EXPLORING THE ROLE AND THERAPEUTIC POTENTIAL OF RAB GTPASES IN HUNTINGTON'S DISEASE

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.

Huntington's disease (HD) is a fatal neurodegenerative disorder caused by the expansion of a polyglutamine tract in the huntingtin (HTT) protein. This mutation causes HTT to misfold and aggregate, leading to widespread cellular dysfunction and death of vulnerable neurons. The pathogenesis of HD has been linked to defects in intracellular vesicle trafficking due to impaired function of Rab GTPases. ~60 Rab GTPases in mammalian cells are critical for nearly every step of vesicle trafficking. We and others have shown that increased expression of Rab5, Rab8 or Rab11 protects against mutant HTT (mHTT) toxicity in cell and fruit fly models of HD.

We have performed a systematic siRNA screen in mammalian cells to identify additional Rabs and related proteins that modulate mHTT toxicity and identified 8 robust candidate disease modifiers. Here we propose to define the role of selected Rabs in HD and assess their therapeutic potential. We will validate protection by candidate Rabs in fruit fly models of HD and human pluripotent stem cell-derived (hPSC) neurons and myeloid cells. We will also test the efficacy of potential Rab modulating compounds, and explore alternative targeting approaches.

The extent of Rab-specific defects in HD and their mode of protection we will be assessed using a range of phenotypes in mammalian cells, fruit flies, and mice. Metrics used include mHTT expression and aggregation, autophagy, vesicle trafficking, as well as Rab activity and localisation. Interesting findings will be confirmed in hPSC derived neurons/myeloid cells, and ex vivo patient myeloid cells. In total, this work will inform the role of Rab GTPases in HD pathogenesis and better define their therapeutic potential and mechanism(s) of protection, informing strategies for exploiting their protective properties. As dysfunctional Rab activity has been implicated in several diseases these findings may have broader significance.

This proposal has direct implications for individuals suffering from Huntington's disease (HD), and their families. Though several drugs are available for symptomatic management of HD, no treatments are available that halt progression or onset of HD and very few new disease-modifying therapies are in the clinical pipeline. It is estimated that HD costs up to $1 billion per year in medical and nursing costs in the United States alone. Furthermore the common neurodegenerative disorders affect more than 700,000 people in the UK, and cases are predicted to double in the next 25 years, costing the UK economy a staggering £50 billion. In addition to the obvious patient benefits, the identification of an effective treatment for HD and other neurodegenerative disorders could dramatically cut these expenditures world-wide.

The laboratories of FG and SJ have considerable expertise in the use of several model systems for studying HD, including flies, mammalian cell lines, human pluripotent stem cells, and patient-derived myeloid cells. We have used these models to understand the mechanisms underlying this disease, as well as identifying novel candidate therapeutic targets as part of wider efforts to develop and test new disease-modifying therapies for HD. One such promising therapeutic approach is huntingtin gene lowering (sometimes called 'gene silencing'), for which SJT is global clinical PI for the Ionis Pharmaceuticals ASO HTT Rx trial which is a first into human clinical trial testing a huntingtin lowering drug for HD. Our previous identification of kynurenine 3-monoxygenase (KMO) as a candidate therapeutic target for Huntington's disease also speaks strongly for our approach. Several inhibitors of KMO have been, and are currently being, developed with the aim of entering clinical trials in the near future. Importantly, KMO inhibitors will likely also have relevance for Alzheimer's disease and Parkinson's disease (PD). In addition, we have also found that a compound which mimics the antioxidant activity of glutathione peroxidases is robustly protective in yeast, fly and mammalian cell models of HD. Notably, this compound - ebselen - is well-tolerated in humans, giving further impetus for future preclinical analyses.

In the work proposed here, we will identify the most effective Rabs for targeting therapeutically in HD, clarify the mechanism(s) underlying protection, and test drug-like chemicals which mimic the beneficial effects of Rab modifiers. To achieve our goals we will utilise multiple models and validate findings in neurons and disease-relevant myeloid cells, in which Rab dysfunction could contribute to altered cytokine/chemokine release. This is in contrast to the majority of past work that has primarily focused on neuronal functions of Rabs in neurodegeneration. We believe the use of multiple models and cell types will better inform the role of Rabs in HD pathogenesis, and may increase the robustness of target validation and prioritisation by identifying model and cell type specific effects.

As outlined above, the proposed research will likely provide additional insights into pathogenesis and treatment for HD, and has a high chance of delivering upon this promise. Importantly, the work proposed here will likely have relevance to other neurodegenerative diseases. Indeed, in published work we have found that modulation of Rab GTPase activity (e.g. Rab8, Rab11) can ameliorate disease phenotypes in models of PD. Therefore, the proposed work should inform therapeutic strategies for HD, and provide the basis for translation into other related disorders.