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

Lead Research Organisation: University College London
Department Name: School of Pharmacy

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

10 25 50
 
Description Overseas 4-year PhD Studentship Grant
Amount £115,000 (GBP)
Organisation University of Leeds 
Department China Scholarship Council
Sector Academic/University
Country United Kingdom
Start 11/2017 
End 10/2021
 
Description Overseas 4-year PhD Studentship Grant.
Amount £130,000 (GBP)
Organisation King Saud University 
Sector Academic/University
Country Saudi Arabia
Start 05/2016 
End 04/2020
 
Description Probing calcium channel dysfunction in LRRK2-Parkinson's
Amount £49,941 (GBP)
Funding ID K-1802 
Organisation Parkinson's UK 
Sector Charity/Non Profit
Country United Kingdom
Start 07/2018 
End 04/2020
 
Description AD and Wnt signalling 
Organisation University College London
Department School of Pharmacy
Country United Kingdom 
Sector Academic/University 
PI Contribution Investigation of Wnt signalling in APP knock-in mice tissue.
Collaborator Contribution Investigation of electrophysiological properties in APP knock-in mice tissue.
Impact Publication: PMID: 30766992
Start Year 2017
 
Description Affect of LRRK2 on Wnt signalling in AD 
Organisation King's College London
Country United Kingdom 
Sector Academic/University 
PI Contribution We provided LRRK2 wild type, mutant and control expression constructs for the investigation of the effect of LRRK2 on non-canonical Wnt signalling with relevance to Alzheimer's disease. Provision of expertise on LRRK2 and late-onset neurodegeneration with a focus on Parkinson's disease.
Collaborator Contribution Provision of expertise of the role of non-canonical Wnt signalling in Alzheimer's disease.
Impact N/A
Start Year 2018
 
Description Collaboration on LRRK2 and Ca2+ signalling 
Organisation University College London
Department Department of Cell and Developmental Biology
Country United Kingdom 
Sector Academic/University 
PI Contribution Provided primary cell cultures of LRRK2 knockout and LRRK2 G2019S knock-in mice. Provided expertise on LRRK2 and signalling. Contribution as Co-I on a submission of a joint grant application to Parkinson's UK February 2018.
Collaborator Contribution Ca2+ analysis in primary neuronal cell cultures from LRRK2 knockout and LRRK2 G2019S knock-in mice.
Impact Grant from Parkinson's UK for 'Probing calcium channel dysfunction in LRRK2-Parkinson's.' Disciplines: pharmacology, gentics, cell biology and imaging
Start Year 2016
 
Description Collaborators at the NIH (Bethesda) 
Organisation National Institute on Aging
Department Laboratory of Neurogenetics
Country United States 
Sector Public 
PI Contribution Three joint PhD studentships between University College London and the NIH (Bethesda) for projects into research on LRRK2 in Parkinson's disease and 1. Endoplasmic Reticulon Function, 2. Microtubule Function and 3. Membrane Trafficking. UCL funds half of these studentships. Provision of cDNA from primary microglia cultures from LRRK2 knockout and LRRK2 G2019S knock-in mice activated with Wnt ligands for RNAseq.
Collaborator Contribution Three joint PhD studentships between University College London and the NIH (Bethesda) for projects into research on LRRK2 in Parkinson's disease and 1. Endoplasmic Reticulon Function, 2. Microtubule Function and 3. Membrane Trafficking. The NIH funds half of these studentships. RNAseq of cDNA from primary microglia cultures from LRRK2 knockout and LRRK2 G2019S knock-in mice activated with Wnt ligands.
Impact Publications: PMID: 27789813 PMID: 29917075 PMID: 27013965 The collaborations involve expertise in cell biology, molecular biology, genetics, protein expression in mouse tissue and advanced microscopy. Even though the collaborations with both partners started before the start of this MRC grant the aims are related. Therefore this award informs/furthers the research of the three currently still active PhD projects and the research of the PhD students informs the research funded by the MRC.
Start Year 2012
 
Description Collaborators at the NIH (Bethesda) 
Organisation National Institutes of Health (NIH)
Department National Institute of Neurological Disorders and Stroke (NINDS)
Country United States 
Sector Public 
PI Contribution Three joint PhD studentships between University College London and the NIH (Bethesda) for projects into research on LRRK2 in Parkinson's disease and 1. Endoplasmic Reticulon Function, 2. Microtubule Function and 3. Membrane Trafficking. UCL funds half of these studentships. Provision of cDNA from primary microglia cultures from LRRK2 knockout and LRRK2 G2019S knock-in mice activated with Wnt ligands for RNAseq.
Collaborator Contribution Three joint PhD studentships between University College London and the NIH (Bethesda) for projects into research on LRRK2 in Parkinson's disease and 1. Endoplasmic Reticulon Function, 2. Microtubule Function and 3. Membrane Trafficking. The NIH funds half of these studentships. RNAseq of cDNA from primary microglia cultures from LRRK2 knockout and LRRK2 G2019S knock-in mice activated with Wnt ligands.
Impact Publications: PMID: 27789813 PMID: 29917075 PMID: 27013965 The collaborations involve expertise in cell biology, molecular biology, genetics, protein expression in mouse tissue and advanced microscopy. Even though the collaborations with both partners started before the start of this MRC grant the aims are related. Therefore this award informs/furthers the research of the three currently still active PhD projects and the research of the PhD students informs the research funded by the MRC.
Start Year 2012
 
Description Interaction between LRRK2 and alpha synuclein 
Organisation University Medical Center Göttingen
Country Germany 
Sector Academic/University 
PI Contribution Expertise and research tools for Wnt signalling
Collaborator Contribution Expertise and research tools for alpha synuclein
Impact Publication: PMID: 30578345
Start Year 2018
 
Description Wnt signalling modulators 
Organisation Alzheimer's Research UK
Department UCL Drug Discovery Institute
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution Assay to investigate changes in canonical Wnt signalling. Provided lentiviral biosensors for in vivo Wnt and Ca2+ signalling assays.
Collaborator Contribution Provided Wnt signalling modulators for testing Wnt signalling activation in different model systems.
Impact No outputs yet. Disciplines: drug discovery, pharmacology, signalling, live imaging, cell signalling
Start Year 2016
 
Description Highlighting MRC-funded research on the UCL School of Pharmacy website 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact MRC-funded research resulting in notable publications or achievements is routinely highlighted on the home page of the UCL School of Pharmacy website (http://www.ucl.ac.uk/pharmacy/research) in a Cluster specific 'News' sections e.g. http://www.ucl.ac.uk/pharmacy/research/disease-models-and-clinical-pharmacology/news. Previously website (http://www.pharmacy.ac.uk/latest_news.html) and the following exmples are no longer available.
Examples: http://www.pharmacy.ac.uk/2276.html
http://www.pharmacy.ac.uk/1520.html

Raising awareness of MRC-funded research with the general public, staff, other stakeholders and M.Pharm. Students.
Year(s) Of Engagement Activity 2008,2009,2010,2011,2012
URL http://www.ucl.ac.uk/pharmacy/research/disease-models-and-clinical-pharmacology
 
Description Parkinson's UK patient conference participation 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Patients, carers and/or patient groups
Results and Impact Recent engagement with patient groups and charities include contribution to the meeting for patients and carers entitled 'Parkinson's - from new Discoveries to new treatments.' Henley Business School, University of Reading, London, 14th July 2016. In addition, I have participated in patient events from Parkinson's UK and also gave oral and poster presentations at Parkinson's UK Research Conferences with patient participation. During these events patients and carers are encouraged to ask questions and contribute to discussions on current and future research aims providing their perspective on important research needs.
Year(s) Of Engagement Activity 2012,2014,2016
 
Description Research conference with patient and industry involvement 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Patients, carers and/or patient groups
Results and Impact 1. Presentation of basic research to patient, carers and industry.
2. Discussion with patient, carers and industry of possible active involvement of patient and carers in the design of basic research projects.
Year(s) Of Engagement Activity 2018