The neurotransmitter dopamine (DA) is involved in motivation, reward mechanisms, and many central nervous system diseases. DA is transported into the presynaptic neurons by a protein called dopamine transporter (DAT). The psychostimulant cocaine is an inhibitor of DAT. When DAT is inhibited by cocaine, the DA in the synaptic cleft increases and this leads to amplified downstream dopaminergic signaling primarily in the mesolimbic pathway. This increase in cocaine-induced DA signaling is responsible for the addictive properties of cocaine. Because DAT is the primary target of cocaine, compounds acting on the DAT in novel ways could potentially treat cocaine use disorders. We previously found a novel compound-KM822 and characterized it as an allosteric modulator of DAT function. KM822 significantly decreases cocaine-induced locomotive response in planarians. To test if KM822 has similar effects in mammals, we administered KM822 and cocaine intracranially into nucleus accumbens (NAc) area in Long Evans rats and measured the locomotion changes. We targeted the NAc as it is a part of the brain that plays a crucial role in the mesolimbic dopaminergic pathway and has been recognized by its high density of DAT. Our results showed that KM822 significantly decreased hyper-locomotion induced by cocaine and notably did not cause any locomotion changes by itself. We also examined KM822's ability to interfere with cocaine's rewarding effect using the conditioned place preference (CPP) assay. In this assay, animals are tested for their preference for a cocaine-associated environment as a model of cocaine seeking. Unlike the locomotion assay, this assay is more relevant to behaviors associated with cocaine addiction and therefore has higher translational value. CPP is also utilized to determine the addictive liability of KM822. c-Fos expression is frequently utilized as a functional marker to investigate neuronal processes in response to a stimuli. To determine how KM822 affects neuronal activity in the NAc, c-Fos staining was conducted, and the expression of c-Fos was compared between animals pre-treated with KM822 and those pre-treated with vehicle before cocaine infusion. In the future, we plan to further assess the impact of KM822 in extinction and relapse utilizing CPP. Overall, these studies demonstrate the ability of KM822 to block DAT inhibitor-induced behaviors in rats and provide strong evidence that the novel allosteric DAT modulator KM822 has significant potential for treating cocaine addiction.

Dopamine is a neurotransmitter that plays a critical role in movement, motivation, and reward. Dopamine neurotransmission is regulated by the dopamine transporter (DAT), which translocates dopamine from the extracellular space to the presynaptic neuron. DAT is a target for psychostimulants such as cocaine, giving DAT a key role in psychostimulant‐use disorders. DAT cell‐surface expression regulated by trafficking to and from the plasma membrane is an important mechanism for controlling DAT function. Two signaling pathways that contribute to DAT trafficking are the protein kinase C (PKC) and mitogen activated protein kinases (MAPKs), including the extracellular signal‐regulated kinase (ERK1/2) and p38 MAPK. ERK1/2 is inactivated by the MAP kinase phosphatase MKP3, which dephosphorylates and inactivates ERK1/2. Previous data from the lab demonstrated that activation of PKC by administration of phorbol 12‐myristate 13‐acetate (PMA) in an in vitro system results in decreased DAT cell‐surface expression, and overexpression of MKP3 attenuates this effect. Furthermore, other studies have shown that ERK1/2 activation, downstream of PKC activation, may lead to phosphorylation of DAT on residue threonine 53 (Thr53), however the significance of this phosphorylation is not completely understood. In order to characterize the contributions of MKP3 and ERK1/2 to DAT phosphorylation and trafficking in vivo, we have developed two viral constructs based on adeno‐associated viral (AAV) vectors that enable cre recombinase (cre)‐dependent expression. One vector cre‐dependently expresses MKP3. The other vector is based on a genetic tool named OptoSOS. This vector enables cre‐dependent, blue‐light induced activation of ERK1/2, and as a result, can spatiotemporally activate intracellular ERK1/2 signaling. Both viral vectors are employed with transgenic rats that express cre in tyrosine hydroxylase‐positive cells (TH‐Cre rats) to drive expression in dopaminergic neurons. From these studies, we find that MKP3 overexpression and resulting ERK1/2 inactivation increases DAT surface expression by blocking the internalization and degradation of the transporter leading to an accumulation of DAT on the cell‐surface. Interestingly, MKP3 overexpression also decreases phosphorylation at Thr53. While we have shown that ERK inactivation results in increased DAT cell‐surface expression, we have employed the OptoSOS system to activate ERK1/2 and assess resulting changes in DAT trafficking. We have confirmed that ERK1/2 is activated by blue‐light in a cre‐dependent manner, and similar to what we observe with MKP3 overexpression, ERK1/2 activation results in changes in DAT cell‐surface expression and phosphorylation. Taken together, our results indicate that MKP3 and ERK1/2 serve critical physiological roles in DAT regulation and can be utilized to investigate the mechanistic relationship between DAT trafficking, phosphorylation, and transporter activity.

The dopamine transporter (DAT) is a transmembrane protein responsible for reuptake the neurotransmitter dopamine back into the presynaptic neurons. Dopamine (DA) is involved in driving motivation, it is increased when people expect a reward, and it is also involved in many central nervous systems (CNS) disorders. The inhibition of the reuptake process prevents dopamine in the synaptic cleft from getting back into the presynaptic neuron. Cocaine and amphetamine both inhibit DA reuptake by binding to the orthosteric site but through different mechanisms. Cocaine is a classical inhibitor that blocks the transporter through competitive inhibition. On the other hand, amphetamine is a substrate of DAT and causes competitive inhibition of DA reuptake and reversal of the transporter leading to the release of DA. As a result of the DA reuptake inhibition, the DA in the synaptic cleft increases, leading to amplified downstream dopaminergic signaling primarily in the mesolimbic pathway. Because DAT is a primary target of psychostimulants, compounds acting on the DAT in novel ways could potentially treat psychostimulant use disorders. We previously found a novel compound—KM822 and characterized it as an allosteric modulator of DAT function that significantly decreases cocaine‐induced locomotive response in planarians. To test if KM822 has similar effects in mammals, we administered KM822 and cocaine through intracranial infusions into Long Evans rats' nucleus accumbens (NAc) and measured locomotion. We targeted the NAc as it is a part of the brain that plays a crucial role in the mesolimbic dopaminergic pathway and has been recognized by its high density of DAT. Our results showed that KM822 significantly decreased hyper‐locomotion induced by cocaine and notably did not cause any increase in locomotion by itself. KM822's ability to block hyper‐locomotion induced by amphetamine, another psychostimulant with a different mechanism of action, was also examined. Finally, we examined KM822's ability to interfere with cocaine's rewarding effect using the conditioned place preference (CPP) assay. In this assay, animals are tested for their preference for a cocaine‐associated environment. Unlike locomotion assay, this assay is more relevant to behaviors associated with cocaine addiction and therefore has high translational value. Overall, these studies demonstrated KM822's ability to block DAT inhibitor‐induced behaviors in rats and provided strong evidence that the novel allosteric DAT modulator KM822 has significant potential for treating psychostimulant addiction.

ID 16668Poster Board 69 Dopamine (DA) is a neurotransmitter that plays a critical role in motivation, reward, and learning. The dopamine transporter (DAT) is responsible for the reuptake of released DA, making it a central regulator of DA neurotransmission. DAT is a target for psychostimulants such as cocaine, giving DAT a key role in psychostimulant-use disorders. Two signaling pathways that contribute to DAT regulation and trafficking to and from the plasma membrane involve the protein kinase C (PKC) and the mitogen activated protein kinase (MAPK) ERK1/2. ERK1/2 is inactivated by the MAPK phosphatase MKP3, which specifically dephosphorylates ERK1/2. Prior in vitro data demonstrated that activation of PKC by phorbol 12-myristate 13-acetate (PMA) results in decreased DAT surface-expression; MKP3 overexpression attenuates this effect. Furthermore, related studies have shown that ERK1/2 activation may lead to phosphorylation of DAT on threonine 53 (Thr53), however, the significance of this phosphorylation is not completely understood, but it is thought to play a significant role in DAT function and trafficking. In vivo data from our lab shows exogenous overexpression of MKP3 and resulting ERK1/2 inactivation increases DAT surface-expression, but interestingly reduces DAT transcript levels. This suggests that post-transcriptional regulation of DAT, such as proteasomal degradation, may be affected by ERK1/2. Additionally, MKP3 overexpression reduced Thr53 phosphorylation levels despite the overall increase of DAT surface-expression. To further characterize the contributions of ERK1/2 to DAT regulation and trafficking in vivo, we will use two viral constructs: AAV9-FLEX-MKP3-R (MKP3) and AAV9-FLEX-OptoSOS-R (OptoSOS). OptoSOS is an optogenetic tool that enables blue-light activation of ERK1/2. Both constructs enable cre recombinase-dependent expression in DA neurons of the VTA, allowing for spatiotemporal inactivation or activation of ERK1/2. We have confirmed that ERK1/2 is activated by blue-light in a cre-dependent manner, and similar to our results with MKP3 overexpression, ERK1/2 activation results in changes in DAT surface-expression and phosphorylation. Additionally, ERK1/2 activation results in increased phosphorylation of tyrosine hydroxylasethe rate limiting enzyme in DA synthesis. We will utilize these constructs to better understand the role of ERK1/2 signaling in regulation of DAT phosphorylation and membrane trafficking, and DA neurotransmission. Taken together, our results indicate that ERK1/2 serves a critical physiological role in DAT regulation, and these studies will provide important information regarding the mechanistic relationship between DAT trafficking, phosphorylation, and activity. These tools could help to reveal novel therapeutic strategies for psychostimulant-use disorders.