Glutamate Transporters and Mitochondria: Signaling, Co-compartmentalization, Functional Coupling, and Future Directions.

In addition to being an amino acid that is incorporated into proteins, glutamate is the most abundant neurotransmitter in the mammalian CNS, the precursor for the inhibitory neurotransmitter γ-aminobutyric acid, and one metabolic step from the tricarboxylic acid cycle intermediate α-ketoglutarate. Extracellular glutamate is cleared by a family of Na+-dependent transporters. These transporters are variably expressed by all cell types in the nervous system, but the bulk of clearance is into astrocytes. GLT-1 and GLAST (also called EAAT2 and EAAT1) mediate this activity and are extremely abundant proteins with their expression enriched in fine astrocyte processes. In this review, we will focus on three topics related to these astrocytic glutamate transporters. First, these transporters co-transport three Na+ ions and a H+ with each molecule of glutamate and counter-transport one K+; they are also coupled to a Cl- conductance. The movement of Na+ is sufficient to cause profound astrocytic depolarization, and the movement of H+ is linked to astrocytic acidification. In addition, the movement of Na+ can trigger the activation of Na+ co-transporters (e.g. Na+-Ca2+ exchangers). We will describe the ways in which these ionic movements have been linked as signals to brain function and/or metabolism. Second, these transporters co-compartmentalize with mitochondria, potentially providing a mechanism to supply glutamate to mitochondria as a source of fuel for the brain. We will provide an overview of the proteins involved, discuss the evidence that glutamate is oxidized, and then highlight some of the un-resolved issues related to glutamate oxidation. Finally, we will review evidence that ischemic insults (stroke or oxygen/glucose deprivation) cause changes in these astrocytic mitochondria and discuss the ways in which these changes have been linked to glutamate transport, glutamate transport-dependent signaling, and altered glutamate metabolism. We conclude with a broader summary of some of the unresolved issues.

Brain endothelial cells induce astrocytic expression of the glutamate transporter GLT-1 by a Notch-dependent mechanism.

Neuron-secreted factors induce astrocytic expression of the glutamate transporter, GLT-1 (excitatory amino acid transporter 2). In addition to their elaborate anatomic relationships with neurons, astrocytes also have processes that extend to and envelop the vasculature. Although previous studies have demonstrated that brain endothelia contribute to astrocyte differentiation and maturation, the effects of brain endothelia on astrocytic expression of GLT-1 have not been examined. In this study, we tested the hypothesis that endothelia induce expression of GLT-1 by co-culturing astrocytes from mice that utilize non-coding elements of the GLT-1 gene to control expression of reporter proteins with the mouse endothelial cell line, bEND.3. We found that endothelia increased steady state levels of reporter and GLT-1 mRNA/protein. Co-culturing with primary rat brain endothelia also increases reporter protein, GLT-1 protein, and GLT-1-mediated glutamate uptake. The Janus kinase/signal transducer and activator of transcription 3, bone morphogenic protein/transforming growth factor ß, and nitric oxide pathways have been implicated in endothelia-to-astrocyte signaling; we provide multiple lines of evidence that none of these pathways mediate the effects of endothelia on astrocytic GLT-1 expression. Using transwells with a semi-permeable membrane, we demonstrate that the effects of the bEND.3 cell line are dependent upon contact. Notch has also been implicated in endothelia-astrocyte signaling in vitro and in vivo. The first step of Notch signaling requires cleavage of Notch intracellular domain by γ-secretase. We demonstrate that the γ-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester blocks endothelia-induced increases in GLT-1. We show that the levels of Notch intracellular domain are higher in nuclei of astrocytes co-cultured with endothelia, an effect also blocked by N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester. Finally, infection of co-cultures with shRNA directed against recombination signal binding protein for immunoglobulin kappa J, a Notch effector, also reduces endothelia-dependent increases in enhanced green fluorescent protein and GLT-1. Together, these studies support a novel role for Notch in endothelia-dependent induction of GLT-1 expression. Cover Image for this issue: doi. 10.1111/jnc.13825.