Dopamine (DA) signaling in the CNS is essential for modulating complex behaviors such as movement, cognition, reward, and motivation. Aberrant DAergic transmission is directly linked to Parkinson's disease, attention-deficit hyperactivity disorder, schizophrenia and addiction. The plasma membrane dopamine transporter (DAT) is central to DAergic signaling, and both limits extracellular DA availability and maintains presynaptic DA stores. Importantly, DAT is the primary target for both addictive and therapeutic psychoactive drugs, such as cocaine, amphetamine, methylphenidate (Ritalin), and the antidepressant bupropion (Wellbutrin), all of which competitively inhibit DAT activity. A wealth of data supports the premise that DAT constitutively traffics to and from the cell surface, and that protein kinase C (PKC) activation rapidly decreases DAT cell surface expression by acutely modulating DAT trafficking rates. While some progress has been made investigating the molecular mechanisms that govern regulated DAT trafficking, a detailed understanding of this complex process has yet to be achieved. Further, the physiological relevance that DAT trafficking imposes onto DAergic function is poorly understood. Our laboratory reported that neuronal GTPase, Rin (RIT2), specifically binds to the DAT carboxy terminus and is required for PKC-stimulated DAT internalization. In the proposed studies, I plan to investigate the molecular underpinnings required for DAT/Rin interactions, and how these interactions potentially mediate synergy between cytosolic DAT domains.
Aim 1 studies will use co-immunoprecipitations and FRET microscopy approaches with chimeric DAT proteins to define the intracellular DAT domains that are necessary and sufficient to confer DAT/Rin interactions. Chimeric and point mutant transporters will further reveal the potential synergistic requirement of DAT amino- and carboxy-termini for PKC-stimulated DAT internalization.
Aim 2 studies will directly test the potential role of Rin-mediated DAT trafficking in DAergic behaviors in vivo, using cell-specific, AAV2-mediated Rin knockdown in DAergic terminal regions. Taken together, these studies will provide the first investigations that directly examine how DAT trafficking potentially impacts rewarding behavior. Moreover, completion of this investigative line will provide me with outstanding training in molecular and behavioral neuroscience.
Psychotic drugs are often used as therapeutic agents, such as methylphenidate prescriptions for ADHD, and the number of prescriptions for these drugs has increased drastically over the past few decades in the United States. Interestingly, psychostimulant abuse rates have also increased over this same time frame. While prescription psychotic drugs have clear therapeutic benefits, the abuse potential for these drugs in addition to other drugs of abuse, such as cocaine and amphetamines, remains a chronic problem in our county. Further, the mechanisms underlying the rewarding properties for abusive psychostimulants remain poorly understood. The proposed studies will directly examine the molecular targets in the brain of these therapeutic and addictive psychoactive drugs. My hope is that the results obtained will lead to greater understanding of the molecular underpinnings behind psychostimulant-induced reward. Having a greater understanding of these mechanisms will hopefully lead to novel therapeutic approaches for treating abuse disorders.
|Sweeney, Carolyn G; Tremblay, Bradford P; Stockner, Thomas et al. (2017) Dopamine Transporter Amino and Carboxyl Termini Synergistically Contribute to Substrate and Inhibitor Affinities. J Biol Chem 292:1302-1309|