Traumatic brain injury (TBI) is a progressive disorder, in which the primary injury results in the initiation of a complex cascade of secondary biochemical and metabolic changes resulting in lasting neurological dysfunction and cognitive impairment. The heterogeneous nature of the disease has complicated the development of pharmacological agents to improve the outcomes of TBI; to date, no therapeutic treatment has been shown to be effective in clinical trials. Treatments targeting multiple secondary outcomes (cell death, axonal degeneration, and inflammation) may provide enhanced therapeutic efficacy following TBI. Small Ras family GTP-binding proteins govern diverse cellular processes by directing the relay of extracellular stimuli to the activation of select intracellular signaling pathways. Rin (RIT2) is a member of the Rit subfamily of Ras-related family of GTPases, and is expressed solely within neurons of the CNS. Early cell culture models demonstrated that Rin signaled upstream of the stress-activated protein kinase, p38, and lacked the transformative abilities displayed by other members of the Ras family, suggesting functions for Rin other than cell growth and proliferation. To begin to define the physiological function of Rin, we generated a RIT2 knockout mouse and examined the impact of Rin loss in the CNS following brain trauma. Our data demonstrates that Rin deficiency is neuroprotective following a controlled cortical impact (CCI) injury, reducing both acute hippocampal neurodegeneration and promoting sustained neuronal survival, without affecting post-CCI neurogenesis. Hippocampal neuroprotection achieved by Rin loss was accompanied by improved cognitive function in injured mice. Furthermore, we demonstrated that Rin loss led to blunting of axonal degeneration and microglial activation in the optic nerve following optic nerve stretch injury. The molecular interaction between Rin and dual leucine zipper kinase suggested a potential role for Rin in the regulation of a novel stress MAPK-dependent neuronal death cascade. Lastly, we demonstrated through diffuse closed head injury, that Rin loss mitigates cytokine release as a result of injury without altering glial activation. Together, these studies establish Rin as a novel regulator of neuronal cell death, cognitive decline, axonal degeneration, and cytokine production following traumatic brain injury.

Dopamine (DA) governs movement, sleep, reward, and cognition. The presynaptic dopamine transporter (DAT), clears released DA, controlling DA signaling and homeostasis. Genetic DAT ablation causes hyperactivity, sleep reduction, and altered psychostimulant response. DAT surface expression is dynamic; DAT constitutively internalizes and recycles to and from the plasma membrane, and acute PKC activation stimulates DAT endocytosis. Cell line experiments demonstrated that PKC-stimulated DAT endocytosis requires Ack1 inactivation and the GTPase, Rit2. How Rit2 controls PKC-dependent DAT internalization, or whether regulated DAT endocytosis impacts behavior, is unknown. Here, I present data supporting that PKC activation stimulates Rit2/DAT dissociation, mediated by the DAT N-terminus. Further, Ack1 and Rit2 function independently to facilitate PKC-stimulated DAT internalization. Moreover, PKC-stimulated DAT endocytosis was limited to ventral striatum in ex vivo slice preparations, and required Rit2. Our lab previously demonstrated that certain DA-dependent behaviors required DAergic Rit2 in mice, however whether this was due to perturbed PKC-stimulated DAT internalization, or DAT-independent Rit2 function(s) remains untested. To address this, I turned to Drosophila and its Rit2 homolog Ric. I found that Ric and dDAT proteins interact in cell lines, and that constitutively active Ric (RicQ117L) increased dDAT function in cultured cells and ex vivo whole fly brains. However, neither DAergic Ric knockdown nor RicQ117L altered overall locomotion or sleep, suggesting that these fundamental behaviors do not require DAergic Ric. Together, these results expand our understanding of intrinsic mechanisms controlling DAT endocytosis, and their impact on behavior.

Dopamine (DA) is a modulatory neurotransmitter required for movement, learning, and reward. Several neuropsychiatric disorders exhibit DAergic dysfunction, including Parkinson’s disease (PD). The presynaptic DA transporter (DAT) constrains DAergic signaling via DA reuptake. Acute PKC activation drives DAT endocytosis, however, endogenous receptor-mediated DAT trafficking in striatal terminals remains ill-defined. Here, I present data supporting biphasic Gq-receptor-mediated DAT trafficking in striatum. Gq-receptor activation drives initial DAT insertion, which requires DA release, DAergic DRD2auto activation, and intact retromer. Subsequent DAT retrieval requires PKC and the neuronal GTPase Rit2. Furthermore, I demonstrate that the endogenous Gq-coupled metabotropic glutamate receptor, mGluR5, expressed on DAergic neurons exerts biphasic DAT regulation. DAergic mGluR5 silencing revealed that mGluR5 is required for motor learning and coordination. DAergic mGluR5 cKO motor deficits were rescued by DAT inhibition, suggesting mGluR5-mediated DAT trafficking is required for these behaviors. Apart from its requisite role in DAT trafficking, Rit2 is a PD associated risk allele. We previously demonstrated that Rit2 is required for psychostimulant response and generalized anxiety, but not basal locomotion. However, Rit2’s roles in more complex motor behaviors and PD pathology remain unknown. DAergic Rit2 silencing revealed that Rit2 is required for male motor learning and prolonged Rit2 suppression leads to progressive manifestation of PD biomarkers, coordination deficits, and decreased DAergic tone. Motor learning deficits were rescued by boosting DA availability, echoing Rit2-mediated hypodopaminergia. Together these results identify receptor-mediated DAT trafficking mechanisms in DA terminals, demonstrate that DAT surface dynamics are required for motor function, and implicate DAergic Rit2 loss in progressive PD-like phenotypes.