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  • In summary activated A AR exacerbated the reverse transport

    2021-10-22

    In summary, activated A2AR exacerbated the reverse transport function of endothelial EAATs through a direct or indirect pathway depending on PKA and glutamate levels in response to OGD in vitro, but A2AR inhibition quickly restored the normal transport function. Moreover, the key mechanisms by which A2AR regulates endothelial EAATs were also verified in a mouse TBI model. Based on our results, the important role of A2AR in regulating glutamate homeostasis in the brain was closely related to its ability to regulate endothelial EAAT function. Additionally, the early intervention in the function of endothelial A2AR after brain injury may represent a very promising treatment.
    Acknowledgements We thank Prof. Jiang-Fan Chen (Boston University) for kindly providing the A2AR KO mice.
    Introduction Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system (CNS) (Komuro and Rakic, 1996), and this neurotransmitter is important in synaptic plasticity, learning, and development under physiological conditions (LoTurco et al., 1991). However, excessive glutamate exposure results in neuronal death, termed excitotoxicity. Excitotoxicity might lead to neuronal damage in various neurological disorders including inflammation, ischemia, and neurodegenerative diseases (Bruijn et al., 2004, Schwartz et al., 2003). Previous studies have demonstrated that activated microglia are a significant source of redundant extracellular glutamate that induces excitotoxic neuronal death (Barger and Basile, 2001, Barger et al., 2007, Piani et al., 1992, Takeuchi et al., 2005), and thus the regulation of such microglial glutamate may be a key therapeutic strategy against excitotoxicity-driven neurological diseases. A family of sodium-dependent excitatory amino lipid metabolism transporters (EAATs) is of prominent importance for glutamate uptake and for regulating glutamate homeostasis in the CNS (Auger and Attwell, 2000, Takeuchi et al., 2006, Yamashita et al., 2006). Thus, EAATs are considered to be a critical buffer against excitotoxicity in CNS disorders. To date, five high-affinity, EAATs have been cloned from human and animal tissue, and they are identified as excitatory amino acid transporters 1–5 (EAAT1–5): EAAT1 (Shashidharan and Plaitakis, 1993, Storck et al., 1992), EAAT2 (Bristol and Rothstein, 1996, Shashidharan et al., 1994), EAAT3 (Kanai and Hediger, 1992), EAAT4 (Fairman et al., 1995), and EAAT5 (Arriza et al., 1997). According to previous studies, EAAT1 and EAAT2 are predominantly expressed in glial cells, EAAT3 and EAAT4 are typically present only in neurons (Danbolt, 2001, O'Shea, 2002), and EAAT5 is located in retinal ganglion cells (Arriza et al., 1997). A recent study showed that astrocytes also express EAAT4 (Hu et al., 2003). However, the precise profile of EAATs expression among glial cells remains unclear, and it is not fully understood which glial cell type takes the key neuroprotective function via EAATs.
    Results
    Discussion In this study, we clarified that astrocytes express EAAT1–5 whereas microglia expressed all EAATs except EAAT4. It has been generally accepted that EAAT1 and EAAT2 are glial while EAAT3, EAAT4, and EAAT5 are neuronal (Anderson and Swanson, 2000, Maragakis and Rothstein, 2001, Rothstein et al., 1994). However, this interpretation has been challenged by recent studies. For example, EAAT1 is also detectable in cultured rat hippocampal neurons (Perego et al., 2000, Plachez et al., 2000). While EAAT2 is primarily an astrocytic transporter, a splice variant of EAAT2 is also preferentially expressed in rat neurons and in non-astrocytic glial cells with the same cellular and subcellular distribution as EAAT3 (Schmitt et al., 2002). EAAT2 is also the predominant nerve terminal glutamate transporter in mouse (Suchak et al., 2003). Protein expression of EAAT2 has been described in cultures of rat primary hippocampal and cortical neurons (Mennerick et al., 1998, Wang et al., 1998) and in the NT2 cell line (Dunlop et al., 1998). In contrast, EAAT3 expression has been shown in rat astrocytes (Conti et al., 1998), in the rat C6 glioma cell line (Dowd et al., 1996, Palos et al., 1996), and in the human U373 astrocytoma cell line (Dunlop et al., 1999). EAAT4 has been found in cultured rat cortical astrocytes (Schlag et al., 1998) and in mouse astrocytes from the spinal cord and the brain (Hu et al., 2003). Here we demonstrated that EAAT1–3 were also expressed in microglia. We also provided the first evidence that microglia do not express EAAT4. However, as a well-characterized antibody against EAAT4 is not commercially available at this time, the precise expression of EAAT4 protein in both glial cell types awaits further elucidation. Although a number of studies have reported that EAAT5 is expressed exclusively in rod photoreceptor and bipolar cells of the retina (Arriza et al., 1997, Wersinger et al., 2006), our findings indicate that EAAT5 is also located in microglia and astrocytes. Taken together, the segregation of neuronal and glial EAATs might be no longer tenable.