Dissecting the molecular mechanisms of lysosome:ER contact site formation and their relevance to neurodegenerative disease

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Human cells contain membrane enclosed compartments, called organelles, that perform specialised tasks. Membrane contact sites, that bridge the membranes of neighbouring organelles, provide platforms for communication between organelles. Cholesterol is an important component of membranes. Cells have two sources of cholesterol: it can either be derived from our diet or newly synthesised by the cell. Dietary cholesterol is taken up into the cell into a compartment called the endosome, from where it is trafficked to the lysosome for onward transport to another compartment, the endoplasmic reticulum (ER). The ER is the site of cholesterol synthesis within the cell and the arrival of dietary cholesterol from the lysosome switches off the synthesis of new cholesterol. When dietary cholesterol fails to be transported from the lysosome to the ER, it accumulates in the lysosome, while cholesterol synthesis in the ER still continues. The resulting imbalance in cellular cholesterol is toxic to the cell and is associated with neurodegenerative disease. We have recently shown an important role for membrane contact sites between the ER and lysosomes in regulating the cholesterol balance by transporting cellular cholesterol to the right location. The aim of this research proposal is to determine exactly how these contact sites are formed, if they transport other lipids as well as cholesterol and if they can be targeted for therapeutic benefit in neurodegenerative disease.Niemann Pick disease type-C (NPC) is a rare but devastating progressive neurodegenerative disease that often starts in early childhood. We know that most (95%) cases of NPC are caused by mutations in a gene that encodes the NPC protein NPC1. NPC1 is on the lysosome membrane and is required for dietary cholesterol to exit the lysosome for transport to the ER. Previous work by our group has found that NPC1 forms part of the lysosome-ER bridge that cholesterol can travel across. In cells from NPC patients, the lysosome fails to connect properly with the ER. As a result, cholesterol accumulates in the lysosome and the lysosome stops working properly. In this proposed work we intend to find out how NPC1 bridges the two organelles and if its interactions with partners on the ER are affected by levels of dietary cholesterol in the lysosome.As well as cholesterol, other lipids also accumulate in lysosomes in NPC that are believed to contribute to the disease progression. Indeed, the only current licensed therapeutic for NPC (miglustat) inhibits the synthesis of these lipids. Although miglustat is not a cure, the fact that it slows progression of the disease shows that imbalance of these lipids is toxic to the cell. Our preliminary data suggests that some of these lipids might also be transported across contact sites, to the ER where they are degraded. In this project we will improve our understanding of the relationship between membrane contact sites and lipid metabolism. We recently showed that artificially bridging lysosomes to the ER can reverse cholesterol accumulation in cells lacking NPC1. This is exciting as it could have therapeutic implications for NPC. We will assess different ways to expand membrane contact sites in cellular models of NPC and build on these studies to see the effect of increased contact sites in animal models. Zebrafish are an excellent model system for studying NPC. NPC zebrafish models mimic many mammalian NPC phenotypes, including lipid accumulation, and movement defects. We will therefore generate zebrafish models of NPC in which to study ER-lysosome contact sites and test our hypothesis that expanding these contact sites will rescue both lipid accumulation and downstream neurodegeneration /movement defects. This may yield novel therapeutic strategies for the treatment of NPC and other neurodegenerative diseases with lipid storage defects.