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. 2017 Mar;74(5):937-950.
doi: 10.1007/s00018-016-2397-5. Epub 2016 Oct 19.

CREB decreases astrocytic excitability by modifying subcellular calcium fluxes via the sigma-1 receptor

A Eraso-Pichot  1 , R Larramona-Arcas  1 , E Vicario-Orri  1   2 , R Villalonga  1 , L Pardo  1 , E Galea  3   4 , R Masgrau  5
Affiliations

CREB decreases astrocytic excitability by modifying subcellular calcium fluxes via the sigma-1 receptor

A Eraso-Pichot et al. Cell Mol Life Sci. 2017 Mar.

Abstract

Astrocytic excitability relies on cytosolic calcium increases as a key mechanism, whereby astrocytes contribute to synaptic transmission and hence learning and memory. While it is a cornerstone of neurosciences that experiences are remembered, because transmitters activate gene expression in neurons, long-term adaptive astrocyte plasticity has not been described. Here, we investigated whether the transcription factor CREB mediates adaptive plasticity-like phenomena in astrocytes. We found that activation of CREB-dependent transcription reduced the calcium responses induced by ATP, noradrenaline, or endothelin-1. As to the mechanism, expression of VP16-CREB, a constitutively active CREB mutant, had no effect on basal cytosolic calcium levels, extracellular calcium entry, or calcium mobilization from lysosomal-related acidic stores. Rather, VP16-CREB upregulated sigma-1 receptor expression thereby increasing the release of calcium from the endoplasmic reticulum and its uptake by mitochondria. Sigma-1 receptor was also upregulated in vivo upon VP16-CREB expression in astrocytes. We conclude that CREB decreases astrocyte responsiveness by increasing calcium signalling at the endoplasmic reticulum-mitochondria interface, which might be an astrocyte-based form of long-term depression.

Keywords: CEPIA indicators; Calcium signalling; Endoplasmic reticulum; MCU; Mitochondria; Mitochondria-associated membranes; VP16-CREB.

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Figures

Fig. 1
Fig. 1
Pre-stimulation with CREB-activating transmitters reduces transmitter-induced calcium responses. a Schematic representation of the protocol used. b Activation of CREB-dependent transcription by different transmitters measured with a luciferase gene-reporting assay. Control is cells not treated with any transmitter but with vehicle; otherwise cells were stimulated with 100 µM ATP, 10 µM noradrenaline (NA), 10 nM endothelin-1 (ET-1), or 100 µM glutamate (Glu) and CREB activity measured after 6 h. Representative calcium traces (c, e, g) and quantification (d, f, h) of maximum increases after the addition of 100 µM ATP (c, d), 10 µM NA (e, f), or 10 nM ET-1 (g, h) after CREB activation with ATP, NA, ET-1, or Glu, identified with color codes. *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 2
Fig. 2
Transmitter-elicited reduction of cytosolic calcium responses is transient and CREB-dependent. Astrocytes were treated with 10 µM NA to induce CREB activation. Representative traces of calcium responses induced by 100 µM ATP at 12 h (a) and 24 h (b) after CREB stimulation. c Quantification of peak calcium responses induced by ATP after 6, 12, and 24 h after CREB stimulation. The data are the mean ± SEM of 3–4 independent experiments. *P < 0.05, ***P < 0.001. d, e Representative 100 µM ATP-induced calcium traces in single cells and f quantification of calcium responses after 6 h of stimulation of CREB with 10 µM NA in Null and A-CREB-infected astrocytes. The data are the mean ± SEM of 3–4 independent experiments. **P < 0.01
Fig. 3
Fig. 3
VP16-CREB decreases calcium responses induced by transmitters. a VP16 immunocytochemistry and b western blot of VP16 in astrocytes infected with VP16-CREB or Null viral vectors. c Schematic representation of the protocol used to measured calcium responses in astrocytes infected with VP16-CREB or Null viral vectors. Representative single cell traces of calcium responses induced by 100 µM ATP (d) or 10 µM NA (f) in VP16-CREB-infected astrocytes (virus MOI 1). e, g Quantification of peak calcium responses at different viral vector loads. Data are normalised for the response of Null-infected astrocytes at the same viral vector load. The data are the mean ± SEM of 4–7 (d) or 3–4 independent experiments (f). *P < 0.05, **P < 0.01
Fig. 4
Fig. 4
VP16-CREB action on calcium signalling pathways. ATP-elicited elevation of calcium was tested in astrocytes transducing VP16-CREB with no added calcium and presence of 0.5 mM EGTA (0Ca2+), or in the presence of inhibitors of calcium release from acidic lysosomal-related stores: 50 µM GPN or 100 µM Ned-19. Control refers to cells without any treatment other than viral vector infection. Representative traces in single cells (a–c) and quantification (d) of 100 µM ATP-induced calcium responses in Null and VP16-CREB-infected astrocytes. The data are the mean ± SEM of 6–8 independent experiments. *P < 0.05, **P < 0.01. e Representative images of astrocytes transfected with the ER calcium dye G-CEPIA1er before and after 100 µM ATP application. Representative traces (f) and quantification (g) of the decrease in ER calcium. Data are mean ± SEM of four independent experiments. ***P < 0.001
Fig. 5
Fig. 5
VP16-CREB increases mitochondrial calcium uptake. a Representative traces in single cells and b quantification of intracellular calcium responses induced by 100 µM ATP in Null and VP16-CREB-infected astrocytes in astrocytes treated with FCCP to inhibit mitochondrial calcium uptake. The data are the mean ± SEM of four independent experiments. c Representative images of the astrocytes transfected with the mitochondrial calcium dye CEPIA3mt before and after 100 µM ATP application. d Representative calcium traces and e quantification of 100 µM ATP-induced mitochondrial calcium increase in Null and VP16-CREB-infected astrocytes. The data are the mean ± SEM of four independent experiments. *P < 0.05
Fig. 6
Fig. 6
Sigma-1 receptor mediates the effects of VP16-CREB. a Quantification of sigma-1 receptor mRNA expression by quantitative PCR and b representative western blot of sigma-1 receptor in astrocytes infected with Null or VP16-CREB. c Representative calcium traces in single cells and d quantification of 100 µM ATP-induced ER calcium decreases measured using G-CEPIAer in astrocytes infected with null or VP16-CREB viral vectors and in the absence and presence of BD1047, a sigma-1 receptor antagonist. e Representative calcium traces in single cells and f quantification of 100 µM ATP-induced mitochondrial calcium increases using CEPIA3mt in astrocytes infected with Null or VP16-CREB viral vectors and in the absence and presence of BD1047. The data are the mean ± SEM of 3–4 independent experiments. *P < 0.05, ****P < 0.0001
Fig. 7
Fig. 7
MCU expression decreases in VP16-CREB-expressing astrocytes. a Quantification of MCU mRNA expression in Null and VP16-CREB-infected astrocytes by quantitative PCR (a). Data are the mean ± SEM of 3–4 independent experiments. ****P < 0.0001. b Representative western blot of MCU in astrocytes infected by Null and VP16-CREB viral vectors
Fig. 8
Fig. 8
CREB decreases calcium responses and upregulates sigma-1 receptor in adult astrocytes. a Representative traces and b quantification of calcium responses induced by 100 µM ATP after 6 h of CREB stimulation (1 h) with 10 µM NA or vehicle (control). c Representative traces and d quantification calcium responses induced by 100 µM ATP in adult cultured astrocytes infected with Null or VP16-CREB viral vectors. e Quantification of sigma-1 receptor mRNA expression by quantitative PCR in Null and VP16-CREB-infected adult cultured astrocytes. In (b, d, and e), data are the mean ± SEM of 4 independent experiments. *P < 0.05. f Quantification by quantitative PCR of sigma-1 receptor mRNA expression in cortices in WT and VP16-CREB mice before (WT and VP16-CREB) and after cryolesion (WT C and VP16-CREB C). Cryolesions were performed to increase VP16-CREB expression in astrocytes conditional to gliosis. Data are the mean ± SEM of 4–5 animals in each condition. *P < 0.05, **P < 0.01, ****P < 0.0001
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