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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 the expression of GLT-1 in astrocytes. In an unpublished analysis of GLT-1 promoter reporter mice, we identified an evolutionarily conserved domain of 467 nucleotides ~ 8 kb upstream of the GLT-1 translation start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 the expression of Pax6 protein in astrocytes in vivo. Lentiviral transduction of astrocytes with exogenous Pax6 increased the expression of enhanced green fluorescent protein (eGFP) in astrocytes prepared from transgenic mice that use a bacterial artificial chromosome 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 uptake, whereas there was no effect on the levels of the other astroglial glutamate transporter, glutamate aspartate 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 and chromatin immunoprecipitation (ChIP). Together, these studies show that Pax6 contributes to the regulation of GLT-1 through an interaction with these distal elements and identify a novel role of Pax6 in astrocyte biology. The astroglial glutamate transporter GLT-1 shows selective expression in astrocytes and its expression can be induced by neurons. In this study, we demonstrate that Pax6 is expressed in astrocytes and binds to the GLT-1 promoter in vitro and in vivo. Exogenous expression of Pax6 increases GLT-1 and enhanced green fluorescent protein (eGFP) expression in astrocytes from a transgenic mouse line that uses the GLT-1 gene to drive eGFP expression, and an shRNA directed against Pax6 attenuates neuron-dependent induction of GLT-1/eGFP. We therefore conclude that Pax6 contributes to the neuron-dependent induction of GLT-1.

SUBMITTER: Ghosh M

PROVIDER: S-EPMC4837027 | biostudies-literature | 2016 Jan

REPOSITORIES: biostudies-literature

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

Ghosh Mausam M Lane Meredith M Krizman Elizabeth E Sattler Rita R Rothstein Jeffrey D JD Robinson Michael B MB

Journal of neurochemistry 20151124 2

The Na(+) -dependent glutamate transporter GLT-1 (EAAT2) shows selective expression in astrocytes, and neurons induce the expression of GLT-1 in astrocytes. In an unpublished analysis 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 ...[more]

PMID: 26485579

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Project description:AimsType 2 diabetes mellitus (T2DM) is related to an increased risk of postoperative cognitive dysfunction (POCD), which may be caused by neuronal hyperexcitability. Astrocyte glutamate transporter 1 (GLT-1) plays a crucial role in regulating neuron excitability. We investigated if T2DM would magnify the increased neuronal excitability induced by anesthesia/surgery (A/S) and lead to POCD in young adult mice, and if so, determined whether these effects were associated with GLT-1 expression.MethodsT2DM model was induced by high fat diet (HFD) and injecting STZ. Then, we evaluated the spatial learning and memory of T2DM mice after A/S with the novel object recognition test (NORT) and object location test (OLT). Western blotting and immunofluorescence were used to analyze the expression levels of GLT-1 and neuronal excitability. Oxidative stress reaction and neuronal apoptosis were detected with SOD2 expression, MMP level, and Tunel staining. Hippocampal functional synaptic plasticity was assessed with long-term potentiation (LTP). In the intervention study, we overexpressed hippocampal astrocyte GLT-1 in GFAP-Cre mice. Besides, AAV-Camkllα-hM4Di-mCherry was injected to inhibit neuronal hyperexcitability in CA1 region.ResultsOur study found T2DM but not A/S reduced GLT-1 expression in hippocampal astrocytes. Interestingly, GLT-1 deficiency alone couldn't lead to cognitive decline, but the downregulation of GLT-1 in T2DM mice obviously enhanced increased hippocampal glutamatergic neuron excitability induced by A/S. The hyperexcitability caused neuronal apoptosis and cognitive impairment. Overexpression of GLT-1 rescued postoperative cognitive dysfunction, glutamatergic neuron hyperexcitability, oxidative stress reaction, and apoptosis in hippocampus. Moreover, chemogenetic inhibition of hippocampal glutamatergic neurons reduced oxidative stress and apoptosis and alleviated postoperative cognitive dysfunction.ConclusionsThese findings suggest that the adult mice with type 2 diabetes are at an increased risk of developing POCD, perhaps due to the downregulation of GLT-1 in hippocampal astrocytes, which enhances increased glutamatergic neuron excitability induced by A/S and leads to oxidative stress reaction, and neuronal apoptosis.

Dataset Information

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

Publications

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

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