Adenosine A 2A Receptors Modulate Glutamate Uptake in Cultured Astrocytes and Gliosomes MARCO MATOS, 1,2 ELISABETE AUGUSTO, 1,2 ALEXANDRE DOS SANTOS-RODRIGUES, 1 MICHAEL A. SCHWARZSCHILD, 3 JIANG-FAN CHEN, 4 RODRIGO A. CUNHA, 1,2 AND PAULA AGOSTINHO 1,2 * 1 Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal 2 Faculty of Medicine, University of Coimbra, Coimbra, Portugal 3 Molecular Neurobiology Laboratory, Department of Neurology, Massachusetts General Hospital, Charlestown, Massachusetts 4 Department of Neurology, Boston University School of Medicine, Boston, Massachusetts KEY WORDS adenosine A 2A receptor; glutamate transporters; astrocytes; gliosomes; GLAST; GLT-I; EAAC1 ABSTRACT Glutamate is the primary excitatory neurotransmitter in the central nervous system, where its toxic build-up leads to syn- aptic dysfunction and excitotoxic cell death that underlies many neurodegenerative diseases. Therefore, efforts have been made to understand the regulation of glutamate transporters, which are responsible for the clearance of extracellular gluta- mate. We now report that adenosine A 2A receptors (A 2A R) con- trol the uptake of D-aspartate in primary cultured astrocytes as well as in an ex vivo preparation enriched in glial plasmalem- mal vesicles (gliosomes) from adult rats, whereas A 1 R and A 3 R were devoid of effects. Thus, the acute exposure to the A 2A R agonist, CGS 21680, inhibited glutamate uptake, an effect pre- vented by the A 2A R antagonist, SCH 58261, and abbrogated in cultured astrocytes from A 2A R knockout mice. Furthermore, the prolonged activation of A 2A R lead to a cAMP/protein kinase A-dependent reduction of GLT-I and GLAST mRNA and pro- tein levels, which leads to a sustained decrease of glutamate uptake. This dual mechanism of inhibition of glutamate trans- porters by astrocytic A 2A R provides a novel candidate mecha- nism to understand the ability of A 2A R to control synaptic plas- ticity and neurodegeneration, two conditions tightly associated with the control of extracellular glutamate levels by glutamate transporters. V V C 2012 Wiley Periodicals, Inc. INTRODUCTION Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system (CNS), but its ex- cessive extracellular accumulation seems to contribute for the evolution of most neurodegenerative disorders (Benarroch et al., 2010; Lipton and Rosenberg, 1994). As glutamate is not metabolized in the extracellular environ- ment, the maintenance of normal glutamatergic neuro- transmission and the prevention of excitotoxicity depends on the reuptake of glutamate, mainly through the astro- cytic glutamate transporters, glutamate–aspartate trans- porter (GLAST)/human homologue EAAT1 and glutamate transporter-1(GLT-I)/human homologue EAAT2 as well as neuronal and astrocytic excitatory amino acid carrier 1 (EAAC-1)/human homologue EAAT3 and cerebellar EAAT4 and retinal EAAT5 (reviewed in Dunlop, 2006; Swanson, 2005). The importance and relevance of the ac- tivity of glutamate transporters are underscored by the impact of their activity in the control of synaptic plastic- ity as well as in the demise of neurodegeneration (reviewed in Tzingounis and Wadiche, 2007). Adenosine is a classical modulator of synaptic trans- mission in the CNS, exerting its effects via four types of G-protein-coupled receptors: A 1 ,A 2A ,A 2B , and A 3 (Fred- holm et al., 2005). Adenosine mainly controls excitatory transmission through a coordinated action of inhibitory A 1 receptors and facilitatory adenosine A 2A receptors (A 1 R, A 2A R) (Fredholm et al., 2005; Gomes et al., 2011). These two adenosine receptors have also been actively pursued as possible drug targets to manage neurodegen- eration (reviewed in Gomes et al., 2011). Accordingly, bolstering A 1 R activation and inhibiting A 2A R function are two purported strategies to control neurodegenera- tive processes (Cunha, 2005; Schwarzschild, 2007). Albeit the physiological and pathological role of adeno- sine receptors has mainly been assumed to result from their direct action on neurons, adenosine receptors are also present in astrocytes where they control the metab- olism of glucose, astrogliosis, cell proliferation, cell vol- ume changes, cell death, and the release of neurotrophic factors and interleukins (for review see, Boison et al., 2009; Dar e et al., 2007). Given the importance of the activity of glutamate transporters in pathological condi- tions, the ability of A 2A R to afford a sustained control of glutamate transporters could emerge as a novel candi- date mechanism to understand the neuroprotection afforded by A 2A R blockade on different brain insults (Gomes et al., 2011; Lopes et al., 2011). This hypothesis is prompted by previous observations that the control by Additional Supporting Information may be found in the online version of this article. Grant sponsor: Portuguese Foundation for Science and Technology (FCT); Grant number: PTDC/SAU-NEU/108668/2008; Grant sponsor: FCT Fellowship (M.M.); Grant number: SFRH/BD/36289/2007; Grant sponsor: NIH; Grant number: K24NS60991; Grant sponsor: DoD; Grant number: W81XWH-11-1-0150; Grant sponsor: CNPq, Brasil fellowship (A.S.-R); Grant number: 200656/2008-2.;. *Correspondence to: Paula Agostinho, Center for Neurosciences of Coimbra, Institute of Biochemistry, Faculty of Medicine, University of Coimbra, 3004-504 Coimbra, Portugal. E-mail: pagost@cnc.cj.uc.pt Received 23 June 2011; Accepted 5 December 2011 DOI 10.1002/glia.22290 Published online 1 February 2012 in Wiley Online Library (wileyonlinelibrary. com). GLIA 60:702–716 (2012) V V C 2012 Wiley Periodicals, Inc.