Biology subjects

Kearney, P. J.

Publications and source records attributed to Kearney, P. J..

5 recordsLinked to original sources

bioRxiv2023

Rit2 loss in dopaminergic neurons drives a progressive Parkinsonian phenotype

Parkinsons disease (PD) is the second most prevalent neurodegenerative disease and arises from dopamine (DA) neuron death selectively in the substantia nigra pars compacta (SNc). Rit2 is a reported PD risk allele, and recent single cell transcriptomic studies identified a major RIT2 cluster in PD DA neurons, potentially linking Rit2 expression loss to a PD patient cohort. However, it is still unknown whether Rit2 loss itself is causative for PD or PD-like symptoms. Here we report that conditional Rit2 silencing in mouse DA neurons drove motor dysfunction that occurred earlier in males than females and was rescued at early stages by either inhibiting the DA transporter (DAT) or with L-DOPA treatment. Motor dysfunction was accompanied by decreased DA release, striatal DA content, phenotypic DAergic markers, DA neurons, and DAergic terminals, with increased pSer129-alpha synuclein and pSer935-LRRK2 expression. These results provide the first evidence that Rit2 loss is causal for SNc cell death and a PD-like phenotype, and reveal key sex-specific differences in the response to Rit2 loss.

neuroscience↗

bioRxiv2021

Presynaptic Gq-coupled receptors drive biphasic dopamine transporter trafficking that modulates dopamine clearance and motor function

Extracellular dopamine (DA) levels are constrained by the presynaptic DA transporter (DAT), a major psychostimulant target. Despite its necessity for DA neurotransmission, DAT regulation in situ is poorly understood, and it is unknown whether regulated DAT trafficking impacts dopaminergic signaling and/or behaviors. Leveraging chemogenetics and conditional gene silencing, we found that activating presynaptic Gq-coupled receptors, either hM3Dq or mGluR5, drove rapid biphasic DAT membrane trafficking, with region-specific differences in ventral and dorsal striata. DAT insertion required DRD2 autoreceptors and intact retromer, whereas DAT retrieval required PKC activation and Rit2. Ex vivo voltammetry revealed that DAT trafficking impacts DA clearance. Importantly, dopaminergic mGluR5 silencing elevated surface DAT, which abolished motor learning and was rescued by inhibiting DAT. We found that presynaptic DAT trafficking is complex, multimodal, and region-specific, and identify cell autonomous mechanisms governing presynaptic DAT tone. Importantly, the findings suggest regulated DAT trafficking impacts both DA clearance and motor function.

neuroscience↗

bioRxiv2021

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 DATs 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.

neuroscience↗

bioRxiv2019

Dopamine transporter trafficking and Rit2 GTPase:In vivo impact and mechanism of action

Following its evoked release, DA signaling is rapidly terminated by presynaptic reuptake, mediated by the cocaine-sensitive DAT. DAT surface availability is dynamically regulated by endocytic trafficking, and direct 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.

neuroscience↗

bioRxiv2019

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.

neuroscience↗