Astrocytic A2A receptors: Novel targets to manage brain disorders
Vanessa Henriques1,, , Nélio Gonçalves1, Paula Agostinho1,2, Rodrigo A. Cunha1,2
1 CNC – Center for Neuroscience and Cell Biology, University of Coimbra, Portugal
2 Faculty of Medicine, University of Coimbra, Portugal

Aim: To validate astrocytic adenosine A2A receptors (A2AR) as a novel target to prevent abnormal glutamate overexcitation.

Introduction: Astrocytes are responsible for clearance of extracellular glutamate, a process controlled by A2AR, extolling their key role as regulators of synaptic transmission and of the abnormal glutamate overexcitation implicated in both acute and chronic brain diseases. We have previously showed that activation of astrocytic A2AR reduce astrocytic glutamate uptake under physiological and pathological conditions,1–3 and that A2AR are aberrantly up-regulated upon multiple brain insults.4–6

Methods: We incorporated EGFP reporter either alone or combined with either a small hairpin to down-regulate A2AR (shA2AR) or a control sequence (shCTR) into Mokola Lyssavirus (Mok-G) and Vesicular Stomatitis Virus (VSV-G) lentivectors and tested whether Mok-G-coated lentivirus selectively and efficiently transduced astrocytes in primary culture or in mouse brain through stereotaxic administration of lentivectors into striatum [STR], hippocampus [HIPP] and prefrontal cortex [PFC] (compared to neurotropic VSV-G-coated lentivirus as controls). Herein, we evaluated viral spreading and cell-type transduction through immunofluorescent colocalization of EGFP with glial (GFAP and vimentin) and neuronal (NeuN) markers.

Results: After 25 days post-infection, Mok-G_EGFP transduced 68% of cultured astrocytes (EGFP- and DAPI-positive, n=1); 100% of GFAP-positive cells colocalized with EGFP as well as 86% cells expressing Vimentin only and 47% expressing both Vimentin and GFAP. Mok-G shA2AR lentiviruses robustly reduced A2AR immunoreactivity compared to Mok-G shCTR in cultured astrocytes. At 4 weeks post-brain administration, Mok-G_EGFP was expressed mainly in astrocytes (GFAP-positive cells) in both STR and HIPP, and to a lower extent in the PFC, whereas VSV-G-coated lentivirus colocalized with NeuN marker and not with GFAP in any tested brain areas.

Conclusion: These data supports the ability of Mok-G lentivectors to efficiently transduce astrocytes to control A2AR density, paving the way for their application to control pathophysiological processes involving astrocytes.

Acknowledgements: Supported by DARPA grant 09-68-ESR-FP-010, FCT grants PTDC/SAU-NSC/122254/2010, UID/NEU/04539/2013, PTDC/NEU-NMC/4154/2014 and PEst-C/SAU/LA0001/2013-2014, QREN grant CENTRO-07-ST24-FEDER-002006, NARSAD and Santa Casa da Misericórdia.

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