Determining the mechanisms of nigro-striatal dysfunction in SGCE mutation positive Myoclonus Dystonia using an iPSC-derived neuronal cell model

Lead Research Organisation: Cardiff University
Department Name: School of Medicine

Abstract

Aims
1. To develop individual nerve cell models of Myoclonus Dystonia using skin cells (fibroblasts) from patients
2. Detailed study of the nerve cell models to determine what effect SGCE gene mutations have on the cells, in particular the neurotransmitter (chemical allowing communication between nerves) dopamine and its receptors.


Background to the research and its importance
Dystonia is one of the most common movement disorders, affecting 1 in 900 per population head. Well recognised forms include writer's cramp, although more severe forms are frequently observed. Dystonia is associated with significant lifetime disability, which has an impact on education and employment. There are currently no effective treatments for dystonia, necessitating an improved understanding of the underlying disease causing mechanisms in order to allow development of novel and potentially disease-modifying therapies to be developed.

This project is focused on Myoclonus Dystonia, an inherited, childhood-onset movement disorder with significant associated psychiatric symptoms. It is one of the most common forms of inherited dystonia and one of the few subtypes caused by mutations to a specific gene (SGCE). These characteristics make Myoclonus Dystonia an opportune disorder in which to study the underlying mechanisms of dystonia. The SGCE gene encodes the epsilon-sarcoglycan protein whose role in the brain, and how defects in its function give rise to dystonia, remain uncertain. Evidence from human brain imaging studies and animal models indicate that the communication (synapse) between dopaminergic neurons arising in the midbrain and medium spiny neurons in the striatum form a focal point of the neuronal disruption in dystonia.

My previous research work involved recruiting one of the largest worldwide cohorts of patients with Myoclonus Dystonia. All of these patients underwent detailed examination of their movement disorder and any psychiatric symptoms. I have selected fibroblast samples from two patients (with different gene mutations) in order to generate these nerve cell models.

Design and methods
The fibroblasts will be used to generate induced pluripotent stem cell (iPSc), cells that are capable of developing into all tissue types. Using a series of well-established techniques, these cells will be converted into nerve cells, more specifically dopaminergic nerve cells (from the midbrain) and medium spiny nerve cells (in the striatum), two nerve cell types thought to be important in dystonia. Both nerve cell types will be examined to determine the effect of SGCE mutations on the epsilon-sarcoglycan protein. Detailed study will also be carried out of the enzymes involved in producing dopamine, the proteins involved in the transporting it across the cell membrane, and the receptors that signal its effect between the two cell types. The second stage of experiments will involve culturing both cell types together, examining their electrical communication and the structure of the neuronal processes.

Results will be compared to nerve models produced from tissue donated by healthy, unrelated individuals. We also plan to use a new genetic technique (Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)) to 'edit' the cells from the patients with Myoclonus Dystonia, returning the SGCE gene sequence to normal, in order to demonstrate that the changes seen in the original nerve cell model are due to the SGCE mutations.

Detailing these processes will improve our understanding of why dystonia, and potentially some psychiatric symptoms arise, as well as providing a platform for the development of new drugs and therapeutic options.

Technical Summary

Aims
Generate a neuronal cell model for SGCE mutation-positive Myoclonus Dystonia (MD) using patient derived fibroblasts
Characterise this model to determine changes to monoaminergic neurotransmission at the nigro-striatal junction

Background
Dystonia is a common and disabling movement disorder. Poor understanding of its pathophysiology has led to few effective treatment options. MD is one of few genetically determined dystonias caused by fully penetrant, autosomal dominant mutations (epsilon-sarcoglycan gene, SGCE), providing a unique opportunity for investigation. Previous studies suggest that disruption of the nigro-striatal junction, specifically dopamine-2-receptor (D2R) function, is central to pathogenesis. My previous work involved detailed phenotyping of a large MD cohort (n=30). Two mutation types will be used to develop induced pluripotent stem cell (iPSC) lines: nonsense (c.289C>T,p.Arg97X) and missense (c.622G>A,p.Gly441Asp) mutations.

Methodology
IPSC lines will be generated using Sendai virus vector techniques and differentiated using dual SMAD inhibition and Activin A into midbrain dopaminergic and striatal medium spiny neurons (MSNs) respectively. Characterisation of the neuronal models will include measurement of dopamine synthetic enzymes, transport proteins and the density and distribution of dopamine receptors. A co-culture platform of the two neuronal cell types will undergo phenotyping using electrophysiological techniques, calcium imaging and detailing of dendritic spine morphology. Control neuronal models will include those from healthy, unrelated individuals and isogenic 'rescue' cell lines of the MD patient samples by use of CRISPR technology.

Scientific and Medical opportunities
This project provides an excellent model system with which to investigate the cellular mechanisms that underlie dystonia pathogenesis. Findings from this work will inform future studies, particularly the identification of novel therapeutic targets.

Planned Impact

Onset of primary dystonias is typically in the first two decades of life, at a time of great developmental and educational importance. A lack of understanding of the pathophysiology and poor recognition of these disorders in society often leads to late presentation to health services, under recognition and frequent misdiagnosis. The increasing evidence for psychiatric symptoms as a primary component of the disorder phenotype adds to the need for accurate diagnosis and effective management.

This project will have an impact on several significant groups. These benefits will be seen in the short-, medium- and long-term.

1. Individuals and families affected by Myoclonus Dystonia
This project aims to develop a neuronal model for Myoclonus Dystonia that will aid in understanding the cellular mechanisms that underpin the disorder. This will allow patients to have a greater understanding of the cause of their disorder, and why those with one mutation type may differ from another. Ultimately the aim of this work is to develop novel therapeutic strategies that will benefit patient care and potentially lead to the beginnings of personalized medicine.

2. Individuals affected by dystonia
The findings and understanding from this project will have implications for the wider spectrum of dystonic disorders. Points of commonality of pathways and mechanisms may provide areas to target in drug development. Results from this work are also likely to facilitate greater understanding from systems based approaches, such as animal models and human imaging studies.

3. Healthcare Professionals
An improved understanding of cellular mechanisms and underlying pathophysiology will allow clinicians (and other healthcare professionals) to better explain the disorder to their patients. It may also provide some explanation as to why some currently used therapies are more effective in some patients than others, and allow more targeted guiding of treatment.

4. Researchers
Successful development of this neuronal model would be a significant contribution to this field of research. It would provide valuable insights and help guide the work of the International Myoclonus Dystonia Study Group (of which I am a member), facilitate greater understanding of the findings from more systems based approaches, as well as impact the research into other neurodevelopmental disorders.

5. Development of therapeutics
Although beyond the scope of this study, the long-term aim of this project is the identification of novel therapeutic targets and development of large scale clinical trials. This would represent a significant outcome for a group of disorders whose current treatments are extremely limited.

6. Professionals involved with the project
This project will form a platform for the development of an exciting and cutting edge research group at Cardiff University, focused on the development of translational models and effective therapeutics for patients with dystonia. We would form one of very few groups internationally with this as their primary focus, and contribute significantly to the many ongoing collaborative initiatives.

7. Wider society
Dystonia is the third most common form of movement disorder with an estimated prevalence of 1/900 (dystonia.org.uk) and significant social, educational and health economic implications. Improved therapeutic strategies could have a widespread impact on these and workforce employment. Positive results in this project would also raise the public profile of Dystonia and potentially lead to further fundraising and charity-led funding of research. Policy makers have recognized the importance of this with the establishment of the European COST Dystonia initiative (BMBS COST Action BM1101) and the NIH funded Dystonia Coalition in North America (rarediseasesnetwork.org).

Publications

10 25 50
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Carroll LS (2018) Dentatorubral-pallidoluysian Atrophy: An Update. in Tremor and other hyperkinetic movements (New York, N.Y.)

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Peall KJ (2017) Low CSF 5-HIAA in Myoclonus Dystonia. in Movement disorders : official journal of the Movement Disorder Society

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Smith MD (2018) Repurposed drugs for use in Parkinson's disease. in Journal of neurology

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Van Egmond ME (2017) A post hoc study on gene panel analysis for the diagnosis of dystonia. in Movement disorders : official journal of the Movement Disorder Society

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Van Egmond ME (2019) Variable Interpretation of the Dystonia Consensus Classification Items Compromises Its Solidity. in Movement disorders : official journal of the Movement Disorder Society

 
Description Medical Advisor to The Dystonia Society UK
Geographic Reach National 
Policy Influence Type Participation in a advisory committee
Impact Involvement with The Dystonia Society has highlighted the shortfall in therapeutic services provided to patients with Dystonia in Wales. This has lead to discussions with clinical leads and Heath Board directors in several of the regions to review access to services, delivery and future development. This is an ongoing advisory role.
 
Description Member of the International Myoclonus Dystonia Study Group
Geographic Reach Multiple continents/international 
Policy Influence Type Membership of a guideline committee
 
Description A comprehensive assessment of motor and non-motor symptoms in primary blepharospasm to aid stratification of disorder phenotypes
Amount £15,000 (GBP)
Organisation Fight for Sight 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2019 
End 01/2020
 
Description Dystonia Medical Research Foundation
Amount $70,000 (USD)
Organisation Dystonia Medical Research Foundation Canada (DMRFC) 
Sector Charity/Non Profit
Country Canada
Start 04/2017 
End 04/2019
 
Description KESS2 East PhD Fellowship
Amount £70,000 (GBP)
Organisation European Union 
Sector Public
Country European Union (EU)
Start 07/2019 
End 07/2022
 
Description Myoclonus Dystonia Project Grant
Amount $15,000 (USD)
Funding ID 512415 
Organisation Dystonia Medical Research Foundation Canada (DMRFC) 
Sector Charity/Non Profit
Country Canada
Start 04/2017 
End 12/2018
 
Description Neuroscience and Mental Health Research Institute Seedcorn Funding
Amount £5,000 (GBP)
Organisation Cardiff University 
Department Neuroscience and Mental Health Research Institute
Sector Academic/University
Country United Kingdom
Start 09/2017 
End 07/2018
 
Title Establishment on an international Myoclonus Dystonia Registry and online non-motor symptoms study 
Description This is an entirely electronic registry and study, whereby individuals are able to registry and consent to involvement via an online portal. For those over 18 years of age we have developed an online series of questionnaires covering memory, mood, sleep, pain and quality of life to be able to be completed at their convenience. This portal also facilitates repeated annual information collection to allow of longitudinal data collection. 
Type Of Material Database/Collection of data 
Year Produced 2018 
Provided To Others? Yes  
Impact This registry is available to the International Myoclonus Dystonia Study Group to help guide the future direction of work. 
 
Description Collaboration with Betsi Cadwaladr University Health Board 
Organisation Betsi Cadwaladr University Health Board
Department Oncology
Country United Kingdom 
Sector Hospitals 
PI Contribution Betsi Cadwaladr University Health Board have been established as a further recruitment site for our longitudinal dystonia phenotypic study. This has involved the research delivery network within Wales.
Collaborator Contribution Our partners are involved in participant identification, recruitment and blood sample collection.
Impact We are anticipating several future publications.
Start Year 2019
 
Description Collaboration with Dr Manju Kurian's group - UCL 
Organisation University College London
Department Institute of Child Health
Country United Kingdom 
Sector Academic/University 
PI Contribution The genetic findings from this work have provided some input to the ongoing work of this group. It has also led to further work between the two groups on CSF neurotransmitter analysis.
Collaborator Contribution Dr Kurian's group principally focuses on the identification of novel disease-causing genes and their translation into cellular and animal models, mainly in relation to disruption to monoaminergic neurotransmission and movement disorders. Collaboration with group has greatly helped with development of my skills and interpretation of whole exam sequencing analysis.
Impact Outputs include one article in submission and another in preparation.
Start Year 2014
 
Description Collaboration with Movement Disorders Research and Clinical Teams in Dublin, Ireland 
Organisation Trinity College Dublin
Department Neurology
Country Ireland 
Sector Academic/University 
PI Contribution I was invited to speak at one of their lay information days in Dublin. From here we realised that there was significant overlap in the work that we were doing, and that there would be significant value in working together. We are currently looking to gain ethical approval for our work in Ireland.
Collaborator Contribution They have been involved in participant identification, and notification of study to patients who potentially ma be interested in participating.
Impact Nil as yet.
Start Year 2018
 
Description Collaboration with Paediatric Movement Disorders Team, Barcelona, Spain 
Organisation Hospital Sant Joan de Deu
Country Spain 
Sector Hospitals 
PI Contribution We ar working with the team in Barcelona to facilitate participation of the Spanish Myoclonus Dystonia community to participate in the international registry and online study.
Collaborator Contribution They have provided assistance with local ethical applications and ensuring that the language of the questionnaires is appropriate for a clinical study. They have also actively advertised the study to their patients and the wider neurological community.
Impact No formal outcomes as yet. We anticipate several high-impact publications.
Start Year 2017
 
Description Collaboration with Professor Marina Tijssen, The Netherlands. 
Organisation University Medical Center Gronigen
Country Netherlands 
Sector Hospitals 
PI Contribution We are collaborating in developing an international database of patients with Myoclonus Dystonia. In addition a PhD student from The Netherlands will be visiting Cardiff to undertake further clinical research.
Collaborator Contribution Data and visiting PhD student.
Impact No outcomes as yet.
Start Year 2017
 
Description Collaboration with Sydney Adult and Paediatric Movement Disorders Teams as part of the Global MD Registry 
Organisation University of Sydney
Department Obstetrics Sydney
Country Australia 
Sector Academic/University 
PI Contribution We have instigated this collaborative partnership as part of the Global Myoclonus Dystonia Registry, for which we are the lead centre. The local teams are involved in identifying potential participants and directing them towards are website, where they are able to consent to involvement and complete a series of questionnaires.
Collaborator Contribution Identificaion of study participants, and advertisement of the study throughout Australia.
Impact No outputs as yet, although we anticipate a number of publications one the registry has completed the first full data set.
Start Year 2018
 
Description The International Myoclonus Dystonia Study Group 
Organisation Dystonia Medical Research Foundation Canada (DMRFC)
Country Canada 
Sector Charity/Non Profit 
PI Contribution This is a recently established group. I was invited to speak at the Dystonia Medical Research Foundation event on Myoclonus Dystonia (Miami, USA February 2016), talk entitled: Psychiatric co-morbidty in Myoclonus Dystonia
Collaborator Contribution This group is made up of the principal movement disorder centres throughout the US, Canada, France, Netherlands and the UK. A commitment to regular meetings has been made. Funding is now available as seed corn grants every two years for projects that relate directly to Myoclonus Dystonia. This group also has strong links with the lay community and the Dystonia Coalition (NIH Rare Disorders project)
Impact Outcomes: 1. Clinical update to be reported in Movement Disorders Clinical Practice journal 2. Meeting set for June 2016 3. Development of therapeutic guidelines
Start Year 2016
 
Description Lay Advisory Group for ongoing research projects 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Patients, carers and/or patient groups
Results and Impact We are establishing a lay advisory group to work alongside the movement disorder research group, to provide advice, reading of participant information and develop research ideas.
Year(s) Of Engagement Activity 2016,2017
 
Description Non-motor symptoms in Dystonia (DBS Conference, London) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Presentation of the evidence for non-motor symptoms in dystonia, the more common symptom groups that are identified and how best to ensure that these are not missed in routine clinical practice. Talk given to >100 professionals working in the DBS field (predominantly specialist nurses), industry partners and members of lay organisations.
Year(s) Of Engagement Activity 2018
 
Description Parkinson's UK Excellence Group meting - lay and professional members 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact Talk given about genetic testing in Parkinson's disease. This needed to cover a broad group of attendees including patients, their relatives, medical practitioners in general and professional fields.
Year(s) Of Engagement Activity 2017
 
Description Talk given at Parkinson's Disease Specialist Nurses Annual Meeting 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Talk given regarding the genetic underpinning of Parkinson's Disease, and how and when to undertake genetic testing. This was to a national audience of Parkinson's Disease Specialist Nurses.
Year(s) Of Engagement Activity 2018
 
Description Talk on non-motor symptoms in primary dystonia to lay meeting organised by Dystonia Ireland. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Patients, carers and/or patient groups
Results and Impact I gave a talk giving an overview of our previous research into the non-motor symptoms in dystonia, our findings and how this had influenced our current longitudinal study. This was also an opportunity to establish further collaborations, and recruitment of participants to our study.
Year(s) Of Engagement Activity 2018
 
Description Talk to the British Neurotoxin Network Annual Meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact I gave a talk at the annual BNN meeting discussing our ongoing research, and in particular our longitudinal study exploring the non-motor phenotype in primary dystonia.
Year(s) Of Engagement Activity 2018