Studies with ENT1 null mice indicate that ENT1 regulates ethanol intoxication and preference, as well as motivational effects of ethanol. These behaviors appear to be attributed to the increased glutamate signaling in the striatum where cortical glutamatergic axons mainly send their signaling to control motor functions, habits and motivations. We found that EAAT2 expression was reduced in ENT1 null mice by proteomics, and confirmed it by Western blot analysis, indicating that the increased glutamate levels in ENT1 null mice may be partly due to the reduced synaptic glutamate uptake by EAAT2 in astrocytes. Despite evidence demonstrating that a genetic variant of EAAT2 (G603A) is implicated in alcoholism, regulation of astrocytic EATT2 expression and function in response to ethanol is poorly understood in molecular, cellular and behavioral levels. Considering that ENT1 and EAAT2 are predominantly expressed in the astrocytes, our main hypothesis is that inhibition of ENT1 causally reduces EAAT2 expression through intracellular mechanisms in the astrocytes. This hypothesis will be tested using cultured astrocytes, ENT1 null mice, and a novel bi-transgenic mouse line that expresses green fluorescent protein in astrocytes in ENT1 null background. First, we will determine if ethanol alters ENT1 and EAAT2 expression and function in the astrocytes. We will also investigate astrocyte-specific signaling pathways involved in ENT1 and EAAT2 expression and function using a proteomic technique. Using tandem mass spectrometry and a newly developed electrophysiological method, we will determine if ethanol alters adenosine and glutamate releases from the astrocytes. Secondly, to investigate the physiological role of ENT1 and EAAT2 in the astrocytes, we will examine astrocytes function in ENT1 null mice using bi-transgenic mice, GFAP-EGFP/ENT1 null mice, or GFAP-EGFP/ENT1 wild-type mice. We will isolate the astrocytes using FACS (fluorescent-assisted cell sorting), and then will examine protein profiles using a mass-tag labeling proteomic technique, iTRAQ, to compare between genotypes with saline or different ethanol doses. Following this, we will validate the altered protein expressions using a functional proteomics such as Western blot and immunofluorescence experiments. Finally, we will determine whether pharmacological or genetic regulations of EAAT2 alter alcohol preference and reward in mice. The overall goal of this project is to identify a novel ENT1 and EAAT2 signaling pathway that regulates ethanol responses in the astrocytes and neuro-glial interactions which may contain targets for the development of new therapeutics to treat alcohol use disorders in humans.

Public Health Relevance

We will investigate the role of ethanol-sensitive adenosine transporter, ENT1 and a glutamate transporter, EAAT2, in the striatal astrocytes. We will use cultured astrocytes, ENT1 null mice, and astrocyte-specific marker expressing mice to carry out cellular, molecular, biochemical, pharmacological, and behavioral studies. The results of our studies are expected to define a novel ENT1 and EAAT2 signaling pathway that regulates ethanol response in the astrocytes and neuro-glial interactions, which may lead us to develop new therapeutics to treat alcohol use disorders.

Agency
National Institute of Health (NIH)
Institute
National Institute on Alcohol Abuse and Alcoholism (NIAAA)
Type
Research Project (R01)
Project #
5R01AA018779-03
Application #
8135634
Study Section
Special Emphasis Panel (ZAA1-CC (03))
Program Officer
Cui, Changhai
Project Start
2009-09-30
Project End
2014-08-31
Budget Start
2011-09-01
Budget End
2012-08-31
Support Year
3
Fiscal Year
2011
Total Cost
$341,489
Indirect Cost
Name
Mayo Clinic, Rochester
Department
Type
DUNS #
006471700
City
Rochester
State
MN
Country
United States
Zip Code
55905
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Oliveros, Alfredo; Starski, Phillip; Lindberg, Daniel et al. (2017) Label-Free Neuroproteomics of the Hippocampal-Accumbal Circuit Reveals Deficits in Neurotransmitter and Neuropeptide Signaling in Mice Lacking Ethanol-Sensitive Adenosine Transporter. J Proteome Res 16:1445-1459
Oliveros, A; Cho, C H; Cui, A et al. (2017) Adenosine A2A receptor and ERK-driven impulsivity potentiates hippocampal neuroblast proliferation. Transl Psychiatry 7:e1095
Oliveros, Alfredo; Choi, Doo-Sup (2017) Repurposing Tigecycline for the Treatment of Alcohol Use Disorder. Alcohol Clin Exp Res 41:497-500
Ayers-Ringler, Jennifer R; Oliveros, Alfredo; Qiu, Yanyan et al. (2016) Label-Free Proteomic Analysis of Protein Changes in the Striatum during Chronic Ethanol Use and Early Withdrawal. Front Behav Neurosci 10:46
Lindberg, Daniel; Choi, Doo-Sup (2016) Disruption of Integrated Neuronal and Astrocytic Signaling Contributes to Alcohol Use Disorder. Alcohol Clin Exp Res 40:2309-2311
Ayers-Ringler, Jennifer R; Jia, Yun-Fang; Qiu, Yan-Yan et al. (2016) Role of astrocytic glutamate transporter in alcohol use disorder. World J Psychiatry 6:31-42
Ho, Ada Man-Choi; Qiu, Yanyan; Jia, Yun-Fang et al. (2016) Combined Effects of Acamprosate and Escitalopram on Ethanol Consumption in Mice. Alcohol Clin Exp Res 40:1531-9
Vadnie, Chelsea A; Ayers-Ringler, Jennifer; Oliveros, Alfredo et al. (2016) Antipsychotic-like effects of a neurotensin receptor type 1 agonist. Behav Brain Res 305:8-17
Lindberg, D; Shan, D; Ayers-Ringler, J et al. (2015) Purinergic signaling and energy homeostasis in psychiatric disorders. Curr Mol Med 15:275-95

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