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

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.

In sorting an array of cargoes, for example proteins, lipids, nutrients, and solutes, to a variety of cellular destinations, the endosomal network performs an essential function in regulating and fine-tuning numerous processes within the cell. Retromer complexes are evolutionarily conserved orchestrators of endosomal sorting. The canonical SNX-BAR-retromer is composed of a stable 'cargo-selective' trimer composed of VPS26, VPS29 and VPS35, and a 'membrane-deforming sub-complex' composed of a heterodimer of the sorting nexin (SNX)-Bin/Amphiphysin/Rvs (BAR) domain-containing proteins (SNX-BARs) SNX1 or SNX2 with SNX5 or SNX6. The non-canonical SNX3-retromer shares the VPS26:VPS29:VPS35 trimer but contains a distinct membrane-bound sub-complex composed of the non-BAR domain-containing SNX3. These endosomal sorting complexes regulate parallel endosome-to-TGN retrieval pathways and, in the case of the SNX-BAR-retromer, endosome-to-plasma membrane recycling.

Recently a missense D620N mutation in VPS35 has been linked with late-onset, autosomal dominant Parkinson's Disease (PD). Although VPS35(D620N) cases show an earlier onset than commonly observed, they are otherwise similar to classic sporadic PD suggestive of considerable overlap in disease pathways. Elucidating not only the VPS35(D620N) mutation but more broadly the role of retromer in neuronal endosomal sorting, may ultimately lead to new insight into the causes of PD. Based on extensive preliminary data we shall test the following hypothesis: The underlying defect for patients with VPS35(D620N)-linked PD is an uncoupling of retromer-mediated cargo sorting from WASH-dependent actin polymerization, which is manifested as a decrease in the efficiency of processing of tubular profiles into isolated transport carriers, and a resultant defect in retromer-mediated endosomal sorting of neuronal cargo proteins, that include specific glucose transporters that normally function to suppress PD neuropathology.

Who will benefit from this research?
Given the fundamental nature of the proposed research, the principal beneficiaries will be national and international researchers with interests ranging from retromer complexes, phosphoinositides, sorting nexins, and endosomal sorting complexes through to those studying the assembly of multi-protein complexes, membrane re-modelling, regulation of actin dynamics, and the molecular basis of membrane trafficking. By extending research into neuronal-like cultures and the molecular basis of Parkinson's Disease, data will influence neuroscientists studying endosomal sorting in neurones, and interest the wider community of basic and applied scientists currently trying to elucidate the complex cellular basis of Parkinson's Disease. The later will include members of the commercial private sector who are tackling the growing issue of Parkinson's Disease within the ageing population.

Further afield, in choosing to take an unbiased global view of retromer-dependent endosomal sorting, data from the proposal has the potential to influence a much wider sphere of scientists, far beyond those described above. For example, Table 2 (see proposal) shows that besides affecting glucose uptake through altered sorting of GLUT-1, VPS35 suppression leads to reduced cell surface expression of transports involved in zinc and copper homeostasis (including ATP7A, mutations in which cause Menkes syndrome), cell signaling (e.g. the PDGF receptor), cell adhesion and various proteins of unknown functions. One anticipates that a parallel analysis in neuronal cells will generate a similar broad data set, with far reaching significance.

Understanding the etiology of Parkinson's Disease forms an important research goal in order to address the quality of life and wellbeing of an ageing population. With the investment that has been made in genetic epidemiology and the resultant data that has been generated, fundamental medical research that seeks to translate these findings into a greater understanding of human disease will interest and benefit the general population as well as government and non-government policy-makers. Our proposed analysis of the VPS35(D620N) missense mutation in Parkinson's Disease is one such example.

How will they benefit from this research?
Parkinson's Disease is the most common neurodegenerative movement disorder, affecting about 6 million people worldwide with a slow progression of the symptoms. Its prevalence is expected to double in the most populated areas within the next two decades, according to increasing aged population. Consequently, Parkinson's Disease is a socio-economic issue and a major challenge for the public health system. Understanding the etiology of this disease forms an important research goal given the ageing population. By generating a quantified global 'map' of the alteration in endosomal sorting observed in VPS35 suppressed and VPS35(D620N) expressing neuronal cells, the current proposal seeks to formulate testable hypotheses on how the altered map may ultimately influence age-related neurodegeneration in Parkinson's Disease. Longer term, it is hoped that by validating or refuting individual hypotheses, research will enhance our understanding of Parkinson's Disease and influence how this disease may be prevented and/or treated.