Role of the Amyloid Precursor Protein in the cellular phosphoinositide metabolism through its interaction with the PIKfyve complex

While the role of the Amyloid Precursor Protein APP in Alzheimer's disease is well established, its normal role in organisms is ill defined. As most proteins that fulfill complex functions do so in concert with other proteins that they physically bind (so-called interaction partners), knowing binding partners can give valuable insights into protein function. We have established a large number of binding partners of APP, among them a protein complex consisting of the subunits PIKfyve, Vac14 and Fig4. These three proteins are well known to regulate the levels of an important signalling lipid, phosphatidylinositol-3,5-bisphosphate (PI3,5P2). Interestingly, loss of Vac14 or Fig4 lead to profound neurodegeneration in mice as well as humans. The fact that both APP and the PIKfyve complex are implicated in neurodegeneration and the fact that they bind each other is novel and surprising. This suggests that both participate in the same cellular process. We suggest that APP is a regulator of PI3,5P2 via its binding of the PIKfyve complex. We now need to test whether this hypothesis is correct.

We will test this idea using the genetic model organism C. elegans. In this simple organism there are genes that are highly similar to human APP, PIKfyve, Vac14 and Fig4. The powerful advantage of using C. elegans is the availability of numerous mutants. We have obtained mutants in which APP, PIKfyve and Vac14 are defective. We will characterise the consequences of these defects in animals in terms of appearance of the animals, their behavior and the integrity of their neuronal system. In the next step, through crossing the APP mutants with PIKfyve or Vac14 animals we can combine the mutations in one animal. This then allows to compare the consequences of the double mutants with the single mutants and allows to deduce whether genes act in concert to fulfill a biological function (termed a genetic interaction). We predict that the double mutants will have a strongly enhanced phenotype compared to the single mutants. This type of phenotypic enhancement is only observed when several genes are functionally connected. This type of approach provides invaluable information that cannot be obtained by any other approach.

We will also analyse the levels of the signalling lipid PI3,5P2 that appears to be of key importance for the integrity of the central nervous system. Correlating the levels of this with the characteristics of the animals (appearance, behavior etc.) will allow us to understand what role APP and the PIKfyve complex play in its regulation.

Our work suggests that the transmembrane receptor (APP) is able to module the production of PI3,5P2 by a direct interaction with the PIKfyve complex. Loss of function of the PIKfyve complex is known to lead to neurodegeneration in mammals. Our work for the very first time links APP with PI3,5P2 regulation and thus may provide an entirely novel and unexpected mechanism how loss or aberrant processing of APP can instigate neurodegeneration.

The Amyloid Precursor Protein APP is a type-I transmembrane protein with still largely unknown function. We established the interactome of its intracellular domain and identified Vac14 of the PIKfyve complex as a direct interactor. The PIKfyve complex regulates the signalling phosphoinositide PI3,5P2. We will analyse the function of the interaction between Vac14 of the PIKfyve complex and APP using the genetic model organism C. elegans. The advantage of this system is that there is only one homologue of APP in C. elegans in contrast to the three mammalian APP and APP-like genes, eliminating the problem of functional redundancy present in mammals. We will use control animals, single mutations in APP, PIKfyve, Vac14 and double mutants in APP/PIKfyve and APP/Vac14. Furthermore we will utilise APP-GFP as a rescue strain. We will perform the following phenotypic analysis on these strains:
1.) Morphology and behaviour of the worms.
2.) Integrity and functionality of the endosomal system in different tissues (hypodermal cells, intestine, coelomocytes, body wall muscle, neurons), as PIKfyve has been shown to play a crucial role in endosomal homeostasis. For this we will use established endosomal markers (Rab5, Rab7, Rab11, LampI). Furthermore we will analyse receptor sorting (ced-1) by the endosomal system and autophagy (lgg-1).
3.) Integrity and functionality of the neuronal system using fluorescent markers to lable synapses (pre- and post-synaptic) and behavioural assays for analysing motor function. Both APP and the PIKfyve complex are known to play key roles in the central nervous system.
4.) Measure the content of the endosomal lipids PI3P, PI3,5P2 and PI5P and correlate them with the phenotypic analysis.
These data will allow us to test whether the genes for APP, Vac14 and PIKfyve interact on a genetic level as suggested by their biochemical interaction. This will allow us to test whether APP is a novel regulator of the phospho-inositide metabolism.

Alzheimer's disease has for a long time been viewed as a disease caused by the accumulation of the beta amyloid peptide, leading to the deposition in plaques, thus forming sites of inflammation and necrosis in patients' brains. At the core of this hypothesis is a toxic gain-of-function mechanism of the unphysiological accumulation of beta amyloid. However, all strategies targeting beta amyloid production or deposition have failed to produce positive effects on mental health and disease progression, raising fundamental doubts about whether we really understand the disease mechanism of Alzheimer's. Therefore it is urgent to explore alternative models for the disease. The main hindrance for this is our limited understanding of the physiological function of the Amyloid Precursor Protein APP. With the establishment of the interactome of the intracellular domain of APP we have made a major step towards understanding the multiple roles that APP plays in cells.

The next step is to identify the functional significance of the interaction partners identified. In this proposal we will dissect the functional connection between APP and the PIKfyve complex as suggested by their biochemical interaction and the pilot data we have obtained using C. elegans as a model system (our unpublished data). Our data so far allow us to propose an entirely novel mechanism for neurodegeneration for Alzheimer's disease, namely an impaired function of the PIKfyve complex caused by aberrant APP processing and a change in PI3,5P2 metabolism. The data obtained with this proposal could indeed change the way we think about the disease with extremely wide ranging consequences.

First and foremost the impact of our work will be realised by publicising our results and conclusions to a large scientific audience in the form of original research articles, a review and presentation at congresses (as outlined in Academic Beneficiaries). However, the interaction between APP and Vac14 of the PIKfyve complex may also constitute a suitable drug target. Prior to publication we will explore with the help of the Aston Business Partnership Unit whether the knowledge of this interaction and its functional significance on PI3,5P2 production can be patented for drug screening. This could reap huge rewards in economic terms given the funds that are currently spent by pharmaceutical companies on drug screening for treating Alzheimer's disease.

The findings of this project will also be of interest to the general public due to the importance of APP in Alzheimer's disease. For public engagement we have in collaboration with Alzheimer Research UK successfully publicised the pilot project "Interaction between the Alzheimer pre-protein and the mTOR signalling complex: Implications for Alzheimer's disease." that was awarded to Dr. Wassmer's lab previously by Alzheimer Research UK with a press release followed by broadcasting on a local radio show and announcement on Twitter. The interest in the project led also to a lab visit of members of the public (organised by Alzheimer Research UK) that want to learn more about the project in April 2012.
We will also publicise this project and findings thereof using the Aston University press office and Twitter. This will raise public awareness of the focus and scope of publicly funded research in the UK.

The technical and intellectual capabilities that will be acquired by the postdoctoral researcher working on this project will enable him/her to pursue a career in academic science or industry and thus will benefit other labs/companies with experience and input, thus benefitting the UK research base and economy.