Dissecting the role of LRRK2 in the regulation of autophagy

Parkinson's disease is an incurable brain disease that afflicts over 125,000 people in the UK, a number that will increase as the population ages. It is caused by cells dying in the part of the brain that helps control movement. At the moment we do not have a treatment that can be used to slow down or treat the cause of the disease, and so an important aim of scientific research into Parkinson's is to develop new drugs. Over the past decade we have learned a lot about the processes in the brain that lead to cells dying in Parkinson's disease by looking at rare cases where the disease is inherited in families. By identifying mutations in the genes that cause Parkinson's in these families we can study the changes in the brain that can lead to disease. This proposal looks at one of these genes, called LRRK2 (pronounced lurk2). Mutations in LRRK2 are the most common genetic cause of Parkinson's, responsible for about 6000 cases in the UK. We now know that subtle changes in LRRK2 (not as potent as mutations) can increase the risk of developing the more common, sporadic type of Parkinson's. This makes LRRK2 a very interesting gene as a potential new drug target. What makes it even more attractive as a drug target is that it has two pockets, called enzyme domains, which can be occupied by small chemical compounds (drugs) to change how it functions. This project focuses on one aspect of what LRRK2 does - looking at how it might be involved in a process called autophagy (pronounced ort-oh-fay-gee). Autophagy is one of the ways in which cells in the body get rid of waste, and we already know that when there are problems with autophagy rubbish builds up and can cause cells to die - a process that is very important in brain diseases like Parkinson's and Alzheimer's. Work from my laboratory shows that when you use a drug to turn LRRK2 "off", binding to one of those enzyme pockets, autophagy is switched on in human cells that we can grow in a dish. What we are looking at with this project is exactly how this happens, and what other genes are involved in the process. We are also going to look at what mutations in LRRK2 do to change autophagy. We think that mutations might slow down autophagy, which could lead to rubbish building up in the brain over the years and eventually causing enough brain cells to die to make the person with the mutation develop Parkinson's. Finding out what LRRK2 does, and how these mutations change its behavior, is going to be very important for the drug companies who are trying to develop new compounds to treat Parkinson's. It will give them ways in which to measure how active LRRK2 is in cells, which in turn can be used to judge how successful a new drug is at changing that activity, and might uncover new ways to target LRRK2 in the brain. All of this will be critical for developing new drugs for Parkinson's - something that is of huge importance to all of those people who already have the disease and those who will develop it in the future.

Mutations in Leucine Rich Repeat Kinase 2 (LRRK2) are the most common genetic cause of Parkinson's disease (PD). Recent genome wide association studies have identified variation at the LRRK2 locus as a significant risk factor for idiopathic PD, linking the Mendelian and sporadic forms of Parkinson's. In combination with the presence in the LRRK2 open reading frame of two enzymatic active sites (a kinase and a GTPase), these facts make LRRK2 one of the most attractive drug targets for PD. This proposal investigates the role of LRRK2 in the regulation of autophagy. Data from my group and from other laboratories around the world have revealed that altering LRRK2 function, either by inhibition, knockout, knockdown or mutation, impacts on autophagy although the precise mechanism remains obscure. We have preliminary data that localizes LRRK2 downstream of mTORC1 in the regulation of autophagy, as chemical inhibition of LRRK2 kinase activity results in an induction of autophagy in the absence of any impact of translational targets of mTORC1 (S6 and 4E-BP1). In this new investigator research grant, I will use a combination of cellular and chemical approaches to manipulate LRRK2 in order to establish the exact point at which LRRK2 intersects with the induction of autophagy, specifically examining the phosphorylation of ULK1 and ATG13 as potential effectors for LRRK2s inhibitory impact on autophagy. In parallel to this, I will examine the impact of mutations in LRRK2 upon the regulation of autophagy at the endogenous level using fibroblasts isolated from individuals harbouring mutations in LRRK2 and age/sex matched controls. Preliminary data suggests that mutations result in a decreased ability to enter autophagy upon starvation, and so in this proposal I will carry out a detailed examination of autophagic flux, along with an examination of the impact of LRRK2 mutations on key regulators of the induction of autophagy using in vitro and cellular models.

The data generated by this proposal will have implications for a wide range of beneficiaries beyond an immediate academic audience. LRRK2 is a priority drug target for Parkinson's disease (PD), and understanding the normal function of LRRK2 will have major implications for drug discovery programs directed at this gene. A number of multinational pharmaceutical companies (including GSK, Pfizer, Genentech and Eli Lilly) have programs to develop compounds that modify the activity of LRRK2, with the bulk of this effort directed towards the development of kinase activity inhibitors. By the end of this project, we will have a clear idea of how inhibiting LRRK2's kinase activity alters autophagy, data that will be of great importance to the pharmaceutical sector in their efforts to target this gene. According to a recent estimate, PD costs between £449 million and £3.3 billion to the UK economy (Findley LJ, Parkinsonism and Related Disorders 13: S8-S12, 2007), and it is clear that a therapy for this disorder would be of great economic significance within the UK and around the globe. In more human terms, a disease modifying therapy for PD will have a huge impact on the 125,000 people in the UK who have PD and the tens of thousands who will go on to develop the disorder in the coming decade, including a large number with mutations in LRRK2.

Beyond PD, the LRRK2 locus has also been implicated in Inflammatory Bowel disease, cancer and leprosy. Although our understanding of the links between LRRK2 and these disorders is not as advanced as for PD, a clearer understanding of LRRK2's biology and role in autophagy has potential impacts in terms of drug development efforts in each of these three disease areas. Given the efforts being directed towards developing drugs to target LRRK2, repurposing of these compounds for testing in other disorders would be a natural follow up to studies in PD - and the data generated by this proposal will have implications for our understanding of how LRRK2 may be linked to these disorders, aiding this process. There are an estimated 120,000 people in the UK living with Ulcerative Colitis, with a further 60,000 living with Crohn's disease (the two most common forms of inflammatory bowel disease, estimates from Crohn's and Colitis UK). Although not life threatening, these disorders are extremely debilitating and there is an active ongoing research effort to develop new treatments - into which this research can feed.

LRRK2 has been linked to oncogenesis by a number of studies, although the exact nature of these links is unclear. Developing novel drugs to target cancer is, however, a key medical goal and the information generated by this proposal will be of relevance to understanding LRRK2's role in cancer by illuminating its basic biological function, data which in the longer term will impact on the efforts to develop drugs to target cancer in the future.

Leprosy is a chronic bacterial infection of the peripheral nerves and mucosa, and if untreated can be severely disabling. It is extremely rare in the UK, but is still an ongoing health problem in South Asia, Africa and South America with a worldwide incidence of over 200,000 (data from WHO). Thankfully multidrug therapy for leprosy is very effective, but given the ongoing issues with multidrug resistance in Tuberculosis, caused by a bacterium closely related to the causative agent of leprosy, the development of further candidate drugs for Leprosy should be encouraged, with repurposed LRRK2 drugs falling into this category.

Finally, autophagy more broadly is an area of increasing interest from a biomedical drug discovery point of view. A greater understanding of the mechanisms governing the initiation of autophagy, an aspect of this process that the data generated by this proposal will be directly relevant to, will have an impact across a wide swathe of drug discovery for the disorders to which autophagy has been linked.