Role of microtubule acetylation in Parkinson's disease

Nerve cells (neurones) transmit signals in the brain. They have a cell body and long string-like extensions (up to tens of inches) that connect to other neurones. These extensions are called axons. In Parkinson's disease the axons of neurones that produce a chemical called dopamine break down and connections are lost. This causes the neurones to die and as a result there is less dopamine in the brain. This shortage of dopamine in the brain causes the typical tremor, walking and talking problems associated with Parkinson's disease.
The research in this project is to find out how neurones die in Parkinson's disease. We concentrate particularly on a process called "axonal transport". Axonal transport is like the Royal Mail's Parcel-Force but in neurones; it delivers all kinds of goods to their destinations in the axon. Technically axonal transport is like a train journey: Molecular motors ("the locomotives") hook up to cargoes ("the carriages"), and they ride on protein tracks called microtubules ("the rails") and use a "fuel" called ATP. When axonal transport breaks down the axon starves because no deliveries are being made, and eventually the neurone dies.
Mutations in a gene called LRRK2 are the most common cause of familial Parkinson's disease (~7 in 100) and are also found in the sporadic, more common form of the disease (~3 in 100). We have found that mutant LRRK2 stops axonal transport of a cargo called mitochondria (which produce energy in the cell and are known to be involved in Parkinson's disease). Our investigations revealed that mutant LRRK2 most likely stops axonal transport by damaging the microtubule rails. Using this information we tested a number of drugs that act on microtubules and found one that was able to repair the defective axonal transport. This drug is called TSA (short for trichostatin-A). To test if this finding held up in a whole living organism we turned to a model of Parkinson's disease in fruit flies (Drosophila in Latin). These flies have the human disease causing LRRK2 mutations and have difficulties climbing and flying. So in their own way these flies have movement difficulties similar to those of human Parkinson's patients. We fed these flies TSA and we found that not only did TSA rescue the axonal transport defect but it also improved the movement problems of the flies. So, at least in the fruit fly a drug that modifies microtubules is working. However, flies are not humans and for this possible therapy to make it to the clinic more work is needed.
In this project we want to investigate how TSA restores transport and why it protects neurones from dying. The most likely explanation is that TSA works by increasing a modification of microtubules called acetylation. Our first aim is to investigate if this is so. Secondly we don't know if the drug works on a specific LRRK2 related pathway or if it acts on an unrelated, but still beneficial, level. You can compare this with a painkiller such as paracetamol that relieves pain but doesn't cure the cause of the pain. We think that LRRK2 may act on proteins that regulate the acetylation of microtubules. This is what we want to investigate in our second aim. Finally, we want to find out if this novel mechanism is also involved in other forms of Parkinson's disease that are not caused by mutant LRRK2. This is important to establish the possible benefits of drugs that target microtubules as a therapy for all Parkinson's disease.
In summary with this project we want to make significant inroads into understanding the reasons why neurones die in Parkinson's disease. We have already found a drug that may be beneficial and we now want to find out exactly how the drug does this. This is necessary to design the best therapeutic strategies and to try to avoid the disappointment of yet another failed clinical trial. If we are successful we will be one step closer to develop drugs such as TSA as a therapy for Parkinson's disease.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common genetic cause of Parkinson's disease (PD). LRRK2 is a multifunctional protein comprising GTPase and kinase activities that affects many cellular processes. How mutations in LRRK2 cause PD is not known.
We have found that pathogenic mutations in the LRRK2 Ras of complex proteins (Roc) GTPase domain (R1441C) and the carboxy-terminal of Roc (COR) domain (Y1699C) domain preferentially associate with deacetylated microtubules, and inhibit axonal transport in vitro in primary neurones and in vivo in transgenic Drosophila larvae. Moreover these LRRK2 Roc-COR domain mutants caused locomotor deficits in vivo in adult transgenic Drosophila. Treating neurones with the histone deacetylase inhibitor Trichostatin A (TSA) to increase microtubule acetylation restored axonal transport in vitro and systemic administration of TSA to transgenic mutant LRRK2 flies was able to restore axonal transport in vivo. Furthermore post hoc administration of TSA reversed the locomotor deficits in adult LRRK2 Roc-COR mutant transgenic flies. Thus, our findings reveal a novel pathogenic mechanism for mutant LRRK2 and a potential therapeutic intervention for PD.
In this project we will test the hypothesis that disruption of MT acetylation by mutant LRRK2 is a neurotoxic event in PD that leads to neuron death by disrupting axonal transport.
Our aims are to investigate:
(1) the therapeutic potential of MT acetylation and the (de)acetylase pathways involved in LRRK2 Roc-COR mutant associated familial PD;
(2) how LRRK2 regulates MT acetylation;
(3) the involvement of MT acetylation and LRRK2 in models of a-synuclein-related familial PD and sporadic PD
In summary, this study aims to investigate how LRRK2 regulates MT acetylation and its involvement in PD pathogenesis. This will allow us to pinpoint and test more precise drug targets and to determine the therapeutic potential of MT acetylation drugs in PD.

In the short term, the main beneficiaries of knowledge arising from this research will be the people employed on this grant, and academic and clinical neuroscientists, particularly those with an interest in PD and neurodegenerative disease. The employment and training of research staff involved in the project will benefit their careers both by learning specific research skills a well as a range of transferable skills such as analysis and problem solving, interpersonal and leadership skills, project management and organisation, information management, self-management, and written and oral communication.
In the medium to long term our research will have a wider impact and benefit the pharmaceutical industry, PD patients and their families and society as a whole.

PD is the second most common neurodegenerative disease after Alzheimer's disease. PD affects up to 1% of the population above the age of 60 and 4-5% above the age of 85. There are about 127,000 people that suffer from PD at any given time in the UK (source: Parkinson's UK). At present the total yearly cost of treating PD in the UK is estimated at £2.0 billion. As advancing age is the main risk factor for PD, the socio-economic impact of PD is set to rise significantly in the future because of the aging population. In addition to care provided within the NHS, a significant amount of care for patients with PD takes place in the community, often with support of charitable organisations such as the Parkinson's UK. Thus PD places a significant public and private socio-economic burden on the UK.

Our research seeks to identify novel therapeutic targets in PD that can be developed into better PD therapies. The effect of better therapies will be most tangible for patients and their immediate families. Indeed improving the symptoms and halting or slowing the progression of disease will have an enormous impact on their quality of life. In terms of economics this research could free up considerable resources in the NHS that are currently allocated to PD and are set to rise in the future. Clearly the wider public would benefit from this. Furthermore this work has the potential to influence government policy by allowing the refocusing of resources. This research will also impact on the public sector. The University of Sheffield could benefit from licensing of any patents granted based on our findings, enhanced REF performance and increased reputation (via publications, press communiqués etc.). The Parkinson's UK charity would benefit as they funded our original study.

In addition to its impact on health and wellbeing, the public, and the public sector our research may also have an economical impact via the commercialisation of our results (e.g. therapies based on (de)acetylase drugs) and/or spinout companies. Accordingly the main commercial beneficiaries would be the pharmaceutical industry with whom we will engage in the later stages of drug development.

Finally, as the outcome of this research may also be applicable to other neurodegenerative disease such as Alzheimer's disease and ALS the potential impact may be much higher.