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

Abstract

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 Pitx3 IRES2-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.

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

“…PKC activation drives DAT and Rit2 to dissociate at the cell surface, and failure of DAT and Rit2 to dissociate correlates with increased DAT stability at the plasma membrane, and inability to undergo PKC-stimulated endocytosis 43 . Importantly, we also found that conditional Rit2 knockdown (Rit2-KD) in DA neurons significantly increases acute cocaine sensitivity in vivo in male mice 45 . These data raise the possibility that Rit2-mediated changes in psychostimulant sensitivity are due to inability of DAT to undergo regulated trafficking.…”

RIT2 reduces LRRK2 kinase activity and protects against alpha-synuclein neuropathology

“…In fact, the genetic deletion of DAT in rats increases extracellular DA lifetime, resulting in locomotor hyperactivity (Leo et al, 2018). Interestingly, the conditional KO of Rit2 in mouse midbrain DA neurons decreases total DAT protein in the striatum, strengthening the possible modulation of DAT by RIT2 (Sweeney et al, 2019).…”

GeneCup: mine PubMed for gene relationships using custom ontology and deep learning

“…For example, RIT2 is associated with smoking initiation 1 and autism 23 . Recent publications indicated that RIT2 is involved in dopamine transporter trafficking 24 and plays a sex-specific role in acute cocaine response 25 . SYT4 is expressed in the hippocampus and entorhinal cortex 26 and regulates synaptic growth 27,28 .…”

“…Recent publications indicated that RIT2 is involved in dopamine transporter trafficking (Fagan et al . 2020) and plays a sex-specific role in acute cocaine response (Sweeney et al . 2020).…”