Defining the role of retromer-like in endolysosomal cargo sorting in health and disease.

All human 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 defined 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 organelle within this maze of membranes - organelle being another term for describing a 'membrane defined compartment' that performs a particular cellular function(s). Not surprisingly, if such transport is perturbed, so that the wrong proteins and lipids are delivered to the incorrect organelle, the function of that organelle can be adversely affected leading to de-regulated cell, tissue and organism level physiology. In turn this leads to the development of various 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 over ten years our laboratory has focused on describing the mechanistic details that control regulated transport of proteins and lipids with a specific aspect of the cell's membraneous maze termed the endolysosomal network. In particular, we have studied an ancient, highly evolutionary conserved protein complex called retromer. Our research, and that of others, is defining retromer function and in so doing is 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 age-related neurodegenerative diseases such as Parkinson disease and Alzheimer disease.

While the importance of retromer is increasingly recognised, a major unanswered question relates to the mechanism(s) of retromer-independent protein and lipid transport. Addressing this question would constitute a major advance for the field: (i), it is an essential step towards achieving a detailed mechanistic understanding of these processes; (ii), increased mechanistic understanding will provide further insight into de-regulated protein and lipid transport in human disease; and (iii), as evidence continues to define retromer de-regulation in neurodegenerative disease, understanding the integration of retromer-dependent and retromer-independent pathways is likely to provide rationale routes for therapeutic strategies that exploit the flexibility within the endolysosomal network to compensate for retromer dysfunction.

In the current programme, we aim to build on our recent identification of a previously unrecognised protein assembly that orchestrates retromer-independent protein and lipid transport within the endolysosomal network - we have termed this 'retromer-like'. From the existing literature there appears to be links between retromer-like de-regulation and neurodegenerative disease, and in preliminary studies we have revealed additional links to hypercholesterolaemia (high blood cholesterol). The study of retromer-like will therefore provide new insight into these, and other, human diseases.

In the programme, we describe a holistic approach supported by national and international collaborations, to apply a broad but focused array of cutting-edge techniques to address two inter-related aims:

1). The fundamental question of how retromer-like functions as a 'machine' to regulate protein and lipid sorting through the endolysosomal network.

2). The application of acquired knowledge to define how retromer-like's activity is required for in vivo cell, tissue and organism-level physiology and how this is de-regulated in human disease.

The endolysosomal network sorts thousands of integral membrane proteins (termed cargos), and is essential for organelle biogenesis, maintenance, and quality control. Our research seeks to achieve a thorough mechanistic understanding of endolysosomal cargo sorting, specifically how cargo are retrieval from the lysosomal degradative fate and export and recycling to various organelles. Retromer is a heterotrimer of VPS35, VPS29 and VPS26, that has emerged as a key regulator of cargo sorting that is de-regulated in Parkinson and Alzheimer disease.

In the current programme we build on our discovery of 'retromer-like' (RETL), an evolutionary conserved heterotrimer of C16orf62, DSCR3 and VPS29 that orchestrates retromer-independent endolysosomal sorting of cargos that include integrins and low-density lipoprotein receptors. Using an array of cutting-edge techniques, from protein biochemistry, quantitative proteomics and in vitro analysis in cell culture through to disease-associated studies and in vivo analysis in Drosophila, we will address two inter-related aims (in conceptual order).

First, the fundamental question of how RETL functions to regulate endolysosomal cargo sorting. Here we will define the underly principles behind assembly of the RETL heterotrimer, establish the cargo proteins sorted via the RETL complex, and how RETL assembles accessory proteins to form a function sorting coat.

Secondly, we apply acquired knowledge to establish how RETL-mediated cargo sorting contributes to cell, tissue and organism-level physiology in health and disease. Here we focus on RETL-mediated integrin sorting in cell migration, including the in vivo analysis of Drosophila hemocyte migration, and the role of RETL in neuronal function and viability using in vitro iPS cells and Drosophila photoreceptor as model systems. For the latter, we explore the potential plasticity between retromer-dependent and RETL-dependent endolysosomal cargo sorting.

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 endolysosomal sorting complexes through to those studying the assembly of multi-protein complexes, membrane re-modelling, and the molecular basis of membrane trafficking. By extending research into neuronal-like cultures and in vivo analysis in Drosophila, data will influence neuroscientists studying endolysosomal sorting in neurons, and interest the wider community of basic and applied scientists currently trying to elucidate the complex cellular basis of neurodegenerative disease. This will include members of the commercial private sector who are tackling this growing issue within the ageing population.

Further afield, in choosing to take an unbiased global view of retromer-like dependent endolysosomal sorting, data from the proposal has the potential to influence a much wider sphere of scientists, far beyond those described above. For example, within the programme we stress the importance of retromer-like for the recycling of integrins and the in vitro and in vivo significance of this for cell migration, and extend the functional importance of this complex into cholesterol homeostasis (LDL receptor sorting). One anticipates that a parallel analysis in neuronal cells will generate a similar broad data set, with far reaching significance.

Understanding the aetiology of neurodegenerative 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 nongovernment policy-makers. Our proposed collaborative neurogenetic analysis of the retromer-like in Parkinson disease and other neurological disorders is one such example.

How will they benefit from this research?
Neurodegenerative disease is a major health issue and its prevalence is expected to increase, according to increasing aged population. Consequently, these diseases are a socioeconomic issue and a major challenge for the public health system. Understanding disease aetiology forms an important research goal. By generating a quantified global 'map' of the alteration in endolysosomal sorting observed in retromer-like suppressed neuronal cells, the current proposal seeks to formulate testable hypotheses on how the altered map may ultimately influence age-related neurodegeneration in neurodegenerative disease, especially in the context of Parkinson disease. Longer term, it is hoped that by validating or refuting individual hypotheses, research will enhance our understanding of Parkinson disease and influence how this disease may be prevented and/or treated.