MRC Transition Support award CSF Kathryn Peall

先前研究的目的包括从诊断为肌阵挛性肌张力障碍的患者收集的组织样本中开发神经细胞模型。使用这些模型，工作的目的是详细了解这些神经细胞的发育和功能，特别是与神经递质（允许神经之间交流的化学物质）多巴胺的关系，以及这些变化如何引起肌挛缩性肌张力障碍和肌张力障碍。研究背景及其重要性肌张力障碍是最常见的运动障碍之一，每900人中就有1人受到影响，并与严重的终身残疾有关，目前尚无有效的治疗方法。肌阵挛性肌张力障碍是肌张力障碍的一种遗传形式，是由特定基因（SGCE）突变引起的少数类型之一，使其成为研究肌张力障碍潜在机制的合适疾病。来自人脑成像研究和动物模型的证据表明，皮层中的神经元以及mDA和msn之间的通信（突触）形成了肌张力障碍中神经元破坏的关键区域。迄今为止的进展包括开发三种患者细胞系，以及对其SGCE突变进行基因校正，以提供“正常”（野生型）基因匹配的对照（可比较）样本。为了进一步证明我们观察到的变化是由SGCE突变引起的，我们对一种胚胎干细胞系（经常用于研究）进行了基因编辑，去除了SGCE基因的两个拷贝。对这些细胞系的研究表明，没有SGCE的皮质神经元更容易兴奋（电活性），并且具有更复杂的分支模式，而mDA和MSN表明大脑中这些细胞类型的相对比例和性质发生了发育变化。已开发的mDA和MSN细胞系在单个细胞水平（膜片钳技术）和细胞网络（多电极阵列）上进行详细的电分析。影响多巴胺能神经传递的化合物将被用来确定多巴胺水平高低的影响，以及多巴胺受体和转运体活性的增加和减少。由于SGCE突变导致的基因表达变化将使用rna测序进行分析。该方法使用在细胞发育过程中特定时间点提取的遗传物质（RNA），然后评估这些基因在每个阶段的相对表达，指出在驱动导致肌阵挛性肌张力障碍的机制中重要的基因和基因途径。最后，mDA和msn将一起培养，使不同类型的细胞分别生长，但彼此形成突触（通信）。我们将使用有或没有SGCE突变的细胞类型组合来确定这些变化是由于神经元内部的差异还是受到周围分子的影响。这些详细的见解将提高我们对肌张力障碍症状出现的原因的理解，帮助开发新的治疗方法。临床科学家奖学金为我提供了一个重要的平台，让我开始发展自己的独立研究小组，并提高了我在肌张力障碍研究领域的国际领导者地位。然而，这种可信性很大程度上仍然与我的临床专业知识有关。这段额外的支持期将使我能够发表迄今为止的干细胞工作，并完成并发表剩余的工作，不仅对该领域做出了重大贡献，而且还使我在干细胞生物学方面不断增长的专业知识得到认可和发展。为了最大限度地利用这项工作，通过进一步的拨款和高级奖学金申请，以及有意义的临床应用，这是必不可少的。

Aims of previous fellowshipThese 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 importanceDystonia 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 dateThis 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 awardThe 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 leadershipThe 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.