Dopamine transporter trafficking and Rit2 GTPase: Mechanism of action and in vivo impact.

Following its evoked release, dopamine (DA) signaling is rapidly terminated by presynaptic reuptake, mediated by the cocaine-sensitive DA transporter (DAT). DAT surface availability is dynamically regulated by endocytic trafficking, and direct protein kinase C (PKC) activation acutely diminishes DAT surface expression by accelerating DAT internalization. Previous cell line studies demonstrated that PKC-stimulated DAT endocytosis requires both Ack1 inactivation, which releases a DAT-specific endocytic brake, and the neuronal GTPase, Rit2, which binds DAT. However, it is unknown whether Rit2 is required for PKC-stimulated DAT endocytosis in DAergic terminals or whether there are region- and/or sex-dependent differences in PKC-stimulated DAT trafficking. Moreover, the mechanisms by which Rit2 controls PKC-stimulated DAT endocytosis are unknown. Here, we directly examined these important questions. Ex vivo studies revealed that PKC activation acutely decreased DAT surface expression selectively in ventral, but not dorsal, striatum. AAV-mediated, conditional Rit2 knockdown in DAergic neurons impacted baseline DAT surface:intracellular distribution in DAergic terminals from female ventral, but not dorsal, striatum. Further, Rit2 was required for PKC-stimulated DAT internalization in both male and female ventral striatum. FRET and surface pulldown studies in cell lines revealed that PKC activation drives DAT-Rit2 surface dissociation and that the DAT N terminus is required for both PKC-mediated DAT-Rit2 dissociation and DAT internalization. Finally, we found that Rit2 and Ack1 independently converge on DAT to facilitate PKC-stimulated DAT endocytosis. Together, our data provide greater insight into mechanisms that mediate PKC-regulated DAT internalization and reveal unexpected region-specific differences in PKC-stimulated DAT trafficking in bona fide DAergic terminals.

Conditional, inducible gene silencing in dopamine neurons reveals a sex-specific role for Rit2 GTPase in acute cocaine response and striatal function.

Dopamine (DA) signaling is critical for movement, motivation, and addictive behavior. The neuronal GTPase, Rit2, is enriched in DA neurons (DANs), binds directly to the DA transporter (DAT), and is implicated in several DA-related neuropsychiatric disorders. However, it remains unknown whether Rit2 plays a role in either DAergic signaling and/or DA-dependent behaviors. Here we leveraged the TET-OFF system to conditionally silence Rit2 in Pitx3IRES2-tTA mouse DANs. Following DAergic Rit2 knockdown (Rit2-KD), mice displayed an anxiolytic phenotype, with no change in baseline locomotion. Further, males exhibited increased acute cocaine sensitivity, whereas DAergic Rit2-KD suppressed acute cocaine sensitivity in females. DAergic Rit2-KD did not affect presynaptic TH and DAT protein levels in females, nor was TH was affected in males; however, DAT was significantly diminished in males. Paradoxically, despite decreased DAT levels in males, striatal DA uptake was enhanced, but was not due to enhanced DAT surface expression in either dorsal or ventral striatum. Finally, patch recordings in nucleus accumbens (NAcc) medium spiny neurons (MSNs) revealed reciprocal changes in spontaneous EPSP (sEPSP) frequency in male and female D1+ and D2+ MSNs following DAergic Rit2-KD. In males, sEPSP frequency was decreased in D1+, but not D2+, MSNs, whereas in females sEPSP frequency decreased in D2+, but not D1+, MSNs. Moreover, DAergic Rit2-KD abolished the ability of cocaine to reduce sEPSP frequency in D1+, but not D2+, male MSNs. Taken together, our studies are among the first to acheive AAV-mediated, conditional and inducible DAergic knockdown in vivo. Importantly, our results provide the first evidence that DAergic Rit2 expression differentially impacts striatal function and DA-dependent behaviors in males and females.

Convergent Balancing Selection on the Mu-Opioid Receptor in Primates.

The mu opioid receptor is involved in many natural processes including stress response, pleasure, and pain. Mutations in the gene also have been associated with opiate and alcohol addictions as well as with responsivity to medication targeting these disorders. Two common and mutually exclusive polymorphisms have been identified in humans, A118G (N40D), found commonly in non-African populations, and C17T (V6A), found almost exclusively in African populations. Although A118G has been studied extensively for associations and in functional assays, C17T is much less well understood. In addition to a parallel polymorphism previously identified in rhesus macaques (Macaca mulatta), C77G (P26R), resequencing in additional non-human primate species identifies further common variation: C140T (P47L) in cynomolgus macaques (Macaca fascicularis), G55C (D19H) in vervet monkeys (Chlorocebus aethiops sabeus), A111T (L37F) in marmosets (Callithrix jacchus), and C55T (P19S) in squirrel monkeys (Saimiri boliviensis peruviensis). Functional effects on downstream signaling are observed for each of these variants following treatment with the endogenous agonist ß-endorphin and the exogenous agonists morphine, DAMGO ([d-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin), and fentanyl. In addition to demonstrating the importance of functional equivalency in reference to population variation for minority health, this also shows how common evolutionary pressures have produced similar phenotypes across species, suggesting a shared response to environmental needs and perhaps elucidating the mechanism by which these organism-environment interactions are mediated physiologically and molecularly. These studies set the stage for future investigations of shared functional polymorphisms across species as a new genetic tool for translational research.

Dopamine Transporter Amino and Carboxyl Termini Synergistically Contribute to Substrate and Inhibitor Affinities.

Extracellular dopamine and serotonin concentrations are determined by the presynaptic dopamine (DAT) and serotonin (SERT) transporters, respectively. Numerous studies have investigated the DAT and SERT structural elements contributing to inhibitor and substrate binding. To date, crystallographic studies have focused on conserved transmembrane domains, where multiple substrate binding and translocation features are conserved. However, it is unknown what, if any, role the highly divergent intracellular N and C termini contribute to these processes. Here, we used chimeric proteins to test whether DAT and SERT N and C termini contribute to transporter substrate and inhibitor affinities. Replacing the DAT N terminus with that of SERT had no effect on DA transport Vmax but significantly decreased DAT substrate affinities for DA and amphetamine. Similar losses in uptake inhibition were observed for small DAT inhibitors, whereas substituting the DAT C terminus with that of SERT affected neither substrate nor inhibitor affinities. In contrast, the N-terminal substitution was completely tolerated by the larger DAT inhibitors, which exhibited no loss in apparent affinity. Remarkably, all affinity losses were rescued in DAT chimeras encoding both SERT N and C termini. The sensitivity to amino-terminal substitution was specific for DAT, because replacing the SERT N and/or C termini affected neither substrate nor inhibitor affinities. Taken together, these findings provide compelling experimental evidence that DAT N and C termini synergistically contribute to substrate and inhibitor affinities.

Quantitative molecular assessment of chimerism across tissues in marmosets and tamarins.

BACKGROUND: Marmosets are playing an increasingly large and important role in biomedical research. They share genetic, anatomical, and physiological similarities with humans and other primate model species, but their smaller sizes, reproductive efficiency, and amenability to genetic manipulation offer an added practicality. While their unique biology can be exploited to provide insights into disease and function, it is also important that researchers are aware of the differences that exist between marmosets and other species. The New World monkey family Callitrichidae, containing both marmoset and tamarin species, typically produces dizygotic twins that show chimerism in the blood and other cells from the hematopoietic lineage. Recently, a study extended these findings to identify chimerism in many tissues, including somatic tissues from other lineages and germ cells. This has raised the intriguing possibility that chimerism may play an increasingly pervasive role in marmoset biology, ranging from natural behavioral implications to increased variability and complexity in biomedical studies. RESULTS: Using a quantitative PCR based methodology, Y-chromosomes can be reliably detected in the females with male fraternal twins allowing for a relative quantification of chimerism levels between individuals and tissues. With this approach in common marmosets (Callithrix jacchus) and cotton-top tamarins (Saguinus oedipus), chimerism was detected across a broad array of tissues. Chimerism levels were significantly higher in tissues primarily derived from the hematopoietic lineage, while they were lower, though still detectable, in tissues with other origins. Interestingly, animals with a characteristic marmoset wasting disease show higher levels of chimerism in those tissues affected. Fibroblast cell lines from chimeric individuals, however, are not found to be chimeric themselves. CONCLUSION: Taken together, the levels of chimerism in tissues of different origins coupled with other lines of evidence suggest that indeed only hematopoietic cell lineages are chimeric in callitrichids. The chimerism detected in other tissues is likely the result of blood or lymphocytic infiltration. Using molecular methods to detect chimerism in a tissue sample seems to have allowed a substantial increase in the ability to detect these minor cell populations.