Astrocyte reactivity and inflammation-induced depression-like behaviors are regulated by Orai1 calcium channels

Abstract

Astrocytes contribute to brain inflammation in neurological disorders but the molecular mechanisms controlling astrocyte reactivity and their relationship to neuroinflammatory endpoints are complex and poorly understood. In this study, we assessed the role of the calcium channel, Orai1, for astrocyte reactivity and inflammation-evoked depression behaviors in mice. Transcriptomics and metabolomics analysis indicated that deletion of Orai1 in astrocytes downregulates genes in inflammation and immunity, metabolism, and cell cycle pathways, and reduces cellular metabolites and ATP production. Systemic inflammation by peripheral lipopolysaccharide (LPS) increases hippocampal inflammatory markers in WT but not in astrocyte Orai1 knockout mice. Loss of Orai1 also blunts inflammation-induced astrocyte Ca²⁺ signaling and inhibitory neurotransmission in the hippocampus. In line with these cellular changes, Orai1 knockout mice showed amelioration of LPS-evoked depression-like behaviors including anhedonia and helplessness. These findings identify Orai1 as an important signaling hub controlling astrocyte reactivity and astrocyte-mediated brain inflammation that is commonly observed in many neurological disorders.

Fig. 1. Orai1 mediates SOCE in astrocytes.

a Immunohistochemistry of WT hippocampal astrocytes in culture showing expression of Orai1 in the soma and processes. Orai1 labeling (with a monoclonal antibody, Abcam, M266.1) is lost in astrocytes from Orai1 KO (Orai1^{fl/fl GFAP-Cre}) mice. Scale bar: 25 µm. b Quantification of Orai(1-3) and STIM(1-2) mRNAs via two-step qPCR in WT and Orai1^{fl/fl GFAP-Cre} astrocytes. The expression of Orai2-3 and STIM1-2 isoforms is not altered in Orai1 cKO astrocytes. Data are normalized to GAPDH and further normalized to the expression of the corresponding WT mRNA in each case. Mean +/-SEM, n = 6 mice for each Orai isoform and n = 3 mice for each STIM isoform. c Orai1 mRNA is unaffected in microglia from Orai1^{fl/fl Aldh1l1-Cre/ERT2} mice. n = 4 cultures from 2 mice/group. d–f SOCE is abolished in Orai1^{fl/fl Aldh1l1-Cre/ERT2} astrocytes exposed to 4-OH tamoxifen (1 µM). SOCE was induced by depleting ER Ca²⁺ stores with thapsigargin (TG, 1 µM) administered in a 0 Ca²⁺ Ringer’s solution. Re-addition of extracellular Ca²⁺ (2 mM) reveals SOCE in WT but not in Orai1^{fl/fl Aldh1l1-Cre/ERT2} astrocytes. SOCE in WT astrocytes is also blocked by La³⁺ (2 µM). Panel E summarizes the rate of Ca²⁺ influx (nM/sec) following re-addition of extracellular Ca²⁺ following store depletion. Panel F summarizes the total amount of calcium over baseline entering the cell for five minutes after Ca²⁺ re-addition. g Ca²⁺ release from stores is unaffected in Orai1^{fl/fl Aldh1l1-Cre/ERT2} astrocytes. The area under the curve (A.U.C.) indicates the total amount of calcium over baseline released for the three minutes following TG application in 0 mM Ca²⁺. Data were analyzed from the same cells for panels E-G. n = 56 cells from 3 WT mice and 66 cells from 3 Orai1 cKO mice. Data are presented as mean values +/- SEM. The sex of the pups was not determined. Statistical tests were conducted by two-tailed, unpaired T-tests. Source data are provided as a Source Data file.

Fig. 2. RNA-sequencing reveals that stimulus-evoked inflammatory pathways are reduced in Orai1 cKO astrocytes.

Orai1^fl/fl (WT) and Orai1^{fl/fl GFAP-Cre} astrocytes were stimulated with a low dose (0.2 µM) of TG + 50 nM PDBu (or DMSO) for 6 h and differentially expressed genes were examined by RNA-seq analysis. a MA plot (log-intensity fold change vs log mean expression) showing differential gene expression between stimulated WT and Orai1 cKO astrocytes. Blue dots denote genes expressed significantly more in WT cells and red indicates genes expressed significantly more in Orai1 cKO astrocytes. Gray dots denote genes that were not significantly different between groups (p < 0.05, adjusted for multiple comparisons). b Pathway enrichment scores of selected reactome pathways related to inflammation. The x-axis indices show enrichment in WT-treated over KO-treated cells. c Heatmap of z-normalized within-sample expression of significantly variable genes in the “Interleukin-1 signaling” pathway from panel b. Both the treatment (TG+PdBu) and the genotype (WT vs Orai1 KO) have a strong influence on the relative expression of genes in this pathway. d–g Gene-set enrichment analysis (GSEA) in astrocytes following cell stimulation. The GSEAs indicate the extent to which all identified genes within the IL-1 signaling (d), NFκB activation (e), IFN-γ signaling (f), and ROS (g) reactome pathways are enriched in the WT or Orai1 cKO cells. Each vertical line on the x-axis represents a gene. h, i Enrichment of genes regulated by NFAT (h) or CREB (i) (shown on log₂ scale). NFAT- and CREB-response genes were collated from previous studies^{, –}. n = 2 female, 3 male WT mice, 1 male, 4 female KO mice. Source data are provided as a Source Data file.

Fig. 3. Orai1 signaling stimulates metabolic reprogramming in astrocytes.

a Differentially expressed genes (shown on Log₂ scale) related to glycolysis and glucose metabolism induced by Orai1 deletion. Astrocytes were stimulated with a low dose (0.2 µM) of TG + 50 nM PDBu for 6 h. b Enrichment highlights (of the 25 most enriched pathways) assessed by Quantitative Enrichment Analysis with the Global Test for statistical analysis (metaboanalyst.ca). Bars denote the enrichment ratio in stimulated control over Orai1 KO cells. c–e Relative metabolite quantities of individual metabolites in the glycolysis (c), glutamate metabolism (d), and urea cycles. Data are shown as a heat map with each box representing values from an individual astrocyte culture from one mouse. Red depicts the maximum and blue minimum for each row. n = 3 mice/group. f Relative levels of ATP, ADP, and AMP. Orai1 cKO astrocytes show reduced levels of ATP and ADP. g Within-sample ATP/AMP ratios. (h, i) Relative levels of NAD⁺ and NADH, and NADP⁺ and NADPH in astrocytes. j Nadk expression using gene counts from the RNAseq experiment in Fig. 2. (n = 5 mice/group). b–i n = 3 mice in each genotype. Bars indicate Mean +/- SEM. The sex of the pups was not determined. Statistical tests were conducted by two-tailed, unpaired T-tests. Source data are provided as a Source Data file.

Fig. 4. Orai1 activation stimulates the production of pro-inflammatory cytokines from astrocytes.

a Induction of inflammatory cytokines and chemokines assessed by real-time PCR. WT (Orai1^fl/fl) and Orai1 KO (Orai^{fl/fl GFAP-cre}) astrocytes were stimulated for 6 h with a low dose (0.2 µM) of TG + 50 nM PDBu and cytokines were measured via two-step real-time PCR. mRNA levels were normalized to GAPDH. (n = 4 mice/group). b PGE-2 release from astrocytes measured by ELISA. (n = 4 WT mice, 3 KO mice.) c, d Anti-inflammatory cytokines assessed by qPCR (c) and ELISA (d) (n = 4 mice/group). e Induction of proinflammatory mediators by thrombin. Cytokines were measured by real-time PCR in astrocytes treated with thrombin (10 U/ml, 6 h) (n = 5 WT mice, 4 KO mice). f, g Activation of CRAC channels induces nuclear translocation of NFAT-GFP. WT (Orai1^fl/fl) (f) or Orai1^{fl/fl GFAP-cre} astrocytes (g) astrocytes were transfected with NFATc3-GFP to monitor NFAT dynamics. Cells were incubated with NucBlue for 20 min prior to imaging to visualize the nucleus. Cells were stimulated for 15 min with TG (1 µM) in 2 mM Ca²⁺ Ringer’s solution to stimulate Orai1 activity. h Quantification of NFAT-GFP nuclear translocation (ratio of the mean GFP intensity in the nuclear and cytosolic regions). Data are displayed as the nuclear/cell GFP fluorescence ratio (n = 8 cells from 3 mice/group). Scale bar = 10 µm. For all summary data, the bars indicate mean +/- SEM. For panel a: data separated by sex are provided in Supplementary Fig. 6. For panels b–h: the sex of the pups was not determined. Statistical tests were conducted by two-way ANOVA followed by Tukey posthoc tests for panels a–e, and paired t-test for panel h. Source data are provided as a Source Data file.

Fig. 5. Expression of neuroinflammatory markers following LPS challenge is reduced in Orai1 cKO mice.

a A schematic of the protocol used for the experiment. Orai1^fl/fl (WT) and Orai1^{fl/fl Aldh1l1-Cre/ERT2} mice were injected with tamoxifen for five days to induce the deletion of Orai1. LPS or saline was delivered as indicated on day 10 following the last tamoxifen injection. Animals were sacrificed 2 days later and analyzed by immunohistochemistry and ELISA. b, c Immunohistochemistry of hippocampal GFAP (to label astrocytes) and IBA1 (to label microglia) in the dentate gyrus and the CA1 regions of the hippocampus. The images shown were obtained from male mice; female mice showed a similar pattern (Supplementary Fig. 8). Scale bar = 50 µm. d Quantification of GFAP and IBA1 expression assessed by measuring the % area of the region of interest occupied by fluorescent signal. Data are given as means +/- SEM for n = 14–18 images from 3 WT saline mice, n = 14–20 images from 5 WT LPS mice, n = 11–13 images from 3 KO saline mice, n = 10–20 images from 4 KO LPS mice. e Levels of IL-1α and IL-6 measured in homogenized hippocampal tissue lysates by ELISA. (IL-1α - WT saline group: n = 5 male, 2 female mice; WT LPS group: n = 6 male, 2 female mice; KO saline group: n = 2 male, 2 female mice; KO + LPS group: n = 3 male, 3 female mice. IL-6 - WT saline group: n = 5 male, 2 female mice; WT LPS group: n = 5 male, 2 female mice; KO saline group: n = 1 male, 3 female mice; KO LPS group: n = 5 male, 2 female mice. f Peripheral LPS administration increases thrombin levels in the brain. Thrombin (A) and SDF-1α (B) were measured via ELISA in homogenized hippocampal tissue lysates in saline (white bars) and LPS-injected (black bars) mice 48 h after injection. Thrombin is significantly elevated in LPS-treated WT and cKO mice, while SDF1α is modestly elevated. (WT saline group: n = 3 female and 5 male mice. WT LPS group, n = 3 female and 5 male mice. Orai1 cKO saline group: n = 3 female, 1 male mice, Orai1 cKO LPS group: n = 4 male, 2 female KO mice). All data are given as mean +/- SEM. Statistical tests were conducted for the pooled data by two-way ANOVA followed by Tukey posthoc tests in each graph. Source data are provided as a Source Data file.

Fig. 6. LPS-evoked increases in astrocyte Ca²⁺ signaling are abolished in Orai1 cKO mice.

a Schematic illustrating experimental protocol. GCaMP6f was expressed in astrocytes of the hippocampus through stereotaxic injections of an AAV5 virus with an astrocyte-specific gfaABC1D promoter. After allowing 2-3 weeks for GCamp6f expression, mice were intraperitoneally injected with either 1 mg/mL LPS or equivalent volume of saline. 24-hours following intraperitoneal LPS administration, Ca²⁺ fluctuations in astrocytes expressing GCaMP6 were imaged using 2PLSM in the Stratum Radiatum region of the CA1 hippocampus. b 2-P images of astrocytes transfected with gCAMP6f. Each image is the maximum intensity projection of the time series (180 s). Scale bar = 20 µm. c Traces of the ∆F/F₀ values from the soma, primary branches, and distal branches of astrocytes in brain slices from WT and Orai1 cKO mice. Mice were administered either saline or LPS as indicated. Sample images and traces are representative of experiments performed on 10–16 cells/group from 3 mice/group. d, e Summary graphs of the frequency (d) and amplitude (e) of calcium transients calculated over three minutes of imaging. Data are presented as mean values +/- SEM. Each dot represents one ROI (amplitude is the average of all ∆F/F₀ peaks in one ROI, frequency is the total # of events/3 min), and statistical tests were performed on the mean amplitude/frequency of all ROIs pertaining to a given cell. WT saline: n = 16 cells from 2 male, 1 female mice; WT + LPS: n = 15 cells from 2 female, 1 male mice; cKO+saline: n = 10 cells from 1 female, 2 male mice; cKO+LPS: n = 13 cells from 1 female, 2 male mice. Statistical tests were conducted by two-way ANOVA followed by Tukey posthoc tests for each graph. Illustrations created with BioRender.com. Source data are provided as a Source Data file.

Fig. 7. Astrocyte Orai1 cKO mice show distinct changes in inhibitory synaptic transmission following LPS challenge.

a Schematic of the experimental timeline. WT and Orai1 cKO mice were injected with LPS intraperitoneally and brains were removed for slice electrophysiology ~18-20 h later. b Schematic of the slice-patch clamp recordings carried out in panels C-H. Whole-cell recordings were carried out in CA1 pyramidal neurons to record excitatory and inhibitory postsynaptic currents. c, d Example traces of sIPSCs in the indicated conditions and genotypes. e–h Summary graphs of the amplitude and frequency of sIPSCs. The amplitude and frequency were reduced in LPS administered Orai1 cKO mice but not WT mice. Data are presented as mean values +/- SEM. (e, g). Cumulative distribution graphs showing amplitude and inter-event interval (IEI) for WT (f) and cKO (h) mice illustrate the effect of LPS on Orai1 cKO mice. The number of recordings were as follows: n = 9 WT cells from 3 male & 1 female mouse, n = 13 WT + LPS cells from 2 female & 3 male mice, n = 11 WT + LPS+Thr cells from 2 female & 3 male mice, n = 5 cKO cells from 4 male mice, n = 6 cKO+LPS cells from 2 male & 1 female mice, n = 6 cKO+LPS+Thr cells from 2 male & 1 female mice. Statistical tests were conducted by one-way ANOVA followed by Tukey posthoc tests for all comparisons. Illustrations created with BioRender.com. Source data are provided as a Source data file.

Fig. 8. Astrocyte-specific Orai1 KO mice are protected in a model of inflammation-induced depression-like behaviors.

a, b Schematics illustrating the timeline of the experiments. Tamoxifen was injected intraperitonially (i/p) for Cre-induced deletion of Orai1. LPS or saline was administered i/p nine days later to induce systemic inflammation. Tests for general locomotion (open-field) and depression-like behaviors (sucrose preference test (SPT), forced swim test (FST), and tail suspension test (TST)) were performed beginning 24 h after the LPS administration. c–e Analysis of depression-like behaviors following LPS administration. WT male mice consistently showed depression-like behaviors after administration of LPS, manifested as significantly decreased sucrose preference scores in the SPT and increased immobility times in the FST and TST. Orai1 cKO mice did not show these changes (n = 13 WT Sal and 13 WT LPS mice, 10 KO Sal mice, n = 8 KO LPS mice). f, g Mouse sickness behavior analysis including murine sepsis scores (MSS) (f) and general locomotion (g). MSS is increased and general locomotion is decreased six hours post LPS administration in both WT and cKO mice but normalizes at 24 h. (n = 14 WT saline, n = 13 WT LPS, n = 10 Orai1 cKO saline, n = 8 Orai1 cKO LPS.) h Astrocyte Orai1 cKO mice do not show deficits in working memory as assessed in the Y-maze (n = 7 WT, n = 8 cKO). i, j Astrocyte Orai1 cKO mice do not show deficits in associative memory as assessed in the fear conditioning test. Fear conditioning was quantified by measuring the freezing duration in the indicated context. (n = 7 WT, n = 5 cKO) k, l cKO mice also show no deficits in general anxiety as measured by time spent in exposed areas of the Open Field (k) and Zero Maze (l). (n = 14 WT, n = 10 cKO (OFT), n = 8 cKO (Zero Maze)). All data are presented as mean values +/- SEM. All data are from male mice. Statistical tests were conducted by two-way ANOVA followed by Tukey posthoc tests in each graph. Illustrations created with BioRender.com. Source data are provided as a Source Data file.