Functional validation of a role for the candidate gene Ctr9 in psychostimulant action

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The mechanisms that mediate substance abuse liability are incompletely understood and therapeutic interventions for psychostimulant addiction remain largely ineffective. We conducted a genome-wide association study in Drosophila and identified the gene Ctr9 to be significantly associated with the locomotor response to amphetamine. Ctr9 is known to play a role in regulating gene expression as part of the Paf1C transcription complex. It was also shown in one publication to interact with the dopamine transporter, the major molecular target of psychostimulants. We have validated the role of Ctr9 in amphetamine-induced hyperactivity in flies; dopamine neuron-specific knockdown of Ctr9 leads to a dramatic increase in the response to amphetamine and methamphetamine, whereas its overexpression blunts the response. We have also shown that Ctr9 co-localizes with dopamine transporter (DAT) outside of the nucleus in dopamine neurons in the adult fly brain. We now aim to characterize the mechanisms underlying its effects on dopamine neurotransmission and methamphetamine- induced behavior in Drosophila and to develop a genetic model to functionally validate the role of Ctr9 in methamphetamine- and cocaine-induced hyperlocomotion in rats, which are uniquely suited to model traits that mimic compulsive drug-seeking behavior in humans and will thus facilitate our long-term goal to investigate the role of Ctr9 in addiction-like behavior. In pursuit of these goals, we propose the following specific aims: (1) To elucidate the mechanisms by which Ctr9 modulates methamphetamine-induced locomotor activity in flies. (a) We will use 2-photon imaging of a dopamine biosensor to determine the impact of Ctr9 knockdown and overexpression on optogenetically-evoked and methamphetamine-induced dopamine release. (b) To determine if the nuclear localization of Ctr9 is essential for its role in dopamine-mediated locomotor activity in Drosophila, we will express a truncated Ctr9 protein that is blocked from translocating to the nucleus (Ctr9ΔSH2) and test its impact on basal and methamphetamine-induced locomotion. (c) We will knock down the expression of Ctr9 strictly during adulthood, and at different times during development, to determine if Ctr9 knockdown interferes with early neurodevelopment to influence susceptibility to methamphetamine later in life or if it acts acutely during adulthood to modulate methamphetamine-induced behavior. (2) To establish a role for Ctr9 in the behavioral response to methamphetamine in rats and determine its impact on dopamine neurotransmission and DAT function. We will (a) generate a conditional Ctr9 knockout rat using a CRISPR/Cas9-mediated “Germline Gene Targeting” approach and (b) test the effects of deleting Ctr9 selectively in dopamine neurons on basal and methamphetamine- and cocaine-induced hyperactivity. (c) We will test the impact of selective knockout of Ctr9 in dopamine neurons on DAT cell-surface localization, evoked dopamine release, uptake and methamphetamine-induced dopamine efflux in striatal brain slices.