3(mc46) animals. Bar, 50μm. (B) Quantification of the relative vacuolated area in ppk-3(mc46) and apl-1::GFP; ppk-3(mc46) animals showed that APL-1 overexpression failed to rescue complete loss of PPK-3 function (n≥38, p = 0.73 (two-tailed t-test), suggesting that APL-1 functions upstream of PPK-3.</p

Overexpression of APL-1 reduces vacuolar pathology caused by hypomorphic PIKfyve complex mutants.

<p>(A, B) Overexpression of APL-1::GFP (established in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130485#pone.0130485.ref032" target="_blank">32</a>]) significantly reduced the number of vacuoles in PPK-3 partial loss-of-function mutant animals (Wilcoxon rank test, p<0.01, n≥25 per strain). Note that in the case of the stronger hypomorphic ppk-3(n2668) mutant APL-1::GFP expression, while strongly ameliorating the phenotype, failed to fully rescue the vacuolar phenotype.</p

APL-1 interacts genetically with the PIKfyve complex genes vacl-14 and ppk-3.

<p>(A) Mutations in apl-1, ppk-3 and vacl-14 led to the formation of vacuoles in the C. elegans intestine and hypoderm (indicated by arrows) apparent in the anterior tips of the worms as observed by Differential Interference Contrast (DIC) microscopy. Combination of apl-1, ppk-3 and vacl-14 mutations in double mutant worms strongly enhanced this phenotype (* indicate very large vacuoles), particularly evident in the apl-1(yn5) ppk-3(n2668) double mutant. (B) Box plots demonstrated that the number of vacuoles in apl-1/ppk-3 and apl-1/vacl-14 double mutants is significantly increased compared to the single mutants (Wilcoxon rank test, p<0.01, n≥20 per strain), demonstrating that APL-1 functionally interacts with the PPK-3 complex. This showed that the C-terminal domain of APL-1 is necessary for suppressing vacuole formation induced by loss of PPK-3 and VACL-14 activity. Bar, 50 μm.</p

Mutations in apl-1 and ppk-3 or vacl-14 lead to defective processing of late endosomes and lysosomes in young adults.

<p>(A) In control animals and single apl-1(yn5), vacl-14(ok1877) and ppk-3(n2835) mutants late endosomes stained using GFP::RAB-7 as a marker were visible as vesicles or small vacuoles. Combination of the mutations in apl-1(yn5); vacl-14(ok1877) or apl-1(yn-5) ppk-3(n2835) double mutants led to a drastic alteration of the morphology of the late endosomal compartment, e.g. late endosomes became tubular and clustered in the hypoderm and intestine when comparing double with single mutants. While in single mutants individual compartments were abundant, they appeared 'squashed' and tubular in the double mutants (indicated by arrows). (B) Analysis of the lysosomal marker GFP::LMP-1 showed that in single apl-1(yn5), vacl-14(ok1877) and ppk-3(n2835) mutants distinct, LMP-1 positive vacuoles of variable sizes were apparent. However, in apl-1(yn5); vacl-14(ok1877) and apl-1(yn5) ppk-3(n2835) double mutants the LMP-1 positive structures appeared aggregated, mimicking the defect observed in GFP::RAB-7 worms, suggesting a defect in late endosomal and lysosomal trafficking. These data showed that APL-1 and the PPK-3 complex are required for late endosomal processing. Bar, 100μm.</p

Working model for the interplay of APP with the PIKfyve complex to control endosomal sorting and homeostasis.

<p>APP is well known to traffic between the trans-Golgi-network (TGN), the plasma membrane (PM) and the endosomal system consisting of early endosomes (EE), late endosomes (LE) and lysosomes ([<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130485#pone.0130485.ref010" target="_blank">10</a>] and references therein). The PIKfyve complex phosphorylates PI(3)P to PI(3,5)P2 (indicated by red labelling of the membrane) which is crucial for endosome-to-TGN transport, endosomal homeostasis [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130485#pone.0130485.ref015" target="_blank">15</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130485#pone.0130485.ref018" target="_blank">18</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130485#pone.0130485.ref025" target="_blank">25</a>] and other endosomal sorting processes during the maturation of endosomes [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130485#pone.0130485.ref047" target="_blank">47</a>]. In this study we have shown that APP can associate via its intracellular domain with Vac14 of the PIKfyve complex and that the C. elegans APP orthologue APL-1 functionally cooperates with the PIKfyve/PPK-3 complex in C. elegans. We propose a working model in which APP/APL-1 traffics through the vesicular transport system and, upon arrival in endosomes, interacts with the PIKfyve/PPK-3 complex via its intracellular domain. The genetic data obtained in C. elegans suggest that APL-1 stimulates PPK-3 activity which is necessary to maintain endosomal function. Compromised PIKfyve/PPK-3 function impacts on endosomal sorting and homeostasis which in C. elegans as well as in mammals has detrimental consequences for neuronal function and integrity [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130485#pone.0130485.ref017" target="_blank">17</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130485#pone.0130485.ref025" target="_blank">25</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130485#pone.0130485.ref026" target="_blank">26</a>].</p

Vac14 associates with the intracellular domain of APP (AICD) biochemically and in HeLa cells.

<p>(A) Proteo-liposome recruitment analysed by mass spectrometry allowed the identification of the intracellular interactome of APP. A Vulcan plot showing that PIKfyve, Vac14 and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130485#pone.0130485.g004" target="_blank">Fig 4</a> of the mammalian PIKfyve complex were significantly enriched in AICD proteo-liposomes (right quadrant) compared to controls (left). The dashed line indicates the 0.05 significance threshold. (B) Western blotting confirmed the enrichment of Vac14 by AICD presenting proteo-liposomes while the intracellular domain of the none-related receptor Sortilin and two additional controls (a non-related control peptide designated as 'control 1' or coupled cysteine 'control 2'–both described in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130485#pone.0130485.ref027" target="_blank">27</a>]) failed to bind Vac14, showing that Vac14 specifically associates with the intracellular domain of APP. (C) APP expressed as a C-terminal CFP fusion displayed strong colocalisation with Vac14-mCit in HeLa cells. Limited colocalisation of APP and Vac14 could be observed on early endosomes (labelled with EEA1) and late endosomes/lysosomes labelled with LampI, as indicated by arrows on the insets. Bar, 20 μm. (D) Line scans (indicated by the red line in (C) demonstrated strong colocalisation between APP and Vac14. (E) In live-cell imaging APP fused to mCherry displayed co-movement with Vac14-mCit positive vesicles and tubular carriers (arrows) that track through the cytoplasm. (F, G) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130485#pone.0130485.s007" target="_blank">S1 Video</a>). AICD fused to mCherry also colocalised with Vac14-mCit in live cell imaging (arrows) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130485#pone.0130485.s008" target="_blank">S2 Video</a>). (G) Four individual vesicles positive for both AICD and Vac14 were tracked and the first image of the sequence overlayed with the traces to illustrate comigration of AICD and Vac14. Note that in live cell imaging due to the delay caused by the change of filters on fast moving vesicles the staining in the red and green channels are slightly displaced. Bar, 5μm.</p