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.

National Institute of Health (NIH)
National Institute on Alcohol Abuse and Alcoholism (NIAAA)
Research Project (R01)
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Special Emphasis Panel (ZAA1-CC (03))
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Cui, Changhai
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Mayo Clinic, Rochester
United States
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Abulseoud, Osama A; Camsari, Ulas M; Ruby, Christina L et al. (2014) Attenuation of ethanol withdrawal by ceftriaxone-induced upregulation of glutamate transporter EAAT2. Neuropsychopharmacology 39:1674-84
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Sari, Youssef; Sreemantula, Sai N; Lee, Moonnoh R et al. (2013) Ceftriaxone treatment affects the levels of GLT1 and ENT1 as well as ethanol intake in alcohol-preferring rats. J Mol Neurosci 51:779-87
Nam, Hyung Wook; Hinton, David J; Kang, Na Young et al. (2013) Adenosine transporter ENT1 regulates the acquisition of goal-directed behavior and ethanol drinking through A2A receptor in the dorsomedial striatum. J Neurosci 33:4329-38
Nam, Hyung Wook; Bruner, Robert C; Choi, Doo-Sup (2013) Adenosine signaling in striatal circuits and alcohol use disorders. Mol Cells 36:195-202
Abulseoud, Osama A; Miller, Joseph D; Wu, Jinhua et al. (2012) Ceftriaxone upregulates the glutamate transporter in medial prefrontal cortex and blocks reinstatement of methamphetamine seeking in a condition place preference paradigm. Brain Res 1456:14-21
Wu, Jinhua; Lee, Moonnoh R; Kim, Taehyun et al. (2011) Regulation of ethanol-sensitive EAAT2 expression through adenosine A1 receptor in astrocytes. Biochem Biophys Res Commun 406:47-52
Nam, Hyung Wook; Lee, Moonnoh R; Zhu, Yu et al. (2011) Type 1 equilibrative nucleoside transporter regulates ethanol drinking through accumbal N-methyl-D-aspartate receptor signaling. Biol Psychiatry 69:1043-51

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