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

多巴胺转运体缺陷综合征(DTDS)是一种常染色体隐性遗传性帕金森病，由人类多巴胺转运体DAT/SLC6A3突变引起。SLC6A3是一种膜转运蛋白，结合了突触前膜上的多巴胺内化和Na+/Cl-转运，是多巴胺能神经传递的主要调节因子，与包括自闭症谱系障碍(ASD)和ADHD在内的多种人类神经疾病有关。最近，在帕金森病患者中发现了10个新的错义变异，这些患者表现出更广泛的帕金森表型，具有不同的发病和严重程度。然而，这些突变对SLC6A3分子功能的影响以及它们在生物体范围内对多巴胺能信号转导的影响目前尚不清楚。为了研究SLC6A3连锁的疾病机制，我们将通过CRISPR-Cas将7个新的错义变体工程到内源秀丽线虫同源基因dat-1中，建立新的DTDS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突变对多巴胺控制的行为和运动功能的影响，并监测神经毒素诱导的线虫多巴胺能神经元的退化。为了验证他们的发现，这名学生将在DTDS的中脑多巴胺能神经元模型中测试选定的疾病SLC6A3变体的影响，该模型将从患者诱导的多能干细胞(IPSCs)发育而来。学生将通过研究SLC6A3的功能、多巴胺毒性、神经退化和突变的多巴胺能细胞中的炎症来评估疾病的细胞机制。此外，还将用药冠酮治疗多巴胺能神经元和线虫模型，这些药物在拯救与疾病相关的SLC6A3折叠缺陷方面一直是有效的。DTDS的建模主要依赖于人类细胞培养技术和小鼠模型。在体外测试多巴胺运输的变化并不容易转化为人脑的帕金森表型。小鼠SLC6a3/DAT基因敲除分析每年发表3-4次研究，每项研究使用约100只小鼠(PMID：34011628)，强调了SLC6A3在多巴胺能脑功能中的关键作用，但没有传达关于患者错义变体的数据。敲入(KI)方法提供了关于患者等位基因的更准确的数据，最近获得了势头，到目前为止已经建立了5个KI小鼠模型来研究SLC6A3错义变异的影响。这些研究估计每项研究使用300只小鼠来产生和描述KI模型。由于缺乏快速、廉价的体内模型来评估日益增多的SLC6A3变异的影响，导致功能分析和潜在治疗方法的开发缓慢而低效。在这里，我们建议在线虫SLC6A3的同源基因中迅速引入7个新的疾病变体，以取代开发Ki小鼠模型的需要。在线虫模型中获得的结果将被转化为患者IPSC模型，用于立即进行治疗研究。因此，我们估计，在主办机构的这个项目中，将替换2100只动物。当秀丽线虫Ki模型应用于其他SLC6A3相关神经疾病，如ADHD或ASD时，我们的替代策略将产生更广泛的3R影响，导致进一步减少小鼠的使用，有可能在全球范围内每年替代300-400只动物进行SLC6A3相关疾病研究。考虑到目前还没有治疗DTDS的方法，结合药理伴侣和IPSCs的快速体内建模将为我们提供一种强大的方法来帮助我们理解新的SLC6A3变异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.