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

目标1.使用患者的皮肤细胞（成纤维细胞）开发肌阵挛肌张力障碍的个体神经细胞模型2。神经细胞模型的详细研究，以确定SGCE基因突变对细胞的影响，特别是神经递质（允许神经之间通信的化学物质）多巴胺及其受体。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，每个人口头中有1/900。公认的形式包括书写痉挛，尽管更严重的形式经常被观察到。肌张力障碍与严重的终身残疾有关，对教育和就业产生影响。目前肌张力障碍尚无有效的治疗方法，因此有必要进一步了解潜在的致病机制，以便开发新的和潜在的疾病修饰疗法。本项目的重点是肌阵挛肌张力障碍，这是一种遗传性的儿童期发作的运动障碍，伴有显著的相关精神症状。它是遗传性肌张力障碍的最常见形式之一，也是由特定基因突变（SGCE）引起的少数亚型之一。这些特点使肌阵挛肌张力障碍成为研究肌张力障碍的潜在机制的一种合适的疾病。SGCE基因编码ε-肌聚糖蛋白，其在大脑中的作用以及其功能缺陷如何引起肌张力障碍仍然不确定。来自人脑成像研究和动物模型的证据表明，中脑中产生的多巴胺能神经元和纹状体中的中型棘神经元之间的通信（突触）形成了肌张力障碍神经元破坏的焦点。我以前的研究工作涉及招募肌阵挛肌张力障碍患者的全球最大队列之一。所有这些患者都接受了详细的运动障碍和任何精神症状的检查。我选择了两个病人的成纤维细胞样本（具有不同的基因突变），以产生这些神经细胞models.Design和methodsThe成纤维细胞将被用来产生诱导多能干细胞（iPSC），能够发展成所有组织类型的细胞。使用一系列成熟的技术，这些细胞将被转化为神经细胞，更具体地说是多巴胺能神经细胞（来自中脑）和中等多刺神经细胞（在纹状体中），这两种神经细胞类型被认为在肌张力障碍中很重要。将检查两种神经细胞类型以确定SGCE突变对ε-肌聚糖蛋白的影响。还将对参与产生多巴胺的酶、参与将其转运穿过细胞膜的蛋白质以及在两种细胞类型之间传递其作用信号的受体进行详细研究。实验的第二阶段将涉及将两种细胞类型一起培养，检查它们的电通信和神经元过程的结构。结果将与健康无关个体捐赠的组织产生的神经模型进行比较。我们还计划使用一种新的基因技术（CRISPR）来“编辑”肌阵挛肌张力障碍患者的细胞，使SGCE基因序列恢复正常，以证明在原始神经细胞模型中看到的变化是由于SGCE突变引起的。详细说明这些过程将提高我们对肌张力障碍的理解，并可能出现一些精神症状，以及为开发新药和治疗方案提供平台。

Aims1. To develop individual nerve cell models of Myoclonus Dystonia using skin cells (fibroblasts) from patients2. 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 importanceDystonia 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 methodsThe 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.