Analysing the cell biology of the Parkinson's Disease-linked missense mutation in the retromer VPS35 subunit.

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Neuronal and non-neuronal cells are composed of an outer boundary that is defined by a complex mixture of protein and lipids called the plasma membrane. This encircles a fluid filled 3-dimensional space, termed the cytosol, which contains additional membrane-encircled compartments each composed of a unique combination of proteins and lipids. For cells to function normally, proteins and lipids must be efficiently transported to the correct membrane-enriched compartment within this maze of membranes. Not surprisingly, if such transport is perturbed, so that the wrong proteins and lipids are delivered to the incorrect membrane-enriched compartment, cell function can be adversely affected which in turn leads to the development of various human diseases. Establishing the mechanisms through which cells achieve regulated protein and lipid transport is therefore a major challenge in cell biology with direct implication for our understanding of human disease. For the last 10 years our laboratory has focused on describing the mechanistic details that govern regulated transport of proteins and lipids within a specific aspect of the cell's membraneous maze termed the endocytic network. In particular, we have studied a family of proteins called the retromers. Our research, and that of others, is beginning to define retromer function and in so doing revealing its importance in a variety of cellular processes that are vital for normal cell function. Furthermore, it has become apparent that defects in retromer underlie a variety of human diseases including neurodegenerative diseases.One central component of retromer is a protein called VPS35. A specific mutation in the VPS35 gene, which encodes for a switch of the aspartic acid amino acid at position 620 for the amino acid asparagine, has been observed as an uncommon (estimated between 0.1 and 1.0%) cause of familial autosomal dominant Parkinson's Disease (this is termed the VPS35(D620N) mutation). With such a discovery, it becomes imperative that functional studies are now performed to determine the role of VPS35(D620N) in the pathogenesis of Parkinson's Disease. In the current proposal we seek to utilize our extensive experience of studying retromer to define the functional implication of the Parkinson's Disease-linked VPS35 mutation for retromer function, with the ultimate aim of revealing how this affects nerve cell function and death.Our proposed research will address the following questions:1). How does the VPS35(D620N) mutation affect the assemble of the multi-protein retromer complex?2). What effect(s) does the VPS35(D620N) mutation have on the ability of retromer to regulate the transport of specific proteins within the endocytic network?3). How does normal VPS35 and the VPS35(D620N) mutation affect protein transport and cellular viability within human dopaminergic-like neuronal cultures that are commonly used to study Parkinson's Disease? Overall, data derived from the proposed research, which will be disseminated through peer-review publications and oral presentation at international meetings, will define the functional implication of the Parkinson's Disease-linked VPS35 mutation for neuronal cell viability, knowledge which may lead to the identification of novel biomarkers and provide a rationale for therapeutic intervention in patients carrying the VPS35(D620N) mutation.