Dopaminergic Ric GTPase activity impacts amphetamine sensitivity and sleep quality in a dopamine transporter-dependent manner in Drosophila melanogaster

Dopamine (DA) is required for movement, sleep, and reward, and DA signaling is tightly controlled by the presynaptic DA transporter (DAT). Therapeutic and addictive psychostimulants, including methylphenidate (Ritalin; MPH), cocaine, and amphetamine (AMPH), markedly elevate extracellular DA via their actions as competitive DAT inhibitors (MPH, cocaine) and substrates (AMPH). DAT silencing in mice and invertebrates results in hyperactivity, reduced sleep, and blunted psychostimulant responses, highlighting DAT’s essential role in DA-dependent behaviors. DAT surface expression is not static; rather it is dynamically regulated by endocytic trafficking. PKC-stimulated DAT endocytosis requires the neuronal GTPase, Rit2, and Rit2 silencing in mouse DA neurons impacts psychostimulant sensitivity. However, it is unknown whether or not Rit2-mediated changes in psychostimulant sensitivity are DAT-dependent. Here, we leveraged Drosophila melanogaster to test whether the Drosophila Rit2 ortholog, Ric, impacts dDAT function, trafficking, and DA-dependent behaviors. Orthologous to hDAT and Rit2, dDAT and Ric directly interact, and the constitutively active Ric mutant Q117L increased dDAT surface levels and function in cell lines and ex vivo Drosophila brains. Moreover, DAergic RicQ117L expression caused sleep fragmentation in a DAT-dependent manner but had no effect on total sleep and daily locomotor activity. Importantly, we found that Rit2 is required for AMPH-stimulated DAT internalization in mouse striatum, and that DAergic RicQ117L expression significantly increased Drosophila AMPH sensitivity in a DAT-dependent manner, suggesting a conserved impact of Ric-dependent DAT trafficking on AMPH sensitivity. These studies support that the DAT/Rit2 interaction impacts both baseline behaviors and AMPH sensitivity, potentially by regulating DAT trafficking.