Investigation of dopamine transporter dysfunction linked to DAT/SLC6A3 mutations in CRISPR-Cas engineered C. elegans and patient derived iPSCs

研究 CRISPR-Cas 改造的线虫和患者来源的 iPSC 中与 DAT/SLC6A3 突变相关的多巴胺转运蛋白功能障碍

• 批准号: 2740761
• 金额: --
• 依托单位: University of Reading
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2022
• 资助国家: 英国
• 起止时间: 2022 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2740761
• 关键词: Investigation; dopamine; transporter; dysfunction; linked

Dopamine transporter deficiency syndrome (DTDS) is an autosomal recessive parkinsonian disorder caused by mutations in the human dopamine transporter DAT/SLC6A3. SLC6A3 is a membrane symporter protein that couples dopamine internalisation and Na+/Cl- transport at the presynaptic membrane and is a major regulator of dopaminergic neurotransmission with relevance to various human neurological disorders including autism spectrum disorder (ASD) and ADHD. Recently, 10 new missense variants have been identified in patients presenting a wider spectrum of parkinsonian phenotypes with varying onset and severity of disease. However, the impact of these mutations on the molecular function of SLC6A3 and their organism wide consequences on dopaminergic signalling are currently unknown.To investigate SLC6A3-linked disease mechanisms, we will establish novel in vivo models of DTDS by CRISPR-Cas engineering of 7 new missense variants into the endogenous Caenorhabditis elegans orthologue dat-1. In humans, dopamine regulates a range of behavioural responses and motor control, most of which are conserved throughout evolution. The student will validate the impact of DTDS dat-1 mutations on dopamine-controlled behaviours and motor function and monitor neurotoxin-induced degeneration of dopaminergic neurons in C. elegans. To validate their findings, the student will test the impact of a selected disease SLC6A3 variant in a midbrain dopaminergic neuron model of DTDS, that they will develop from patient derived induced pluripotent stem cells (iPSCs). The student will assess the cellular mechanisms underpinning disease, by investigating SLC6A3 functionality, dopamine toxicity, neurodegeneration and inflammation in the mutant dopaminergic cells. This will be complemented by treatment of the dopaminergic neuron and the C. elegans models with pharmacochaperones, which have been effective in rescuing disease relevant SLC6A3 folding defects.Modelling DTDS mostly relies on human cell culture technologies and mouse models. Testing the alterations of dopamine transport in vitro is not easily translating to Parkinsonian phenotypes of the human brain. Mice Slc6a3/DAT knockout analysis, of which yearly 3-4 studies are published with the use of around 100 mice/study (PMID:34011628), highlighted the essential role of SLC6A3 in dopaminergic brain function but did not convey data on the patients' missense variants. Knock-in (KI) approach, providing more accurate data on patient alleles, has recently gained momentum, and thus far 5 KI mouse models have been generated for investigating the impact of missense variants in SLC6A3. These studies used an estimated 300 mice/per study to generate and characterize the KI models. The lack of fast, cheap in vivo models to assess the impact of the increasing number of SLC6A3 variants identified render the functional analysis and the development of potential therapeutic approaches slow and inefficient. Here we suggest to rapidly introduce 7 new disease variants into the C. elegans orthologue of SLC6A3, replacing the need for developing KI mouse models. The results obtained in the nematode model will be translated into a patient iPSC model for immediate therapeutic research. Thus, we estimate the replacement of 2100 animals within this project at the host institutes. When C. elegans KI models are applied to other SLC6A3-linked neurological conditions, such as ADHD or ASD, our replacement strategy will have wider 3R impact leading to further reduction in use of mice, with potential to replace yearly further 300-400 animals on SLC6A3-linked disease research worldwide. Considering that there is currently no treatment for DTDS, the rapid in vivo modelling combined with pharmacological chaperones and iPSCs will provide a powerful approach to contribute to our understanding of the functional consequences of novel SLC6A3 variants in patients.

多巴胺转运蛋白缺乏综合征（DTDS）是由人多巴胺转运蛋白DAT/SLC6A3引起的常染色体隐性帕金森病。 SLC6A3是一种膜的共物蛋白，在突触前膜上伴侣多巴胺内在化和Na+/Cl-转运，并且是多巴胺能神经传递的主要调节剂，与包括自闭症谱系（ASD）和ADHD在内的各种人类神经疾病（包括自闭症）和ADHD的各种人类神经疾病相关。最近，在帕金森氏症表型更广泛的患者中，已经发现了10种新的错义变体，疾病的发作和严重程度各不相同。但是，这些突变对SLC6A3的分子功能及其生物体对多巴胺能信号的影响的影响目前尚不清楚。为了研究SLC6A3连锁疾病机制，我们将建立7种新型DTDS的新型DTDS，该模型是由7种新的新型工程秀丽隐杆线虫直系同源物DAT-1的错义变体。在人类中，多巴胺调节了一系列行为反应和运动控制，其中大多数在整个进化过程中都是保守的。学生将验证DTDS Dat-1突变对多巴胺控制行为和运动功能的影响，并监测秀丽隐杆线虫中多巴胺能神经元的神经毒素诱导的变性。为了验证他们的发现，学生将测试所选疾病SLC6A3变体在DTD的中脑多巴胺能神经元模型中的影响，它们将从患者衍生的诱导多能干细胞（IPSC）中发展。该学生将通过研究突变多巴胺能细胞中的SLC6A3功能，多巴胺毒性，神经变性和炎症来评估基础疾病的细胞机制。这将通过治疗多巴胺能神经元和具有药剂蛋白的秀丽隐杆线虫模型来补充，这些模型有效地营救了疾病相关的SLC6A3折叠缺陷。模型DTD主要依赖于人类细胞培养技术和小鼠模型。在体外测试多巴胺转运的改变不容易转化为人脑的帕金森氏症表型。小鼠SLC6A3/DAT基因敲除分析，其中每年的3-4个研究通过使用约100只小鼠/研究（PMID：34011628），强调了SLC6A3在多巴胺能脑功能中的重要作用，但没有传达患者的数据。”错义变体。敲入（KI）方法提供了有关患者等位基因的更准确数据的，最近已经获得了动力，到目前为止，已经生成了5种Ki小鼠模型来研究SLC6A3中错义变体的影响。这些研究使用估计的300只小鼠/每项研究来生成和表征Ki模型。缺乏快速，廉价的体内模型来评估越来越多的SLC6A3变体的影响，从而确定了功能分析和潜在治疗方法的发展缓慢且效率低下。在这里，我们建议在SLC6A3的秀丽隐杆线虫直系同源物中迅速引入7种新的疾病变体，以取代开发Ki小鼠模型的需求。线虫模型中获得的结果将转化为患者IPSC模型，以进行立即治疗研究。因此，我们估计在主机机构中，该项目中2100只动物的替代。当秀丽隐杆线虫Ki模型被应用于其他SLC6A3连接的神经系统状况（例如ADHD或ASD）时，我们的替换策略将具有更大的3R影响，从而进一步减少小鼠的使用，并有可能替换每年进一步的300-400只动物对300-400只动物对SLC6A3连接的疾病研究。考虑到目前尚无DTD的治疗方法，结合了药理学伴侣和IPSC的快速体内建模将提供一种有力的方法，以帮助我们理解患者新型SLC6A3变体的功能后果。