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In vivo studies of the ERK1/2 phosphatase, MKP3 in dopaminergic neurons on gene expression, dopamine signaling and cocaine-associated behaviors
Dissertation   Open access

In vivo studies of the ERK1/2 phosphatase, MKP3 in dopaminergic neurons on gene expression, dopamine signaling and cocaine-associated behaviors

Stacia Irene Lewandowski
Doctor of Philosophy (Ph.D.), Drexel University
Apr 2021
DOI:
https://doi.org/10.17918/00000700
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Abstract

Cocaine abuse Mitogen-activated protein kinases Phosphatases Dopamine
Cocaine use disorder is a chronic and debilitating disease with no FDA-approved pharmacotherapies for treatment. Cocaine exerts its addictive effects by blocking the dopamine transporter (DAT), leading to excess dopamine (DA) in the synaptic cleft, resulting in the euphoric "high" that is often sought-after during addiction. The ERK1/2 Map Kinase signaling pathway has been implicated in the stimulant effects of psychostimulants such as cocaine. However, limitations exist in these previous studies as they were based on systemic or local administration of kinase inhibitors and thus lack the anatomical specificity that is needed for a more detailed investigation of the role of ERK1/2 signaling in specific cell types. Here we describe the modulation of the ERK1/2 pathway in vivo by expressing the ERK1/2 phosphatase MKP3 in DA neurons of the ventral tegmental area (VTA) of Long Evans rats, resulting in a decrease of ERK1/2 signaling specifically in these neurons. We demonstrate that DA neuron specific ERK1/2 inhibition differentially affects high (HCRs) and low (LCRs) cocaine responding animals. This classification is based upon their acute locomotor response to cocaine. Specifically, we find that MKP3 expression and resulting ERK1/2 inhibition only affects HCRs, and we demonstrate that MKP3-expressing HCRs more closely resemble LCRs at both the behavioral locomotor response to cocaine and the DAT protein level. We hypothesize that this results from MKP3's role in regulating intracellular DAT trafficking and surface expression. Furthermore, this is supported by functional studies of the DAT where we demonstrate that inhibition of ERK1/2 signaling in DA neurons influences DA neurotransmission in the nucleus accumbens (NAc) and that the DAT in MKP3-overexpressing animals is resistant to dynamic regulation of its surface expression. Finally, studies utilizing Viral Translating Ribosomal Affinity Purification (vTRAP) and resulting RNA-Seq data demonstrate upregulation of several genes including dopaminergic genes, such as tyrosine hydroxylase and the DAT, which has been further confirmed also to occur on the protein level. We think these studies have established a unique model to understand and identify factors involved in regulating DA neuron activity and associated behaviors and could have potential for identifying novel therapeutic targets for the treatment of cocaine use disorders.

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