Regulation of Wnt and calcium signalling by the Parkinson's disease protein LRRK2

Parkinson's disease (PD) is a currently incurable condition that leads to progressive physical decline and often also cognitive impairment. Replacement therapies with dopamine, the chemical messenger depleted in the brains of PD patients, provide symptomatic relief for a period of time but do not modify disease progression and are associated with debilitating side-effects. PD affects 1% of the population over the age of 60 and 4% over the age of 80. This has clear implications for an aging population with current estimates suggesting seven million PD sufferers worldwide. During the disease process, brain dysfunction and ultimately degeneration of brain cells is apparent but the underlying cell biological and signalling dysfunctions leading to this condition are unknown. In order to develop therapeutics that can halt or slow the progression of PD we need to understand signalling events occurring during the development of the disease.

Mutations in Leucine Rich Repeat Kinase 2 (LRRK2) are a common cause of familial PD. Patients with these mutations develop PD that is clinically indistinguishable from the most common idiopathic, late-onset form of the disease. This fact makes LRRK2 and LRRK2 pathway components particularly attractive as therapeutic targets. Evidence for a role of LRRK2 in two main branches of cell signalling pathways is accumulating. One involves so-called Wnt proteins and the other leads to an increase of calcium ions in the cell. Nonetheless, the effect of LRRK2 on specific sub-branches of these pathways remains elusive. Our goal is to understand how LRRK2 affects Wnt- and calcium-signalling in health and disease. This will allow targeting of specific pathways to find a rationale for disease-modifying treatments for this currently incurable disease.

We have model systems that allow us to investigate normal and mutant LRRK2 signalling with the help of signalling reporters. This will establish normal LRRK2 function and mutant LRRK2 dysfunction in Wnt- and calcium-signalling in response to environmental stimuli or changes in metabolism for example in response to toxins or infections. We will also use confocal microscopy to establish the effect of different stimuli on the localisation of mutant LRRK2 in dopaminergic cells that produce fluorescent normal or mutant LRRK2. Our model systems also allow for confocal imaging of signalling pathway activation in cells. Together these experiments will reveal the function of normal and mutant LRRK2 signalling complexes.

We will then assess the effects of substances that can influence Wnt- and calcium-signalling on cellular phenotypes observed with disease-causing LRRK2 mutations. This will generate data on the likely efficacy of Wnt- and calcium-signalling modulators in PD patients, and help to develop a rationale for novel medication targeting LRRK2-dependent signalling pathways.

Taken together, our experiments will reveal the regulation/deregulation of specific Wnt- and calcium-signalling branches by LRRK2 under normal and disease-causing conditions. Achieving our objectives will increase understanding of how LRRK2 mutations affect signalling pathway regulation during early events leading to PD and inform strategies for therapies that can slow, halt or cure this disease.

Mutations in Leucine Rich Repeat Kinase 2 (LRRK2), a common cause of Parkinson's disease (PD), lead to symptoms indistinguishable from idiopathic late-onset PD. This fact makes LRRK2 and LRRK2 signalling pathways particularly attractive therapeutic targets. Evidence for LRRK2 modulated canonical Wnt-signalling and Ca2+-signalling through NFAT is accumulating. Nonetheless, evidence for LRRK2 effects on additional Wnt- and Ca2+-pathways is also emerging. Our goal, to understand how LRRK2 effects specific Wnt- and Ca2+-pathways, will provide a rationale for disease-modifying treatments for this currently incurable disease.

LRRK2 knockout and familial G2019S mutant mouse embryonic fibroblast (MEF) and primary neuronal cell cultures will be utilised in luciferase signalling reporter and biochemical assays to evaluate Wnt- and Ca2+-dependent pathway regulation by wild-type LRRK2 and deregulation by mutant LRRK2. Confocal imaging of dopaminergic SH-SY5Y cell lines stably over-expressing EGFP-tagged wild-type or G2019S LRRK2 will determine the subcellular localisation of LRRK2 signalling complexes in response to different environmental (e.g. toxin) or metabolic (e.g. insulin) stimuli, allowing conclusions on signalling regulation/deregulation. Lentiviral biosensors will provide a luminescence-based, real-time read-out of the physiological and pathological function of wild-type and mutant LRRK2 in canonical Wnt and NFAT/Ca2+-signalling in brain during development and adulthood. We will also assess the potential of Ca2+- and Wnt-signalling modulators on previously observed G2019S phenotypes in MEF and SH-SY5Y cells to generate data on the likely efficacy of these modulators in PD.

This research will provide key insights into LRRK2-regulated specific Wnt- and Ca2+-pathways in health and disease, increase our understanding of how mutant LRRK2 affects signalling pathways during early events leading to PD and inform therapeutic strategies to halt or cure PD.

Who will benefit from this research?
Our proposed project is likely to have broad-ranging impact on several groups. These include: academics (Academic Impact) and in the longer-term, healthcare professionals associated with neuroscience and neurodegeneration, the general/lay public who are directly or indirectly affected by neurodegenerative diseases (particularly those caused by mutations in PARK genes) and pharmaceutical companies concerned with drug development for Parkinson's disease (Economic/Societal Impact).

How will they benefit from this research?
Academics will gain valuable insight into the biological roles and signalling function of LRRK2 in health and events leading to Parkinson's disease. In addition, we will transfer technical and managerial skills to a new generation of research scientists, either directly (to postdoctoral RA) or indirectly (to undergraduate or Ph.D. students involved in our research). This will also help to develop an international skill base through the subsequent mobility of these new scientists. Former Ph.D. students and postdoctoral scientists have benefitted in terms of their career trajectories by training in our labs and taking on posts at many other Universities (e.g. Bath, Bristol, UCL), healthcare (e.g. Medicines and Healthcare products Regulatory Agency) or funding agencies (e.g. Alzheimer's Research UK). KH previous studies of inhibitory synaptic transmission have provided direct evidence of translation for those suffering with inherited neurological disorders by the provision of molecular diagnostics, monitoring equipment and therapeutics (Rees et al 2006, Nat Genet 38: 801-806; Carta et al 2012, J Biol Chem 287: 28975-28985; James et al 2013, Neurobiol Dis 52: 137-149). This leads to improved quality of life for patients, family members, carers and medical staff and can provide clinicians with insights into treatments for neurological disorders.

What will be done to ensure that they have the opportunity to benefit from this research?
The research we propose will increase our basic knowledge of the biology of LRRK2 signalling and translate across scientific disciplines. In the short term (1-3 years), our research will benefit research scientists and medical practitioners via the presentation of new research findings at invited lectures in the UK and internationally to scientific and lay audiences, by publishing in internationally-recognised scientific journals, and by engaging with national/international press, television and radio. The past impact of our work on neurodegenerative diseases is evidenced by: i) Invited talks and lectures given in this area - 14 for KH; 9 for AR; ii) Recent high-profile publications in this research area (e.g. Berwick and Harvey 2014, J Mol Cell Biol, in press; Law et al 2014, J Biol Chem 289: 895-908; Berwick and Harvey 2013, Front Cell Neurosci 7: 82; Berwick and Harvey 2012, Hum Mol Genet 21: 4966-4979; Rahim et al 2012, Gene Therapy 19: 936-946; Berwick and Harvey 2011, Trends Cell Biol 21: 257-265; Sancho et al 2009, Hum Mol Genet 18: 3955-3968; Rahim et al 2009, Gene Therapy 16: 509-520; Rahim et al 2011, FASEB J 25: 3505-18; Osellame et al 2013, Cell Metab 17: 941-953). We are also working with Argenta to translate research findings into novel drug development initiatives for treating inflammatory pain and rhythmic breathing disorders, based on our previous MRC-funded research (G0500833; Harvey et al 2004, Science 304: 884-887). At UCL, we are ideally placed to develop our findings and realise impact via: i) UCL Advances - a Centre for Entrepreneurship and Business Interactions; ii) UCL Business PLC - a dedicated technology transfer company; iii) UCL Consultants Ltd that facilitates consultancy contracts; iv) UCL Partners - one of five accredited academic health science systems in the UK translating cutting-edge research and innovation into measurable health gain for patients and populations.