Analysing the cell biology of SNX10 in endosomal sorting and signaling: implications for osteoclast function in osteopetrosis

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-enriched compartments each composed of a unique combination of proteins and lipids. For cells to functional 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 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 10 years our laboratory has focused on describing the mechanistic details that control regulated transport of proteins and lipids within a specific aspect of the cell's membranous maze termed the endocytic network. In particular, we have studied a family of ancient, evolutionary conserved proteins, the sorting nexins. Within this family we have extensively analysed the function of retromer complexes, and this is generating new insight into the perturbed function of the endosomal network during cell infection by various pathogens, as well as a variety of diseases including Alzheimer's disease and Parkinson's disease.

In the current proposal we seek to utilize our extensive experience of studying sorting nexins to define the function of sorting nexin-10 (SNX10) in the regulation of bone re-modeling. This stems from research showing that mutations in SNX10 are linked with autosomal recessive osteopetrosis, a genetically heterogenous disorder caused by reduced bone resorption by a specific class of cells termed osteoclasts. However, at present we do not understand the basic function of SNX10, and hence have a very limited appreciation of why its mutation leads to this debilitating disease.

Our proposed research will address the following questions:
1). Is SNX10 required for endosomal sorting of RANK and downstream RANKL signaling?
2). Is the cell surface expression of other receptors also regulated by SNX10?
3). What are the molecular details of SNX10-mediated endosomal sorting?
4). Do the molecular details of SNX10-mediated endosomal sorting generate further insight into diseases of bone homeostasis?

Overall, data derived from the proposed research, which will be disseminated through peer-review publications and oral presentation at international meetings, will define the role of SNX10 in osteoclast function, knowledge that will achieve a greater appreciation of the cellular defects that lead to osteopetrosis and possibly other related bone diseases. Longer term, the research may provide a rationale for therapeutic intervention in patients carrying the SNX10 mutations and other osteopetrosis-linked mutations.

In sorting an array of cargos, for example proteins, lipids, nutrients, and solutes, to a variety of cellular destinations, the endosomal network performs an essential function in numerous processes within the cell. Sorting nexins are an ancient evolutionary conserved protein family that are emerging as key orchestrators of endosomal sorting. In humans, 31 sorting nexins are expressed. For the majority we know very little as to their function. For those that have been studied one common role is emerging: the capture of cargo proteins from lysosomal degradation through sorting into multiple pathways to the plasma membrane and trans-Golgi network.

Recently, mutations in the sorting nexin-10 (SNX10) gene have been linked with autosomal recessive osteopetrosis, a genetically heterogenous disorder caused by reduced bone resorption by osteoclasts. Three independent studies, describing 23 patients of Palestinian, Iraqi and North Swedish origin, have revealed a number of nonsense, missense and splicing defects. Patients show a range of severity with no obvious correlation with the specific molecular defect in SNX10. Supporting the in vivo link between disrupted SNX10 function and osteopetrosis are in vitro data that SNX10 is required for osteoclast differentiation and osteoclast resorption.

Based on these data, and additional preliminary data, we shall test the following hypothesis: SNX10 functions to assemble an endosome sorting complex that mediates the efficient sorting and cell surface expression of various receptor types, including receptor activator of NF-kB (RANK), that together are required to commit osteoclast lineage cells fordifferentiation to functional osteoclasts, and that for these receptors, loss of SNX10 function leads to adverse endosomal sorting, a resultant decrease in cell surface expression and inability to fully commit to osteoclastogenesis. Clinically this defect in endosomal sorting is manifested as autosomal recessive osteopetrosis.

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 sorting nexins, retromer, phosphoinositide, endosomal sorting and associated complexes through to those studying the assembly of multi-protein complexes and the molecular basis of membrane trafficking. By extending research into osteoclasts and the molecular basis of autosomal recessive osteopetrosis, data will influence scientists studying osteoclast function and more broadly basic and applied scientists currently studying bone re-modeling in development and disease. The later will include members of the commercial private sector who are tackling bone related diseases in the ageing population.

Understanding diseases associated with bone re-modeling, including autosomal recessive osteopetrosis, 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 SNX10 nonsense and missense mutations in autosomal recessive osteopetrosis is one such example.

How will they benefit from this research?
Osteopetrosis (marble bone disease) is a group of rare, inheritable disorders of the skeleton characterised by increased bone density, which stems from abnormalities in osteoclast differentiation and function. Mutations in more than ten genes have been identified as causative in humans and account for approximately 70% of cases. Understanding the etiology of this genetically heterogenous disease, with different prognostic and therapeutic implications, forms an important research goal. By generating a quantified global landscape of the alteration in cell surface proteome in SNX10 (and retromer) suppressed osteoclast precursor-like cells, the current proposal seeks to formulate testable hypotheses on how the altered landscape may influence signaling sensitivity during osteoclastogenesis. Longer term, it is hoped that by validating or refuting individual hypotheses, research will enhance our understanding of the defects that contribute to the osteopetrosis observed in patients with SNX10 mutations and more broadly other disease associated with adverse osteoclast mediated bone re-modeling.