Glycosphingolipid recycling in health and disease: the GALC-SapA lipid processing complex

Neurodegenerative disorders such as Alzheimer's and Parkinson's diseases affect a significant proportion of our ageing population. Less well known are the fatal early-onset neurodegenerative diseases such as Krabbe disease that primarily affect young children. Recent research has shown that mutations causing these severe-but-rare diseases are also associated with increased risk of developing late-onset neuromuscular and neurodegenerative disorders, including common ones such as Parkinson's disease. This observation reinforces that by studying rare diseases we can gain crucial insight into a number of diseases that affect a great many people.

We know that several early-onset neurodegenerative diseases occur as a result of defects in the recycling of components of the cell known as lipids that make up our cellular membranes. Accumulation of unprocessed lipid results in cellular damage and cell death. In order to correctly process these lipids our cells require specific enzymes and their "helper" or activating partners. Our research is focussed on the enzyme GALC and its partner protein SapA. Defects in either of these proteins cause Krabbe disease and are implicated in open angle glaucoma, one of the leading causes of age-related blindness, and motor neuron disease. Although it is known that mutations in these enzymes cause disease it is not understood how. Our work aims to understand the interactions between these enzymes and activators and how they work together to correctly process lipids in the cell. By understanding this mechanism we can identify new ways to develop novel therapeutics not just for the early-onset diseases such as Krabbe disease but also for the more common late-onset neurodegenerative disorders.

This proposal describes structural and functional studies to determine how sphingolipid processing occurs in cells. Lysosomal hydrolases require activating saposin proteins to process substrates. Defects in these proteins cause a range of diseases including early-onset neurodegenerative disorders. Specific enzyme:saposin complexes are essential for lipid hydrolysis but it is not understood how these proteins interact or how specificity is determined. Our current work is focused on GALC, a lysosomal enzyme that when defective causes Krabbe Disease. Our preliminary data reveals a direct interaction between GALC and SapA that occurs at low pH, consistent with formation of this complex in the acidic environment of the lysosome. We have determined the structure of a GALC:SapA:detergent complex identifying how these two proteins interact and where detergent molecules bind. We now intend to exploit the insights from this structure to test how bona fide lipid substrates are bound in the complex and processed in cells. We will determine structures of the complex in the presence of lipid substrates as well as custom-made, high-affinity lipidated inhibitors. The composition of these complexes will be verified using biophysical methods. Protein interaction studies will be used to test whether specific mutations at the interaction interface reduce or abolish binding. This will be done using structurally-informed mutations and Krabbe disease variants that may cause disease by interfering with SapA binding. We will transfer these insights into functional assays monitoring lipid-processing in cells by generating GALC deficient cell lines and re-introducing wild-type and mutant GALC. To monitor defective lipid processing in these cells we will determine their lipid composition using mass spectrometry, monitor the processing of lipid substrates by immunofluorescence and electron microscopy and carry out cell-based activity assays when the GALC-SapA interaction is disrupted.

1. Who will benefit from this research?
This work constitutes basic research underpinning mechanisms of human disease. For this reason the primary direct beneficiary is the research community, including academics and clinicians. Due to the direct disease relevance of this work, it will also provide indirect benefit to patients and their families by shedding light on new mechanisms underlying Krabbe disease. In the long term there is the potential that structural details of the GALC:SapA:lipid complex could provide the atomic framework for the development of new rationally-designed drug therapies for Krabbe disease. The postdoctoral researcher trained during this research project will directly benefit from the experience in my lab by developing a broad range of experimental expertise leading to improved future career opportunities.

2. How will they benefit from this research?
The results of our research will be shared with researchers and clinicians via publication in high-impact journals with compliant open-access policies and through national and international conference presentations. These routes will also allow patients and healthcare professionals to benefit from the insight provided by our studies. Deposition of our structural data in the Protein Data Bank public repository will mean this work is available to anyone interested in exploiting these structural insights for experimental design or drug development.

The research and professional skills that staff will develop while working in a research laboratory are of great benefit in the wider community. Well-trained staff develop an understanding of how to tackle complex problems and how to address important healthcare needs. These skills translate into a range of professional sectors including the development of science policy and scientific communication but also more broadly into roles that require multi-tasking, teamwork and problem solving.

Our commitment to public engagement will benefit the public (see Communications Plan). A greater understanding of why we do research and how it can translate into new therapeutics is of enormous benefit not just to patients and their families but to the wider community. They will also gain a better appreciation of the complexities of translating research insight into therapeutic strategies and the long term investment that is required in scientific research.

Mutations in GALC lead not just to Krabbe Disease but are also associated with a growing number of later onset neuromuscular and neurodegenerative disorders. The future impact of the current work is difficult to gauge but by providing a greater understanding of the underlying mechanisms of Krabbe disease this work has the potential to benefit patients themselves by improving the way therapeutics are tailored to specific disease variants.