Store-operated Ca2+ entry-dependent Ca2+ refilling in the endoplasmic reticulum in astrocytes

Biochemical and Biophysical Research Communications xxx (xxxx) xxx

Contents lists available at ScienceDirect

Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc

Store-operated Ca2þ entry-dependent Ca2þ refilling in the endoplasmic reticulum in astrocytes Yohei Okubo a, *, Masamitsu Iino b, Kenzo Hirose a a b

Department of Pharmacology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 133-0033, Japan Division of Cellular and Molecular Pharmacology, Nihon University School of Medicine, 30-1 Oyaguchi Kamicho, Itabashi-ku, Tokyo, 173-8610, Japan

a r t i c l e i n f o

a b s t r a c t

Article history: Received 27 November 2019 Accepted 1 December 2019 Available online xxx

Astrocytes regulate various brain functions, for which Ca2þ release from the endoplasmic reticulum (ER) often play crucial roles. Because astrocytic ER Ca2þ release is robust and frequent, the ER Ca2þ refilling mechanism should be critical for ongoing Ca2þ signaling in astrocytes. In this study, we focused on the putative functional significance of store-operated Ca2þ entry (SOCE) in ER Ca2þ refilling. We expressed the ER luminal Ca2þ indicator G-CEPIA1er in astrocytes in acute cortical slices to directly monitor the decrease and recovery of ER Ca2þ concentration upon spontaneous or norepinephrine-induced Ca2þ release. Inhibition of SOCE significantly slowed the recovery of ER Ca2þ concentration after Ca2þ release in astrocytes. This delayed recovery resulted in a prolonged decrease in the ER Ca2þ content in astrocytes with periodic spontaneous Ca2þ release, followed by the attenuation of cytosolic Ca2þ responses upon Ca2þ release. Therefore, our results provide direct evidence for the physiological significance of SOCE in ER Ca2þ refilling after ER Ca2þ release. © 2019 Elsevier Inc. All rights reserved.

Keywords: Astrocyte Calcium Endoplasmic reticulum Inositol 1,4,5-trisphosphate receptor Store-operated calcium entry

1. Introduction Astrocytes, a major type of glial cell in the brain, show a wide variety of intracellular Ca2þ signaling patterns, which mediate astrocytic functions crucial for the physiological and pathophysiological events in the brain [1]. While Ca2þ release from the endoplasmic reticulum (ER) via the inositol 1,4,5-trisphosphate receptor (IP3R) has been recognized as a major component of spontaneous and Gq-coupled receptor-induced Ca2þ signaling in astrocytes, other Ca2þ mobilization mechanisms have also attracted attention [2]. Store-operated Ca2þ entry (SOCE) is a ubiquitous and major mechanism of Ca2þ influx across the plasma membrane in nonexcitable cells, including astrocytes [3]. A decrease in the luminal Ca2þ concentration within the ER ([Ca2þ]ER) evokes SOCE by inducing an interaction between stromal interaction molecule (STIM), the ER-resident Ca2þ sensor, and Ca2þ channels, including Orai and transient receptor potential (TRP) families [4]. A couple of significant functions in Ca2þ signaling have been postulated for SOCE. One is to boost Ca2þ signaling. SOCE after ER

* Corresponding author. Department of Pharmacology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan. E-mail address: [email protected] (Y. Okubo).

Ca2þ release augments and prolongs an elevation of cytosolic Ca2þ concentration ([Ca2þ]Cyt) to regulate various cellular functions [4,5]. In astrocytes, SOCE is reported to mediate the Ca2þ-dependent release of gliotransmitters, such as glutamate and ATP [6,7]. The other function is to preserve intracellular Ca2þ homeostasis. SOCE has been suggested to be crucial for refilling the ER with Ca2þ after ER Ca2þ release [4,5]. This notion has been experimentally supported by assays of ER Ca2þ refilling using canonical Ca2þ add-back protocols, in which SOCE is elicited by immediate add-back of Ca2þcontaining medium after complete ER Ca2þ depletion in a Ca2þ-free medium. This Ca2þ add-back-induced SOCE resulted in ER Ca2þ refilling via sarco/endoplasmic reticulum Ca2þ-ATPase (SERCA)dependent Ca2þ uptake [8,9]. However, it remains elusive whether SOCE is actually indispensable for ER Ca2þ refilling under physiological conditions [10e12]. To address this issue, it is necessary to directly monitor [Ca2þ]ER dynamics during complex Ca2þ signaling in astrocytes in situ. We recently developed a series of calcium-measuring organelleentrapped protein indicators (CEPIAs) that allow visualization of Ca2þ dynamics within the ER with a high signal-to-noise ratio [13,14]. Using G-CEPIA1er, we recently revealed that IP3R-mediated Ca2þ release results in a significant decrease in [Ca2þ]ER in astrocytes [15,16]. In this study, we evaluated the involvement of SOCE in ER-mediated Ca2þ signaling in astrocytes of acute cortical slices. We

https://doi.org/10.1016/j.bbrc.2019.12.006 0006-291X/© 2019 Elsevier Inc. All rights reserved.

Please cite this article as: Y. Okubo et al., Store-operated Ca2þ entry-dependent Ca2þ refilling in the endoplasmic reticulum in astrocytes, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.006

2

Y. Okubo et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx

found that the inhibition of SOCE significantly slowed the recovery of [Ca2þ]ER after spontaneous or norepinephrine (NE)-induced Ca2þ release from the ER and resulted in a sustained decrease in [Ca2þ]ER. This [Ca2þ]ER decrease attenuated the [Ca2þ]Cyt increase that follows spontaneous ER Ca2þ release. These results indicate the crucial role of SOCE in refilling ER Ca2þ to support ER-mediated Ca2þ signaling in astrocytes. 2. Materials and methods 2.1. Preparation of viral vectors To generate adeno-associated viruses (AAVs) for astrocytespecific expression, the cytomegalovirus promoter of pAAV-MCS (AAV Helper Free Expression System, Cell Biolabs, Inc., San Diego, CA, USA) was replaced with the gfaABC1D astrocyte-specific promoter [17]. AAV5 vectors were packaged using the AAV Helper Free Expression System. The packaging plasmids (pAAV-RC5 and pHelper) and transfer plasmid (pAAV-gfaABC1D-G-CEPIA1er or pAAV-gfaABC1D-GCaMP6f) were transfected into HEK293T cells using the calcium phosphate method. The medium was replaced 18 h after transfection with fresh medium, and the cells were incubated for 48 h. Harvested cells were lysed by repeated freezing and thawing, and AAV5 vector particles were purified by ultracentrifugation with cesium chloride. The purified particles were dialyzed against PBS and then concentrated by ultrafiltration using an Amicon 10K MWCO filter (Merck Millipore, Darmstadt, Germany). The number of copies of the viral genome (vg) was determined by real-time quantitative PCR. 2.2. AAV injection All animal experiments were carried out in accordance with the regulations and guidelines of the Institutional Animal Care and Use Committee at The University of Tokyo and were approved by the Institutional Review Committee of the Graduate School of Medicine, The University of Tokyo. Male C57BL/6 mice (postnatal day 56e98) were anesthetized with isoflurane (induction at 5%, maintenance at 1.5e2%, MK-A100, Muromachi, Kyoto, Japan). The mice were placed in a stereotaxic frame (SR-5M-HT, Narishige, Tokyo, Japan). The skull was thinned above the right parietal cortex using a burr powered by a high-speed drill (ULTIMATE XL-D, NSK, Kanuma, Japan). AAV5-gfaABC1D-G-CEPIA1er (0.98 or 1.3  1013 vg mL1) or AAV5-gfaABC1D-GCaMP6f (1.1  1013 vg mL1) was unilaterally injected into the cortex (1.5e2 mm posterior to the bregma, 1e1.5 mm lateral to the midline, and 300 mm from the surface) through glass pipettes. A viral solution (1 mL) was delivered at a rate of 100 nL min1 using a micropump (Legato 130, KD scientific, Holliston, MA, USA). Glass pipettes were left in place for at least 10 min. Mice were sacrificed at 14e28 days after AAV injection for imaging. 2.3. Preparation and imaging of brain slices Coronal cortical slices (300 mm thick) were prepared as described previously [18]. Slices were prepared in ice-cold artificial cerebrospinal fluid (ACSF) bubbled with 95% O2 and 5% CO2 using a vibrating slicer (PRO7, Dosaka, Kyoto, Japan). Slices were incubated in a holding chamber containing ACSF bubbled with 95% O2 and 5% CO2 at 35  C for 1 h and then returned to room temperature (22e24  C). The ACSF contained 125 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 1 mM MgSO4, 1.25 mM NaH2PO4, 26 mM NaHCO3, and 20 mM glucose. Imaging was carried out with a two-photon microscope (TSC MP5, Leica, Wetzlar, Germany) equipped with a waterimmersed objective lens (25  , NA 0.95, HCS IR APO, Leica) and

Ti:sapphire laser (MaiTai DeepSee, Spectra Physics, Santa Clara, CA, USA). Slices were transferred to a recording chamber under a microscope and continuously perfused with ACSF bubbled with 95% O2 and 5% CO2. Tetrodotoxin (1 mM) was added to ACSF to inhibit neuronal activity throughout imaging. Nominally Ca2þ-free ACSF contained 125 mM NaCl, 2.5 mM KCl, 3 mM MgSO4, 1.25 mM NaH2PO4, 26 mM NaHCO3, and 20 mM glucose. The excitation wavelength was 900e920 nm. Emitted fluorescence was filtered by a barrier filter (500e550 nm) and detected with photomultiplier tubes. Data were acquired in time-lapse XY-scan mode (0.2e0.5 Hz). Experiments were carried out at room temperature.

2.4. Data analysis Data were analyzed using ImageJ software. Fluorescence intensities were normalized by the average of the first 10 frames to calculate the fractional changes in fluorescence intensity (DF/F0). A whole astrocyte was selected as the region of interest to measure DF/F0. For statistical analyses, Student’s t-test was performed.

3. Results 3.1. SOCE in astrocytes in situ Although studies of astrocytic SOCE have been extensively performed using dissociated primary culture, analysis in intact brain samples, such as acute brain slices, has been limited to date [19,20]. Therefore, we first confirmed the presence and properties of SOCE in astrocytes in acute cortical slices. We used a serotype 5 AAV carrying the minimal astrocyte-specific gfaABC1D promoter to express cytosolic Ca2þ indicator GCaMP6f in astrocytes of the adult mouse cortex [17]. GCaMP6f-expressing astrocytes in acute cortical slices were imaged using a two-photon microscope (Fig. 1A). Astrocytes often show cell-wide spontaneous [Ca2þ]Cyt increases mediated by Ca2þ release from the ER via IP3R [15]. In this study, the synchronous changes in GCaMP6f fluorescence throughout a single astrocyte were detected by visual inspection and defined as spontaneous Ca2þ responses. To induce ER Ca2þ depletion, we applied cyclopiazonic acid (CPA, 50 mM), a SERCA inhibitor, with nominally Ca2þ-free ACSF (0Ca, Fig. 1B). The appearance of spontaneous Ca2þ responses were transiently enhanced after CPA treatment presumably due to the exaggeration of [Ca2þ]Cyt increases in the absence of SERCA activity (Fig. 1B). The spontaneous Ca2þ responses then disappeared during 10-min-treatment with CPA, indicating the depletion of ER Ca2þ (Fig. 1B). We subsequently applied Ca2þ by perfusing normal ACSF containing 2 mM Ca2þ (2Ca, Fig. 1B). This Ca2þ add-back treatment induced a [Ca2þ]Cyt increase, confirming the presence of SOCE in astrocytes in situ. We evaluated the potency of typical SOCE inhibitors in our experiment. Treatment with 2-aminoethoxydiphenyl borate (2APB, 100 mM), a widely used inhibitor of SOCE [4], almost completely abolished CPA-induced SOCE in astrocytes (Fig. 1B and C). SKF96365 (100 mM), a SOCE-inhibiting imidazole derivative [4], partially blocked CPA-activated SOCE (Fig. 1B and C). Lanthanides, including La3þ and Gd3þ, have been widely used as a potent SOCE inhibitor [4]. However, Gd3þ (1 mM) did not show significant blockage of SOCE under our experimental conditions (Fig. 1B and C). Although the underlying mechanisms of these pharmacological properties should be addressed in a future study (see also the Discussion), we decided to use 2-APB to inhibit SOCE in astrocytes in cortical slices in subsequent experiments.

Please cite this article as: Y. Okubo et al., Store-operated Ca2þ entry-dependent Ca2þ refilling in the endoplasmic reticulum in astrocytes, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.006

Y. Okubo et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx

3

Fig. 2. SOCE-dependent ER Ca2þ refilling in astrocytes after NE-induced Ca2þ release. (A): A G-CEPIA1er-expressing astrocyte in the acute cortical slice. Scale bar, 10 mm. (B): NE-induced [Ca2þ]ER decrease followed by SOCE-dependent recovery. Individual DF/F0 traces (gray) and average traces (black) of G-CEPIA1er upon NE application (10 mM, 1 min) in the presence or absence of 2-APB are shown. A whole astrocyte was selected as the region of interest to measure DF/F0. 2-APB significantly slowed the recovery. n ¼ 8 cells for control, 8 cells for 2-APB. (C): Summary of peak amplitude of NE-induced response (mean ± SEM). 2-APB showed no significant effect. (D): Summary of recovery rate after NE-induced [Ca2þ]ER decrease (mean ± SEM). Recovery rate was defined as percent recovery from the peak at the time point 6 min after NE application. ***P < 0.001 in unpaired Student’s t-test.

Fig. 1. SOCE in astrocytes in acute cortical slices. (A): A GCaMP6f-expressing astrocyte in the acute cortical slice. Scale bar, 10 mm. (B): SOCE in astrocytes in the absence or presence of SOCE inhibitors. Individual DF/F0 traces (gray) and average traces (black) of GCaMP6f are shown. A whole astrocyte was selected as the region of interest to measure DF/F0. ER Ca2þ depletion was induced by CPA (50 mM) with nominally Ca2þ-free ACSF (0Ca). SOCE was then evoked by applying normal ACSF containing 2 mM Ca2þ (2Ca). SOCE was completely disrupted by 2-APB (100 mM), while SKF96365 (SKF, 100 mM) and Gd3þ (1 mM) showed limited inhibitory effect. n ¼ 11 cells for control, 10 cells for 2-APB (magenta), 9 cells for SKF (cyan), and 11 cells for Gd3þ (green). (C): Summary of pharmacological analysis of SOCE shown in (B) [mean ± standard error of the mean (SEM).] The magnitude of SOCE was measured by the area under the curve of GCaMP6f DF/F0 traces during the Ca2þ add-back period. Asterisks indicate the statistical significance from unpaired Student’s t-tests: **P < 0.01, ***P < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

3.2. SOCE-dependent ER Ca2þ refilling in astrocytes To directly clarify the functional significance of SOCE in ER Ca2þ refilling in astrocytes, we visualized [Ca2þ]ER dynamics using G-

CEPIA1er. G-CEPIA1er was expressed in cortical astrocytes using AAV and imaged with a two-photon microscope as described above (Fig. 2A). To evaluate SOCE-dependent ER Ca2þ refilling after [Ca2þ]ER decrease in astrocytes, we evoked ER Ca2þ release by applying an endogenous ligand of Gq-coupled receptor. Norepinephrine (NE) induces cell-wide Ca2þ release from the ER through adrenergic receptor-IP3 signaling in astrocytes in acute slices and in vivo [21,22]. Bath application of NE (10 mM) induced a cell-wide decrease in [Ca2þ]ER in astrocytes, as shown in our previous study [15] (Fig. 2B). Following washout of NE, a recovery of [Ca2þ]ER was observed (Fig. 2B and D). In the presence of 2-APB, the recovery of [Ca2þ]ER after a NE-induced decrease was significantly slowed (Fig. 2B and D). This result clearly indicates the indispensable role of SOCE in ER Ca2þ refilling after Ca2þ release via IP3R in astrocytes. In contrast, the amplitude of NE-induced [Ca2þ]ER decreases was not affected by 2-APB (Fig. 2C). Although 2-APB had previously been reported to inhibit IP3R [23], our result is consistent with the report showing little or no effect of 2-APB on IP3R [24].

Please cite this article as: Y. Okubo et al., Store-operated Ca2þ entry-dependent Ca2þ refilling in the endoplasmic reticulum in astrocytes, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.006

4

Y. Okubo et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx

3.3. SOCE is essential for astrocytic Ca2þ signaling Astrocytes often show spontaneous ER Ca2þ release via IP3R. These spontaneous responses result in cell-wide [Ca2þ]ER decreases, as in the case of NE-induced responses [15]. Therefore, SOCE-dependent ER Ca2þ refilling should be crucial for the maintenance of ER Ca2þ content and ER-mediated Ca2þ signaling in astrocytes. To address this issue, we inhibited SOCE in astrocytes with and without spontaneous responses. In a subset of astrocytes, no spontaneous [Ca2þ]ER response was observed (8 cells among 33 cells, Fig. 3A). These spontaneousresponse-free astrocytes showed a slight and gradual decrease in [Ca2þ]ER upon the inhibition of SOCE by 2-APB, while no changes were observed in the control condition (Fig. 3A and B). This suggests that a background level of SOCE is present under normal [Ca2þ]ER conditions and at least partially contributes to the maintenance of the ER Ca2þ content in astrocytes. In contrast, the majority of astrocytes showed spontaneous cellwide [Ca2þ]ER decreases (25 cells among 33 cells, Fig. 3A). Here, synchronous changes in G-CEPIA1er fluorescence throughout a single astrocyte were detected by visual inspection and defined as spontaneous responses, as in the case of cytosolic GCaMP6f. These spontaneous-response-positive astrocytes show transient [Ca2þ]ER decreases, followed by recovery to the baseline (Fig. 3A and B). In the presence of 2-APB, only a partial recovery of [Ca2þ]ER was observed after each spontaneous [Ca2þ]ER decrease, which resulted in a stepwise decrease in [Ca2þ]ER over time (Fig. 3A and B). Therefore, inhibition of SOCE leads to prolonged decline of ER Ca2þ content and attenuation of spontaneous responses (Fig. 3B and C). We finally evaluated the effect of SOCE inhibition-induced partial ER Ca2þ depletion on [Ca2þ]Cyt dynamics. Spontaneous [Ca2þ]Cyt fluctuations in astrocytes were visualized using GCaMP6f (Fig. 4A). We inhibited SOCE by 2-APB for 20 min to induce sufficient [Ca2þ]ER decrease in spontaneous-response-positive astrocytes, as observed above (Fig. 3B). The amplitude of spontaneous [Ca2þ]Cyt fluctuations was reduced after the 2-APB treatment (Fig. 4A and B). This 2APB-dependent attenuation of cytosolic responses is not due to the inhibition of IP3R, as shown above (Fig. 2C). Furthermore, the frequency of spontaneous [Ca2þ]Cyt fluctuations was not affected by 2APB, indicating no unexpected effect of 2-APB on astrocytic IP3-IP3R signaling (Fig. 4C). Therefore, these results indicate the crucial role of SOCE for ongoing ER-mediated Ca2þ signaling in astrocytes. 4. Discussion In this study, we clarified the functional significance of SOCE in ER Ca2þ refilling in astrocytes in situ through direct monitoring of ER Ca2þ dynamics using G-CEPIA1er. SOCE-dependent ER Ca2þ refilling was observed after Ca2þ release via IP3R, and we showed it to be crucial for maintaining the ER Ca2þ content and intracellular Ca2þ signaling in astrocytes. Therefore, we believe that this study provides the first direct evidence showing that SOCE-dependent Ca2þ refilling is indispensable for physiological ER-mediated Ca2þ signaling. We observed a modest and gradual decrease in [Ca2þ]ER upon 2APB treatment in spontaneous-response-free astrocytes (Fig. 3A and B). Consistent with this observation, it was recently reported that the SOCE inhibitor DPB162-AE, a 2-APB analogue, gradually decreases the basal [Ca2þ]ER in the resting condition in cultured astrocytes [25]. Although these results imply that the constitutive activity of SOCE is present to support Ca2þ homeostasis, we do not exclude the possibility that 2-APB-dependent inhibition of SERCA [26] and/or enhancement of ER Ca2þ leak [27] would contribute partly to the modest [Ca2þ]ER decrease. However, in contrast to the modest effect of 2-APB on [Ca2þ]ER, the SERCA inhibitor CPA

Fig. 3. SOCE-dependent ER Ca2þ refilling in astrocytes after spontaneous Ca2þ release. (A): 2-APB-induced ER Ca2þ decline in astrocytes with and without spontaneous Ca2þ release. Representative DF/F0 traces of G-CEPIA1er were shown to indicate changes in [Ca2þ]ER. A whole astrocyte was selected as the region of interest to measure DF/F0. Astrocytes without spontaneous G-CEPIA1er response [response ()] showed a slight and gradual decrease in [Ca2þ]ER upon 2-APB treatment. Astrocytes with spontaneous G-CEPIA1er responses [response (þ)] showed step-wise [Ca2þ]ER decreases in the presence of 2-APB. (B): Summary of time course of 2-APB-induced ER Ca2þ decline in astrocytes with and without spontaneous Ca2þ response. G-CEPIA1er DF/F0 values were summarized at each time point (mean ± SEM). Time 0 was set at the start of 2-APB treatment. n ¼ 4 cells for response ()/control, 4 cells for response ()/2-APB, 13 cells for response (þ)/control, and 12 cells for response (þ)/2-APB. *P < 0.05 and ***P < 0.001 in unpaired Student’s t-test. (C): Peak amplitude of repetitive spontaneous responses. The first (1st) responses and the second or third (2nd/3rd) responses during each recording were analyzed. In the presence of 2-APB, the 2nd/3rd responses were reduced compared with the 1st responses. n ¼ 13 responses (1st) and 10 responses (2nd/3rd) from 13 cells for control, 12 responses (1st), and 11 responses (2nd/3rd) from 12 cells for 2-APB. ***P < 0.001 in unpaired Student’s t-test.

induces complete depletion of [Ca2þ]ER in spontaneous-responsefree astrocytes in about 700 s (See Fig. 1b of reference [15]). Therefore, these putative side effects of 2-APB, if present, should have only a minor contribution to the ER Ca2þ dynamics in astrocytes.

Please cite this article as: Y. Okubo et al., Store-operated Ca2þ entry-dependent Ca2þ refilling in the endoplasmic reticulum in astrocytes, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.006

Y. Okubo et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx

5

Abbreviations AAV ACSF CEPIA CPA ER IP3R NE SERCA SOCE STIM TRP 2-APB

adeno-associated virus artificial cerebrospinal fluid calcium-measuring organelle-entrapped protein indicator cyclopiazonic acid endoplasmic reticulum inositol 1,4,5-trisphosphate receptor norepinephrine sarco/endoplasmic reticulum Ca2þ-ATPase store-operated Ca2þ entry stromal interaction molecule transient receptor potential 2-aminoethoxydiphenyl borate

Funding This work was supported by the Japan Society for the Promotion of Science KAKENHI (16K08543 and 19K06936), and by a grant from the Tokyo Society of Medical Sciences. References 2þ

Fig. 4. SOCE supports Ca signaling in astrocytes. (A): 2-APB-induced ER Ca2þ decline attenuates spontaneous ER Ca2þ release in astrocytes. Representative DF/F0 traces of GCaMP6f were shown to indicate spontaneous [Ca2þ]Cyt fluctuations before and after 20-min treatment with 2-APB. A whole astrocyte was selected as the region of interest to measure DF/F0. (B, C): Summary of peak amplitude (B) and frequency (C) of spontaneous responses (mean ± SEM). Treatment by 2-APB reduced the peak amplitude but did not affect the frequency. n ¼ 26 events from 9 cells for control and 33 events from 9 cells for 2-APB. ***P < 0.001 in paired Student’s t-test.

2-APB potently blocked astrocytic SOCE as expected, while SKF96365 and Gd3þ showed only a limited inhibitory effect in our experiments. Previous studies indicated that Orai1e3 and TRPC1, Ca2þ channels that are sensitive to SKF96365 and lanthanides, mediate the majority of SOCE in cultured astrocytes [6,28,29]. One possible explanation for this contradiction could be different molecular identities of SOCE between cultures and acute slices. SKF96365-insensitive SOCE and Gd3þ-insensitive SOCE were reported in some types of smooth muscle cells and breast epithelial cell lines [30e33]. Therefore, it is possible that astrocytes in situ recruit these SOCE mechanisms to provide the observed pharmacological properties. The molecular basis of SKF96365-insensitive SOCE and Gd3þ-insensitive SOCE in astrocytes should be addressed in future studies. Complex Ca2þ signaling, including the mutual interaction between SOCE and ER Ca2þ dynamics, is likely a key regulator of astrocytic functions. Therefore, direct analysis of astrocytic Ca2þ handling mechanisms involving SOCE by applying ER Ca2þ imaging techniques to various physiological and pathophysiological inputs should provide novel insights into various brain functions.

Declaration of competing interest The authors declare no competing financial interests.

Acknowledgements We thank Y. Kawashima, J. Suzuki, K. Kanemaru, and K. Kobayashi for their support in producing AAV vectors.

[1] A. Volterra, J. Meldolesi, Astrocytes, from brain glue to communication elements: the revolution continues, Nat. Rev. Neurosci. 6 (2005) 626e640, https://doi.org/10.1038/nrn1722. [2] N. Bazargani, D. Attwell, Astrocyte calcium signaling: the third wave, Nat. Neurosci. 19 (2016) 182e189, https://doi.org/10.1038/nn.4201. [3] A. Verkhratsky, V. Parpura, Store-operated calcium entry in neuroglia, Neurosci. Bull. 30 (2014) 125e133, https://doi.org/10.1007/s12264-013-1343-x. [4] M. Prakriya, R.S. Lewis, Store-operated calcium channels, Physiol. Rev. 95 (2015) 1383e1436, https://doi.org/10.1152/physrev.00020.2014. [5] A.B. Parekh, J.W. Putney, Store-operated calcium channels, Physiol. Rev. 85 (2005) 757e810, https://doi.org/10.1152/physrev.00057.2003. [6] A.B. Toth, K. Hori, M.M. Novakovic, N.G. Bernstein, L. Lambot, M. Prakriya, CRAC channels regulate astrocyte Ca2þ signaling and gliotransmitter release to modulate hippocampal GABAergic transmission, Sci. Signal. 12 (2019), https:// doi.org/10.1126/scisignal.aaw5450 eaaw5450. [7] E.B. Malarkey, Y. Ni, V. Parpura, Ca2þ entry through TRPC1 channels contributes to intracellular Ca2þ dynamics and consequent glutamate release from rat astrocytes, Glia 56 (2008) 821e835, https://doi.org/10.1002/glia.20656. [8] C.M. Fanger, M. Hoth, G.R. Crabtree, R.S. Lewis, Characterization of T cell mutants with defects in capacitative calcium entry: genetic evidence for the physiological roles of CRAC channels, J. Cell Biol. 131 (1995) 655e667, https:// doi.org/10.1083/jcb.131.3.655. [9] H. Jousset, M. Frieden, N. Demaurex, STIM1 knockdown reveals that storeoperated Ca2þ channels located close to sarco/endoplasmic Ca2þ ATPases (SERCA) pumps silently refill the endoplasmic reticulum, J. Biol. Chem. 282 (2007) 11456e11464, https://doi.org/10.1074/jbc.M609551200. [10] T.R. Cully, B.S. Launikonis, Store-operated Ca2þ entry is not required for store refilling in skeletal muscle, Clin. Exp. Pharmacol. Physiol. 40 (2013) 338e344, https://doi.org/10.1111/1440-1681.12078. [11] D.E. Clapham, Calcium signaling, Cell 131 (2007) 1047e1058, https://doi.org/ 10.1016/J.CELL.2007.11.028. [12] J.W. Putney, N. Steinckwich-Besançon, T. Numaga-Tomita, F.M. Davis, P.N. Desai, D.M. D’Agostin, S. Wu, G.S. Bird, The functions of store-operated calcium channels, Biochim. Biophys. Acta Mol. Cell Res. 1864 (2017) 900e906, https://doi.org/10.1016/J.BBAMCR.2016.11.028. [13] J. Suzuki, K. Kanemaru, K. Ishii, M. Ohkura, Y. Okubo, M. Iino, Imaging intraorganellar Ca2þ at subcellular resolution using CEPIA, Nat. Commun. 5 (2014) 4153, https://doi.org/10.1038/ncomms5153. [14] Y. Okubo, J. Suzuki, K. Kanemaru, N. Nakamura, T. Shibata, M. Iino, Visualization of Ca2þ filling mechanisms upon synaptic inputs in the endoplasmic reticulum of cerebellar Purkinje cells, J. Neurosci. 35 (2015) 15837e15846, https://doi.org/10.1523/JNEUROSCI.3487-15.2015. [15] Y. Okubo, K. Kanemaru, J. Suzuki, K. Kobayashi, K. Hirose, M. Iino, Inositol 1,4,5-trisphosphate receptor type 2-independent Ca2þ release from the endoplasmic reticulum in astrocytes, Glia 67 (2019) 113e124, https://doi.org/ 10.1002/glia.23531. [16] Y. Okubo, M. Iino, Visualization of astrocytic intracellular Ca2þ mobilization, J. Physiol. (2019) JP277609, https://doi.org/10.1113/JP277609. [17] E. Shigetomi, E.A. Bushong, M.D. Haustein, X. Tong, O. Jackson-Weaver, S. Kracun, J. Xu, M.V. Sofroniew, M.H. Ellisman, B.S. Khakh, Imaging calcium microdomains within entire astrocyte territories and endfeet with GCaMPs expressed using adeno-associated viruses, J. Gen. Physiol. 141 (2013) 633e647, https://doi.org/10.1085/jgp.201210949.

Please cite this article as: Y. Okubo et al., Store-operated Ca2þ entry-dependent Ca2þ refilling in the endoplasmic reticulum in astrocytes, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.006

6

Y. Okubo et al. / Biochemical and Biophysical Research Communications xxx (xxxx) xxx

[18] F.A. Edwards, A. Konnerth, B. Sakmann, T. Takahashi, A thin slice preparation for patch clamp recordings from neurones of the mammalian central nervous system, Pflüg. Arch. 414 (1989) 600e612, https://doi.org/10.1007/ bf00580998. [19] W. Kresse, I. Sekler, A. Hoffmann, O. Peters, C. Nolte, A. Moran, H. Kettenmann, Zinc ions are endogenous modulators of neurotransmitter-stimulated capacitative Ca2þ entry in both cultured and in situ mouse astrocytes, Eur. J. Neurosci. 21 (2005) 1626e1634, https://doi.org/10.1111/j.14609568.2005.03926.x. [20] K. Singaravelu, C. Lohr, J.W. Deitmer, Regulation of store-operated calcium entry by calcium-independent phospholipase A2 in rat cerebellar astrocytes, J. Neurosci. 26 (2006) 9579e9592, https://doi.org/10.1523/JNEUROSCI.260406.2006. [21] M. Paukert, A. Agarwal, J. Cha, V.A. Doze, J.U. Kang, D.E. Bergles, Norepinephrine controls astroglial responsiveness to local circuit activity, Neuron 82 (2014) 1263e1270, https://doi.org/10.1016/J.NEURON.2014.04.038. [22] F. Ding, J. O’Donnell, A.S. Thrane, D. Zeppenfeld, H. Kang, L. Xie, F. Wang, M. Nedergaard, a1-Adrenergic receptors mediate coordinated Ca2þ signaling of cortical astrocytes in awake, behaving mice, Cell Calcium 54 (2013) 387e394, https://doi.org/10.1016/J.CECA.2013.09.001. [23] T. Maruyama, T. Kanaji, S. Nakade, T. Kanno, K. Mikoshiba, 2APB, 2aminoethoxydiphenyl borate, a membrane-penetrable modulator of Ins(1,4,5)P3-induced Ca2þ release, J. Biochem. 122 (1997) 498e505, https:// doi.org/10.1093/oxfordjournals.jbchem.a021780. [24] J.W. Putney, Pharmacology of store-operated calcium channels, Mol. Interv. 10 (2010) 209e218, https://doi.org/10.1124/mi.10.4.4. [25] S. Sakuragi, F. Niwa, Y. Oda, K. Mikoshiba, H. Bannai, Astroglial Ca2þ signaling is generated by the coordination of IP3R and store-operated Ca2þ channels, Biochem. Biophys. Res. Commun. 486 (2017) 879e885, https://doi.org/ 10.1016/J.BBRC.2017.03.096.

[26] J.G. Bilmen, L.L. Wootton, R.E. Godfrey, O.S. Smart, F. Michelangeli, Inhibition of SERCA Ca2þ pumps by 2-aminoethoxydiphenyl borate (2-APB), Eur. J. Biochem. 269 (2002) 3678e3687, https://doi.org/10.1046/j.14321033.2002.03060.x. [27] D. Leon-Aparicio, J. Chavez-Reyes, A. Guerrero-Hernandez, Activation of endoplasmic reticulum calcium leak by 2-APB depends on the luminal calcium concentration, Cell Calcium 65 (2017) 80e90, https://doi.org/10.1016/ j.ceca.2017.01.013. [28] J. Kwon, H. An, M. Sa, J. Won, J.I. Shin, C.J. Lee, Orai1 and Orai3 in combination with Stim1 mediate the majority of store-operated calcium entry in astrocytes, Exp. Neurobiol. 26 (2017) 42e54, https://doi.org/10.5607/ en.2017.26.1.42. [29] V.A. Golovina, Visualization of localized store-operated calcium entry in mouse astrocytes. Close proximity to the endoplasmic reticulum, J. Physiol. 564 (2005) 737e749, https://doi.org/10.1113/jphysiol.2005.085035. [30] S.M. Wilson, H.S. Mason, G.D. Smith, N. Nicholson, L. Johnston, R. Janiak, J.R. Hume, Comparative capacitative calcium entry mechanisms in canine pulmonary and renal arterial smooth muscle cells, J. Physiol. 543 (2002) 917e931, https://doi.org/10.1113/jphysiol.2002.021998. [31] C.P. Wayman, I. McFadzean, A. Gibson, J.F. Tucker, Two distinct membrane currents activated by cyclopiazonic acid-induced calcium store depletion in single smooth muscle cells of the mouse anococcygeus, Br. J. Pharmacol. 117 (1996) 566e572, https://doi.org/10.1111/j.1476-5381.1996.tb15228.x. [32] C. Baldi, G. Vazquez, R. Boland, Capacitative calcium influx in human epithelial breast cancer and non-tumorigenic cells occurs through Ca2þ entry pathways with different permeabilities to divalent cations, J. Cell. Biochem. 88 (2003) 1265e1272, https://doi.org/10.1002/jcb.10471. [33] R. Flemming, S.Z. Xu, D.J. Beech, Pharmacological profile of store-operated channels in cerebral arteriolar smooth muscle cells, Br. J. Pharmacol. 139 (2003) 955e965, https://doi.org/10.1038/sj.bjp.0705327.

Please cite this article as: Y. Okubo et al., Store-operated Ca2þ entry-dependent Ca2þ refilling in the endoplasmic reticulum in astrocytes, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.006