Machinery and Mechanisms of Endosomal Protein Sorting - A key Pathway Associated with Alzheimer Disease

Many of the proteins within your cells are in a constant state of flux moving from one location in the cell to another to perform their function. One of the most important sites within a cell that controls the movement of proteins is a structure called the 'endosome'. Many diseases, particularly ones associated with the nervous system, are caused when endosomes don't operate efficiently or correctly to control the movement of one or more particular proteins.

This is especially true for Alzheimer disease that is believed to occur when a protein called Amyloid Precursor Protein is not directed correctly from an endosome to another site called the Golgi. If Amyloid Precursor Protein stays too long in endosomes, it is often converted to a form called Abeta that accumulates overtime and causes Alzheimer disease. To understand precisely what causes Alzheimer disease so that better treatments can be found it is necessary to first study how endosomes work.

In this proposal, experiments designed to understand how endosomes work are described. The proposal is based on the idea that one pathway, the endosome-to-Golgi pathway is especially important in Alzheimer disease and that if the components of this pathway can be identified and studied to reveal their function, it will be possible to better understand Alzheimer disease. Experiments to find some of the components of the endosome-to-Golgi pathway have already be performed but now the question that must be answered is, what do these components do and how do they work in that pathway?

In this proposal, the cellular components that function at endosomes to control where in the cell Amyloid Precursor Protein is sent to will be studied so that new ideas and knowledge can be explored in the search for treatments for Alzheimer disease.

The goal of the proposed research is to identify and characterise genes encoding endosomal protein sorting machinery. The genes that function at endosomes will be tested for a role in regulating the localisation and processing of amyloid precursor protein (APP). Those genes are likely to be most relevant for Alzheimer disease (AD).

The endosome-to-Golgi retrieval pathway has been shown to play a key role in mediating the localisation and processing of APP. This occurs through the association of APP with the Vps10-domain containing protein SorL1. The SorL1 protein is a cargo protein for the retromer complex and requires retromer for its retrieval from endosomes to the Golgi. Loss of SorL1 or retromer function increases processing of APP to the neurotoxic Abeta peptide that is causal in AD.

A genome-wide siRNA screen for genes required for endosome-to-Golgi retrieval has been performed in the SEAMAN lab and has identified 88 genes required for endosome-to-Golgi retrieval including SFT2D2, ZDHHC5 and GRINA. The specific functions of the STF2D2, ZDHHC5 and GRINA proteins will be investigated and proteins that function with them identified by native immunoprecipitation and GST-pulldown experiments.

The requirement for the 88 endosome-to-Golgi retrieval genes in regulating APP localisation and processing will be determined and the role of these genes in regulating SorL1 localisation investigated in neuronal cells.

Genes identified through the siRNA screen as enhancing endosome-to-Golgi retrieval when silenced will be validated and their role in regulating APP processing determined.

Additionally a small molecule screen to identify modulators of endosome-to-Golgi retrieval, both positive and negative, will be performed using an antibody-uptake assay in a high throughput format. Those compounds that affect endosomal protein sorting will be characterised with respect to APP processing and effects on the endo-lysosomal system.

This proposal sets out to identify and characterise components of the endosomal protein sorting machinery that are relevant for Alzheimer disease (AD). As the mechanisms that govern endosomal protein sorting are important in a great many physiological processes, there are wide-ranging and far-reaching implications of the results of the proposed studies.

The identification and characteriation of genes and gene-products that effect endosomal protein sorting will provide new candidates for testing for the presence of single nucleotide polymorphisms that are linked to AD. Ultimately, identification of common variants that pre-dispose individuals to AD will have far reaching implications for society. Those individuals found to carry genes that increase risk may be identified through genetic testing and provided with counseling and guidance regarding lifestyle changes that may lessen their chance of developing AD in the future. It is conceivable that genetic testing of this kind will become common-place in the near future, but before that happens, the candidate genes have to be identified.

In addition to AD, there are several forms of inherited neuropathies (e.g. Charcot-Marie-Tooth disease and hereditary spastic paraplegia) that result from, or are linked to, defects in endosomal protein sorting. Currently however there is little prospect for intervention in these diseases and few candidates for further study aimed at developing therapies for neurodegerative disease. The identification of novel components of the endosomal protein sorting machinery will greatly add to the understanding of these pathways and provide many more potential targets for future therapies. Pharmaceutical companies interested in developing therapies for neurodegenerative disorders will be able to use the outputs from the studies proposed herein to identify new targets for therapies.

Additionally, as the endosomal system is often subverted by pathogens (both bacteria and viruses), a deeper knowledge of how endosomal protein sorting works will aid in the development of treatments aimed at preventing the pathogenic effects of certain bacteria and viruses that employ the endosomal protein sorting pathway to evade detection or exert their effects. Pharmaceutical companies may be reluctant to invest in research directed at treating the effects of some pathogens that modulate endosomal protein sorting due to the complexity of the endosomal pathway, but adding to the knowledge of how these pathways operate may provoke a greater interest in identifying the ways and means to intervene in endosomal protein sorting to treat certain infections.

A significant element of this proposal involves screening a small molecule library in order to identify compounds that can modulate endosomal protein sorting. The small molecule library to be screened comprises ~800 compounds that are FDA(Food and Drug Administration)-approved and in some cases already prescribed to treat certain conditions. Therefore, if a compound that can modulate endosomal protein sorting to affect APP localisation and processing is identified, the pathway to further trials of the compound should be accelerated. The identification of a small molecule that can protect APP from processing to Abeta by modulating the activity of the endosomal protein sorting machinery will be of profound interest to both academic researchers and also pharmaceutical companies. Additionally, if any of the small molecules are shown to modulate endosomal protein sorting resulting in an increase in processing of APP to Abeta then that information could have far-reaching implications for the long-term use of some prescription medicines.