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

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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.