MRC Transition Support award CSF Kathryn Peall

Aims of previous fellowship
These included development of nerve cell models from tissue samples collected from patients diagnosed with Myoclonus Dystonia. Using these models, work was aimed at detailed understanding of the development and function of these nerve cells, particularly in relation to the neurotransmitter (chemical allowing communication between nerves) dopamine, how these changes might give rise to Myoclonus Dystonia and dystonic disorders.

Background to the research and its importance
Dystonia is one of the most common movement disorders, affecting 1 in 900 people, and is associated with significant lifetime disability for which there are no current effective treatments. This project is focused on Myoclonus Dystonia, an inherited form of dystonia and one of the few types caused by mutations to a specific gene (SGCE), making it an opportune disorder in which to study the underlying mechanisms of dystonia. Evidence from human brain imaging studies and animal models indicate that neurons in the cortex, as well as the communication (synapse) between mDA and MSNs form key areas of neuronal disruption in dystonia.

Progress to date
This has included developing three patient cell lines, as well as genetically correcting their SGCE mutations to provide a 'normal' (wildtype) genetically matched control (comparable) sample. To provide further evidence that the changes we're observing are due to SGCE mutations, we have genetically edited an embryonic stem cell line (often used in research), removing both copies of the SGCE gene. Studies of these cell lines have shown cortical neurons without SGCE to be more excitable (electrically active) and to have a more complex branching pattern, while the mDA and MSN suggest developmental changes in the relative proportions and nature of these cell types in the brain.

Work planned for transition award
The already developed mDA and MSN cell lines with undergo detailed electrical analysis on an individual cell level (patch-clamp technique) and as a network of cells (multi-electrode array). Chemical compounds that affect dopaminergic neurotransmission will be used to determine the effect of higher and lower levels of dopamine, as well as increasing and decreasing the activity of dopamine receptors and transporters.
Changes in gene expression as a result of the SGCE mutations will be analysed using RNA-sequencing. This uses genetic material (RNA) taken from the cells at specific time points during their development and then evaluating the relative expression of these genes at each stage, indicating genes and gene pathways that are important in driving the mechanisms that cause Myoclonus Dystonia.
Finally, both mDA and MSNs will be cultured together, allowing the different cell types to grow separately but form synapses (communicate) with each other. We will use combinations of the cell types, with and without SGCE mutations, to determine if the changes are due to differences within the neurons or are influenced by surrounding molecules. These detailed insights will improve our understanding of why dystonia symptoms arise, aiding the development of new therapies.

Platform towards research leadership
The Clinician-Scientist fellowship has provided me with a significant platform to begin to develop my own independent research group, as well as increase my recognition as an international leader within the dystonia research field. However, much of this credibility remains linked with my clinical expertise. This additional period of support would allow me to publish the stem cell work to date, as well as to complete and publish the remaining work, not only making a significant contribution to the field but also allowing recognition and development of my growing expertise in stem cell biology. This is essential in order to maximally capitalise on this work, through further grants and senior fellowship applications, as well as meaningful clinical applications.

Aims
1.Functional characterisation of midbrain dopaminergic (mDA) and striatal medium spiny neurons (MSNs) using already established Myoclonus Dystonia (SGCE mutations) patient derived and genetically edited embryonic cell lines.
2.Transcriptomic analysis of differentially expressed genes in mDA and MSN monocultures.
3.mDA and MSN co-culture evaluation combining wild-type (WT) and knock-out (KO) lines.
Background
Dystonia is a common and disabling movement disorder. Poor understanding of its pathophysiology has led to few effective treatments. MD is one of few genetically determined dystonias caused by autosomal dominant mutations, providing a unique opportunity for investigation. Previous work suggests network disruption underlies dystonia pathogenesis with the nigro-striatal junction and cerebral cortex being regions of importance. Work to date with this fellowship has demonstrated a hyperexcitable and more elaborate dendritic arbor phenotype in cortical neuronal cells, and developmental phenotype in mDA and MSNs lines.
Methodology
mDA and MSN neuronal cultures will undergo electrophysiological analysis using single cell patch-clamp and network-based multi-electrode array approaches. In addition to determining baseline excitability, chemical compounds will be used to manipulate components of the dopaminergic neurotransmission pathway (e.g. Dopamine transporter inhibition with nomifensine). Differential gene expression, using RNA-seq, will provide further insights into the impact of loss of epsilon-sarcoglycan expression. Finally, mDA and MSN co-cultures involving WT/WT, KO/KO and WT/KO combinations will provide further understanding of the contributions of intrinsic neuronal and exogenous factors.
Scientific and Medical opportunities
This functional characterisation will provide detailed insights into the cellular mechanisms in key brain regions underpinning dystonia, informing future studies including identification of novel therapeutic targets.