The transcription factor Pax6 contributes to the induction of GLT‐1 expression in astrocytes through an interaction with a distal enhancer element

Abstract

The Na+-dependent glutamate transporter, GLT-1 (EAAT2), shows selective expression in astrocytes, and neurons induce expression of GLT-1 in astrocytes. In unpublished analyses of GLT-1 promoter reporter mice, we identified an evolutionarily conserved domain of 467 nucleotides ~8 kb upstream of the GLT-1 translation start site that is required for astrocytic expression. Using in silico approaches, we identified Pax6 as a transcription factor that could contribute to the control of GLT-1 expression by binding within this region. We demonstrated expression of Pax6 protein in astrocytes in vivo. Lentiviral transduction of astrocytes with exogenous Pax6 increased expression of enhanced green fluorescent protein (eGFP) in astrocytes prepared from transgenic mice that use a bacterial artificial chromosome (BAC) containing a large genomic region surrounding the GLT-1 gene to control expression of eGFP. It also increased GLT-1 protein, and GLT-1-mediated activity, while there was no effect on the levels of astroglial glutamate transporter, GLAST. Transduction of astrocytes with an shRNA directed against Pax6 reduced neuron-dependent induction of GLT-1 or eGFP. Finally, we confirmed Pax6 interaction with the predicted DNA binding site in electrophoretic mobility assays (EMSA) and chromatin immunoprecipitation (ChIP). Together, these studies show that Pax6 contributes to regulation of GLT-1 through an interaction with these distal elements and identify a novel role of Pax6 in astrocyte biology.

Astroglial glutamate transporters in the brain: Regulating neurotransmitter homeostasis and synaptic transmission

“…As such, glutamate transporters make for good potential therapeutic targets. However, our understanding of the complex regulation of glutamate transporter transcription and translation (Ghosh et al, 2011(Ghosh et al, , 2015(Ghosh et al, , 2016, cell surface expression and stabilisation (Stenovec et al, 2008;Yang et al, 2009Yang et al, , 2010Underhill et al, 2015), and internalisation Sheldon et al, 2008;Underhill et al, 2014;Ib añez et al, 2016) is still rather limited. Deciphering the mechanisms by which glutamate transporters are expressed and stabilised at the cell surface is of utmost importance, as it could reveal innovative targets for the treatment of diseases such as traumatic brain injury or epilepsy where extracellular glutamate is high.…”

Transcriptional Regulation of Glutamate Transporters

“…Two groups essentially simultaneously realized that rat astrocytes in culture express little or no GLT-1 protein, but co-culturing astrocytes with neurons induces expression of GLT-1 in astrocytes (Schlag et al, 1998, Swanson et al, 1997). In fact, several subsequent studies have documented low levels of GLT-1 in mouse astrocyte cultures, but neurons also increase GLT-1 transcription in this system (Apricó, Beart, Crawford & O’Shea, 2004, Ghosh, Lane, Krizman, Sattler, Rothstein & Robinson, 2015, O’Shea, Lau, Farso, Diwakarla, Zagami, Svendsen et al, 2006). The effect of neurons is at least in part dependent upon soluble factors but it may also depend on contact (Drejer, Meier & Schousboe, 1983, Gegelashvili et al, 1997, Gegelashvili et al, 2000, Yang, Gozen, Watkins, Lorenzini, Lepore, Gao et al, 2009, Zelenaia et al, 2000).…”

“…While the proximal 2.5kb promoter of GLT-1 gene is highly conserved and has been well characterized, there are several additional evolutionary conserved domains distal to this region out to ~12.5kb from the translation start site (Figure 1) (Ghosh et al, 2015). Analyses of promoter reporter mice generated by Rothstein and his colleagues have revealed that the proximal 7.9 kb of the promoter is not sufficient to direct astrocytic expression of reporter protein, reporter is observed mostly in neurons (Rothstein unpublished observations; for discussion, see Ghosh et al, 2015).…”

“…Analyses of promoter reporter mice generated by Rothstein and his colleagues have revealed that the proximal 7.9 kb of the promoter is not sufficient to direct astrocytic expression of reporter protein, reporter is observed mostly in neurons (Rothstein unpublished observations; for discussion, see Ghosh et al, 2015). When 8.3 kb of promoter is used to control expression, reporter protein is essentially exclusively found in astrocytes, but not all astrocytes express the reporter (Yang, Vidensky, Jin, Jie, Lorenzini, Frankl et al, 2011).…”

“…In fact, we recently showed that Pax6 interacts with this region in vitro (EMSA) and in vivo (ChIP). ShRNA directed knockdown of Pax6 attenuates neuron-dependent induction of GLT-1 and exogenous expression of Pax6 increases GLT-1 expression (Ghosh et al, 2015). Secondly, these data suggest that different subtypes of astrocytes engage different mechanisms to control expression of GLT-1 in vivo .…”