Huntington's disease (HD) is a fatal neurodegenerative disorder characterised by the loss of vulnerable neurons in the brain. The disease is caused by an increase in the size of a repeated DNA sequence which encodes for the amino acid glutamine in the huntingtin (HTT) protein. If the number of glutamines in the HTT protein increases beyond a critical length, it misfolds and clumps together to form protein aggregates, disrupting many vital cellular processes. Notably, other genes can significantly modify the onset of symptoms. This suggests there are many potential therapeutic targets in the human genome capable of significantly altering disease. Studies in patients and disease models have revealed that mutant HTT (mHTT) affects many cellular pathways, including intracellular vesicle trafficking. This is a vital process for the movement of proteins and nutrients between different parts of the cell. Previous work by our laboratory and others has shown that vesicle trafficking defects in HD are at least partly due to a family of proteins called Rab GTPases. These proteins are vital for nearly every aspect of vesicle trafficking, and the function of several Rabs is impaired in HD and other related disorders, including Parkinson's disease. Interestingly, the non-mutant HTT protein may be important for the normal function of some Rabs, which may contribute to their dysfunction in HD. We and others have noted that increased expression of three Rab GTPases - Rab5, Rab8 and Rab11 - reduces disease-relevant symptoms in cultured mammalian cell and fruit fly models of HD. While the role of these Rabs in HD has been explored, little is known about the importance of the ~60 other mammalian Rabs. To address this question we performed a systematic screen of 130 mammalian Rabs and associated genes and identified 8 that modulated mHTT toxicity in mammalian cells. Several additional Rabs have also been identified in independent screens for genes that alter mHTT toxicity and/or misfolding. In this research proposal we aim to further investigate the role of Rab GTPases in HD and explore their therapeutic potential. Notably, this will include the first characterisation of the role of Rabs in peripheral immune cell dysfunction in HD by analysing patient-derived samples. Immune cells are hyper-reactive in HD, producing inflammatory molecules that may contribute to progression of the disease, and Rabs contribute to the secretion of these molecules. Understanding Rab dysfunction in HD is critical for determining their disease and therapeutic relevance, the mode of action of disease modifying Rabs and for formulating therapeutic approaches. We will address this by investigating whether the amount, function or cellular location of candidate Rabs is altered in HD models and patient samples, and how this impacts upon Rab-dependant processes. To validate the protective properties of candidate Rabs, and prioritise them for further study, they will be tested in fruit fly and mouse HD models, allowing us to study the impact of mHTT and Rabs on the complex interactions that occur between cells in living multicellular organisms. Promising candidates will be tested in physiologically relevant human HD model cells, including neurons and immune cells from patients. Drug-like compounds which alter Rab function will also be tested, and alternative methods for targeting them explored. To further inform potential therapeutic approaches and prioritise candidates the mechanisms by which they modify HD relevant-phenotypes will be studied. Our preliminary findings suggest that many candidates increase the clearance of mHTT from the cell, and we will confirm these findings using additional HD models and approaches. In total, this work will help define the role of Rab GTPases in HD, assess their therapeutic potential and inform therapeutic strategies. As Rab dysfunction has been implicated in several diseases these findings may also have broader significance.