Bidirectional ERK1/2 Modulation in Dopaminergic Neurons Regulates DAT Expression and Function

Abstract

<b>Abstract ID 16668</b> <b>Poster Board 69</b> 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 <i>in&nbsp;vitro</i> 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. <i>In vivo</i> 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 <i>in&nbsp;vivo</i>, 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 hydroxylase – the 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.