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

Abstract

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 sexdependent 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 PKCstimulated 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. _______________________________________ DA neurotransmission is required for motor control, learning, memory, motivation, and reward (1,2). DAergic dysregulation is evidenced in numerous neuropsychiatric disorders, including ADHD, ASD, schizophrenia, bipolar disorder, addiction, and PD (3-8). DA signaling is tightly controlled by the presynaptic DAT, which rapidly clears synaptically released DA. DAT is also the primary target for addictive and therapeutic

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“…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 .…”

“…2016). 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 .…”

Direct dopamine terminal regulation by local striatal microcircuitry

“…All of the above effectors—ERK, PKC, PP1, PP2A, CaMKII, Akt, and phosphoinositide 3‐kinase (PI3K)—are directly linked to activity‐dependent signaling, providing a potential mechanism by which changes in terminal excitability can regulate clearance on a rapid timescale. Indeed, changes in membrane potential alter DAT membrane expression and dopamine clearance via rapid DAT trafficking on a second timescale, through interactions Rit2 (Fagan, Kearney, & Sweeney, 2020; Richardson et al., 2016; Sweeney et al., 2020). Additionally, increasing dopamine neuron activity increased phosphorylated‐ERK, DAT phosphorylation at threonine53 (known to be phosphorylated by ERK), and leads to functional increases in DAT‐mediated dopamine clearance rates (Calipari, Juarez, et al, 2017).…”

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

“…Multiple genome-wide association studies (GWAS) have identified Rit2 as a risk allele selectively in DA-related neuropsychiatric disorders, including PD, ASD, schizophrenia, and speech delay 34-42 . We previously reported that Rit2 binds directly to DAT 43, 44 , and that Rit2 is required for PKC-stimulated DAT endocytosis in both cell lines and striatal DAergic terminals 43, 44 . 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 .…”

“…We previously reported that Rit2 binds directly to DAT 43, 44 , and that Rit2 is required for PKC-stimulated DAT endocytosis in both cell lines and striatal DAergic terminals 43, 44 . 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 .…”