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

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...