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

多巴胺转运蛋白缺乏综合征（DTDS）是由人类多巴胺转运蛋白DAT/SLC 6A 3突变引起的常染色体隐性帕金森病。SLC 6A 3是在突触前膜处偶联多巴胺内化和Na+/Cl-转运的膜同向转运蛋白，并且是与包括自闭症谱系障碍（ASD）和ADHD的各种人类神经障碍相关的多巴胺能神经传递的主要调节剂。最近，10个新的错义变异已被确定在患者提出了更广泛的帕金森病表型与不同的发病和疾病的严重程度。然而，这些突变对SLC 6A 3的分子功能的影响及其对多巴胺能信号传导的生物体范围的后果目前尚不清楚。为了研究SLC 6A 3相关的疾病机制，我们将通过将7个新的错义变体CRISPR-Cas工程化到内源性秀丽隐杆线虫直向同源物dat-1中来建立DTDS的新型体内模型。在人类中，多巴胺调节一系列行为反应和运动控制，其中大部分在进化过程中是保守的。学生将验证DTDS dat-1突变对多巴胺控制的行为和运动功能的影响，并监测C.优雅为了验证他们的发现，学生将测试选定疾病SLC 6A 3变体在DTDS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）中发育。学生将通过研究SLC 6A 3功能，多巴胺毒性，神经变性和突变多巴胺能细胞中的炎症来评估疾病的细胞机制。这将通过治疗多巴胺能神经元和C。目前，用药物分子伴侣构建线虫模型已经有效地挽救了疾病相关的SLC 6A 3折叠缺陷，DTDS的建模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并不容易转化为人类大脑的帕金森症表型。小鼠Slc 6a 3/DAT敲除分析，其中每年发表3-4项研究，使用约100只小鼠/研究（PMID：34011628），强调了SLC 6A 3在多巴胺能脑功能中的重要作用，但没有传达关于患者错义变体的数据。敲入（KI）方法，提供更准确的数据，病人的等位基因，最近获得了动力，迄今为止，已产生5 KI小鼠模型，研究的影响，错义变异体在SLC 6A 3。这些研究使用估计300只小鼠/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鉴定的越来越多的SLC 6A 3变体的影响，使得功能分析和潜在治疗方法的开发缓慢且低效。在此，我们建议将7种新的疾病变体快速引入C。elegans的SLC 6A 3直向同源物，取代了开发KI小鼠模型的需要。在线虫模型中获得的结果将转化为患者iPSC模型，用于立即的治疗研究。因此，我们估计在该项目中，在主办机构更换2100只动物。当C.如果将elegans KI模型应用于其他SLC 6A 3相关的神经系统疾病，如ADHD或ASD，我们的替代策略将具有更广泛的3R影响，从而进一步减少小鼠的使用，并有可能在全球SLC 6A 3相关疾病研究中每年替代300-400只动物。考虑到目前还没有DTDS的治疗方法，快速体内建模与药理学伴侣和iPSC相结合将提供一种强有力的方法，有助于我们了解新型SLC 6A 3变体在患者中的功能后果。

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.