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Norepinephrine induces rapid and long-lasting phosphorylation and redistribution of connexin 43 in cortical astrocytes
Biochemical and Biophysical Research Communications xxx (2018) 1e8
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Norepinephrine induces rapid and long-lasting phosphorylation and redistribution of connexin 43 in cortical astrocytes Mutsuo Nuriya a, b, c, *, 1, Ayaka Morita a, d, 1, 2, Takanori Shinotsuka a, Tomoko Yamada a, Masato Yasui a a
Department of Pharmacology, School of Medicine, Keio University, Shinanomachi, Shinjuku, 160-8582, Japan Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency (JST), Kawaguchi, Saitama, 332-0012, Japan Graduate School of Environment and Information Sciences, Yokohama National University, Japan d College of Engineering, Yokohama National University, Kanagawa, 240-8501, Japan b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 August 2018 Accepted 5 September 2018 Available online xxx
Norepinephrine (NE) modulates brain functions depending on both the internal and external environment. While the neuromodulatory actions of NE have been well characterized, the response and involvement of cortical astrocytes to physiological noradrenergic systems remain largely unknown, especially at the molecular level. In this study, we biochemically characterize the action of NE on astrocytes of the murine neocortex. NE stimulation of acute brain slices rapidly increase phosphorylation of connexin 43 (Cx43) at Serine (Ser) 368, in slices from both juvenile and adolescent animals. The phosphorylation is mediated by the protein kinase C (PKC) pathway under the a1-adrenergic receptor and remains elevated for tens of minutes following brief exposure to NE, well after the intracellular calcium level returns to normal level, suggesting the plastic nature of this phosphorylation event. Importantly, this phosphorylation event persists in the absence of neuronal transmissions, suggesting that the effect of NE on Cx43 phosphorylation is induced directly on astrocytes. Furthermore, these NE-induced phosphorylations are associated with biochemical dissociation of Cx43 from gap-junctional plaques to non-junctional compartments. Finally, we show that pharmacological manipulation of the noradrenergic system using psychoactive drugs modulates phosphorylation of Cx43 in the cerebral cortex in vivo. These data suggest that NE acts directly on astrocytes in parallel with neurons and modulates functionally critical connexin channel proteins in a plastic manner. Thus, plasticity of astrocytes induced by the “gliomodulatory” actions of NE may play important roles in their physiological as well as pharmacological actions in the brain. © 2018 Elsevier Inc. All rights reserved.
Keywords: Connexin Phosphorylation Astrocyte Gap junction Norepinephrine
1. Introduction Astrocytes play pivotal roles across broad aspects of brain function [1]. Astrocytes exist in a tiled fashion, each occupying a non-overlapping area but covering practically the entire cerebral cortex as a whole [2]. Using this extensive coverage, astrocytes appear to monitor and maintain the metabolic state of neurons [3]. In addition, accumulating evidence suggests that astrocytes regulate extracellular ionic conditions and thereby modulate neuronal
* Corresponding author. Department of Pharmacology, School of Medicine, Keio University, Shinanomachi, Shinjuku, 160-8582, Japan. E-mail address: [email protected] (M. Nuriya). 1 These authors contributed equally to this work. 2 Deceased February 11, 2013.
activities [4,5]. Besides the various transporters and ion channels expressed on astrocytes, gap junctions are critical to their function in extracellular environment regulation. Gap junctions allow neighboring cells to communicate with each other in an analogue manner by transmitting small bioactive molecules with the size of less than ~1000 Da, based on the concentration gradient. Since astrocytes in the cerebral cortex are connected to neighboring cells via gap junctions, each occupying non-overlapping areas, this gap junctional network of astrocytes, called astrocytic syncytium, creates a long-range analogue cellular network [6]. Given these unique features, astrocyte gap junctions are critically involved in their physiological and pathophysiological functions [1,7e9]. At the molecular level, connexin 43 (Cx43) is the predominant constituent of functional gap junctions in astrocytes, especially in the juvenile animals [10]. Phosphorylation of Cx43 in cortical
https://doi.org/10.1016/j.bbrc.2018.09.021 0006-291X/© 2018 Elsevier Inc. All rights reserved.
Please cite this article in press as: M. Nuriya, et al., Norepinephrine induces rapid and long-lasting phosphorylation and redistribution of connexin 43 in cortical astrocytes, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.09.021
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Fig. 1. NE induces phosphorylation of Cx43 at Ser368 in cortical brain slices. (A). Dose dependency of phosphorylation. Acutely prepared murine cortical slices were incubated with 1, 5, 20 and 100 mM of NE at 37 C for 5 min and the brain lysates probed for Cx43 phosphorylated on Ser368. The band intensities were normalized to those of b-actin. (B) NE induces phosphorylation of Cx43 at Ser368 without changes in total protein level. Brain slices were treated with 20 mM NE for 5 min at 37 C and probed for phosphorylated Cx43 (left) and total Cx43 (right). The band intensities were normalized to those of b-actin. (C). Plot profile of the phospho- and total-Cx43 band. Intensity profiles of phosphoCx43 (left) and total Cx43 (right) were measured and plotted along the migration axis. Those under control conditions and NE-treated groups are shown in black and red, respectively. (D). Confirmation of the phosphorylation using another antibody. NE-induced change of the Cx43 S368 phosphorylation was confirmed by using another phosphospecific antibody from a different source. The antibody used in this panel was raised against the phosphopeptide corresponding to amino acid residues surrounding the phospho-
Please cite this article in press as: M. Nuriya, et al., Norepinephrine induces rapid and long-lasting phosphorylation and redistribution of connexin 43 in cortical astrocytes, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.09.021
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astrocytes is highly sensitive to various stimuli, including pathological conditions, such as ischemia, and pharmacological manipulations, including the general anesthetic propofol [11,12]. Cx43 is phosphorylated at multiple sites and some of these phosphorylation events are known to regulate the functions of Cx43 at gap junctions [13]. Among them, phosphorylation at Serine (Ser) 368 is known to be the key for functional modifications via changes in channel kinetics [14e16]. However, physiological regulation of Cx43 in cortical astrocytes remains to be understood. Norepinephrine (NE) plays essential roles in brain regulation [17]. In addition to its well-characterized actions as a neuromodulator, previous studies have shown that NE induces robust calcium signaling in cortical astrocytes [18,19]. In fact, NE appears to be the dominant stimuli in vivo [19] and has a modulatory action on astrocyte physiology [20,21], according to calcium imaging studies. However, the molecular details of these modulatory actions remain largely unknown. In this study, we employed biochemical analyses to characterize the potential effect of NE on astrocyte Cx43, and demonstrate that NE directly acts on astrocytes and regulates Cx43 by specific phosphorylation and redistribution at the membrane in a plastic manner. 2. Results First, we asked if NE stimulation alters the phosphorylation of Cx43 in the cerebral cortex. Acutely prepared murine cortical slices were treated with NE at different concentrations for 5 min at 37 C and the lysates were probed with an antibody against Cx43 phosphorylated at Ser368. This analysis revealed that 5 min stimulation with more than 20 mM NE increases phosphorylation of Cx43 at Ser368 (Fig. 1A, n ¼ 4 for each group). NE stimulation at 20 mM induced two changes; increase in the intensity of phosphorylated Cx43 (p ¼ 2.2 103, n ¼ 7 for each group) without changes in total Cx43 protein level (p ¼ 9.0 101, n ¼ 7 for each group) (Fig. 1B), and a change in migration patterns to higher molecular weight (Fig. 1C), both of which indicate increase in the phosphorylation of Cx43 at Ser368. We confirmed that this observation by several lines of investigation. First, NE-induced phosphorylation of Cx43 at Ser368 was confirmed by using an antibody from a different source that was raised against phosphopeptides from different species (human vs. rat) (Fig. 1D, p ¼ 9.4 104, n ¼ 8 for each group). Second, the migration patterns of total Cx43 changed from a broad range extending to higher molecular weights to a sharp range at lower molecular weight upon in vitro dephosphorylation reaction (Supplementary Fig. 1). Finally, the change in the migration pattern of Cx43 upon NE stimulation is abolished when the proteins were dephosphorylated in vitro (Supplementary Fig. 1). Examples of the full range of Western blotting results are shown in Supplementary Fig. 2. All of these results strongly suggest that NE stimulation induces Cx43 phosphorylation on Ser368. As astrocytic responses to neurotransmitters may change during developmental stages [22], we characterized astrocytic responses to NE in adolescent animals. Stimulation of brain slices prepared from adolescent mice (7-8 week-old mice) indicated that NE induces robust Cx43 phosphorylation at this stage (Fig. 1E, p ¼ 1.5 103, control: n ¼ 8, NE: n ¼ 7). Next, to characterize the kinetics, we monitored the phosphorylation level of Cx43 across various recovery periods following 5 min pulsatile stimulation by NE. This analysis revealed that the phosphorylation level of Cx43
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remains elevated for tens of minutes before returning to the basal level after one hour (Fig. 1F) (n ¼ 8, 6, 6, 6, 4 for control, 0 min, 5 min, 20 min and 60 min, respectively). In contrast, we found that the same 5 min NE stimulation increases intracellular calcium concentration, but it rapidly returns to basal levels within minutes when analyzed by two-photon microscopy (Supplementary Fig. 3). Therefore, these data combined suggest that when astrocytes receive transient NE stimulation, the information is conferred to phosphorylation of Cx43 that decays with its own kinetics, in much longer timescale than that of calcium dynamics. To further examine the molecular mechanisms underlying this event, we pharmacologically dissected the signaling pathway involved in NE-induced phosphorylation of Cx43. Since Cx43Ser368 is known to be phosphorylated by protein kinase C (PKC) in vitro [14,15], we hypothesized that NE may phosphorylate Cx43 at Ser368 via activation of the a1-adrenoceptor that is coupled to Gq-type G-proteins in the brain. Indeed, we found that specific activation of a1-adrenergic receptors using phenylephrine induced phosphorylation in the same manner as NE stimulation (Fig. 2A, p ¼ 1.2 102, control: n ¼ 8, NE: n ¼ 6). Furthermore, NEinduced phosphorylation of Cx43 at Ser368 was blocked by preincubation of brain slices with the a1-adrenoceptor antagonist prazosin (Fig. 2B, p ¼ 1.0, n ¼ 4 for each group). Finally, blockade of PKC activation by bisindolylmaleimide I (Go6850) abolished the phosphorylation (Fig. 2C, p ¼ 1.0, n ¼ 5 for each group). As was the case for normal NE stimulation (Fig. 1B), there was no difference in the total Cx43 level under any of these pharmacological manipulations (Supplementary Fig. 4). All of these results strongly suggest that NE-induced phosphorylation of Cx43 at Ser368 is mediated by activation of a1-adrenergic receptors and the following PKC pathway. As astrocytes communicate with neurons, NE's action on Cx43 in astrocytes may be mediated by modulation of neuronal activities and following changes in neuronal regulation on astrocytes. To test if the effect of NE is directly on astrocytes or indirectly mediated by neuronal responses, the same experiment was performed in the presence of tetrodotoxin, a blocker of voltage-gated sodium channel and therefore action potential-dependent neuronal activities. NE-induced phosphorylation of Cx43 on Ser368, however, persisted in the presence of tetrodotoxin, indicating that the effect is likely to be a direct result of astrocytic activation (Fig. 2D, p ¼ 3.1 102, n ¼ 6 for each group). To exclude the possibility of potential involvement of action potential-independent neuronal transmissions, the same experiment was performed in the presence of a glutamate receptor antagonist cocktail (CNQX, AP5 and MCPG) [21]. Again, even in the absence of glutamatergic transmissions, NE induced robust phosphorylation of Cx43 (Fig. 2E, p ¼ 2.2 103, n ¼ 7 for each group). Taken together, these data strongly suggest that the action of NE on the phosphorylation of Cx43 at Ser368 is due to the direct action on astrocytes but not indirectly through the modulation of neuronal transmission. We next assessed the consequences of NE-induced phosphorylation on Cx43 proteins. Two biochemical analyses were performed to examine gap junctional assembly (Supplementary Fig. 5) [23]. First, solubility of proteins in 1% triton X-100 was examined. When stimulated by NE for 5 min, a significantly higher fraction of Cx43 became triton X-100 soluble (Fig. 3A, p ¼ 4.9 102, n ¼ 6 for each group). As connexins at the functional gap junction become resistant to solubilization by triton [23], this result indicates that NE stimulation induce relocation of Cx43 from gap junction plaques to
Ser368 of rat Cx43, whereas the one used for the rest of figures was against the human sequence. Band intensities were normalized to those of b-actin. (E). Phosphorylation in adolescent animals. Brain slices prepared from adolescent animals (7-8 week-old mice) were treated with 20 mM NE for 5 min 37 C and probed for phosphorylated Cx43.Band intensities were normalized to those of b-actin. (F). Time course of NE-induced phosphorylation of Cx43 at Ser368. Brain slices were treated with 20 mM NE for 5 min and recovered in normal ACSF for 0, 5, 20 and 60 min. Band intensities were normalized to those of b-actin. NE was applied to brain slices from 5 to 0 min.
Please cite this article in press as: M. Nuriya, et al., Norepinephrine induces rapid and long-lasting phosphorylation and redistribution of connexin 43 in cortical astrocytes, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.09.021
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Fig. 2. Pharmacological characterization of NE-induced phosphorylation of Cx43. (A).a1-Adrenoceptor agonists mimic NE-induce phosphorylation of Cx43. Cortical brain slices were treated with 20 mM (R)-()-phenylephrine for 5 min at 37 C and the lysates were probed for Cx43 phosphorylated at Ser368. The band intensities were normalized to those of b-actin. (B) a1-Adrenoceptor antagonist blocks NE-induced phosphorylation of Cx43. Brain slices were stimulated by 20 mM NE for 5 min at 37 C in the presence of prazosin (10 mM). (C). Inhibition of PKC activity blocks NE-induced phosphorylation of Cx43. Brain slices were stimulated with 20 mM NE for 5 min at 37 C in the presence of bisindolylmaleimide I (200 nM). (D). Blockade of neuronal activities by tetrodotoxin does not alter NE-induced phosphorylation of Cx43. Brain slices were stimulated with 20 mM NE for 5 min at 37 C in the presence of tetrodotoxin (TTX, 1 mM). (E). Blockade of glutamatergic transmissions does not alter NE-induced phosphorylation of Cx43. Brain slices were stimulated with 20 mM NE for 5 min at 37 C in the presence of glutamate receptor antagonist cocktail (50 mM AP5, 50 mM CNQX and 1 mM MCPG).
non-gap junctional sites. This can be achieved by either lateral diffusion out from the gap junction plaques or by internalization (Supplementary Fig. 5). To distinguish these possibilities, a surface biotinylation assay was performed [23]. Interestingly, 5 min NE treatment significantly increased Cx43 biotinylation, suggesting a shift of Cx43 from gap junction plaques to freely accessible nonjunctional positions at the plasma membrane (Fig. 3B, p ¼ 8.2 103, n ¼ 6 for each group). In contrast, other cell surface proteins such as postsynaptic glutamate receptor GluA2 or the astrocytic protein b-dystroglycan are stable during this period (Fig. 3B and Supplementary Fig. 6, p ¼ 5.8 101 for GluA2, p ¼ 4.7 101 for b-dystroglycan, n ¼ 6 for each group). Importantly, when slices were incubated in normal artificial cerebrospinal fluid (ACSF) after 5 min of NE stimulation, changes in biotinylation efficacy remains at 20 min but returns to original levels at 60 min, suggesting that the translocation of Cx43 is a
transient and reversible process (Fig. 3C). Taken together, these results suggest that phosphorylation of Cx43 on Ser368 upon NE stimulation disassemble them from functional gap-junctional plaques to a non-functional pool at the plasma membrane. Finally, we examined whether the phosphorylation of Cx43 is regulated in vivo. First, the phosphorylation of Cx43 was examined using brain lysates. This revealed that Cx43 is phosphorylated at Ser368 in the basal state both in juvenile (2-week-old) as well as adolescent (8-week-old) mice (Fig. 4A). Dephosphorylation of proteins abolished the immunoreactivity of the phospho-specific antibody, confirming the specificity of both phosphorylation and detection (Fig. 4A). Next, the effect of the a1-adrenoceptor antagonist prazosin was examined. Treatment of animals with prazosin significantly reduced the phosphorylation of Cx43 at Ser368 in the cerebral cortex (Fig. 4B, p ¼ 1.1 102, n ¼ 7 for each group). This result confirms the conclusions obtained from in vitro experiments
Please cite this article in press as: M. Nuriya, et al., Norepinephrine induces rapid and long-lasting phosphorylation and redistribution of connexin 43 in cortical astrocytes, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.09.021
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Fig. 3. Biochemical characterization of NE-induced changes of Cx43 proteins. (A). NE stimulation changes the solubility of Cx43 to triton X-100. Cortical brain slices were treated with 20 mM NE for 5 min at 37 C and the lysate were fractionated into non-DRM (1% triton X-100 soluble) and DRM (1% triton X-100 insoluble) fractions. The right panel shows the normalized Non-DRM/DRM ratio. B. NE stimulation changes the efficacy of Cx43 surface biotinylation. Brain slices were treated with 20 mM NE for 5 min at 37 C and labeled with NHS-SS-biotin to label the surface proteins. Biotinylated proteins were then collected and probed for Cx43 (left) and GluA2 (right). The graph shows the normalized biotinylation ratio. (C). Time course of NE-induced changes in the Cx43 surface biotinylation. Brain slices were treated with 20 mM NE for 5 min at 37 C and incubated in the normal ACSF for 20 and 60 min. The slices were then biotinylated and biotinylated proteins were quantified as above.
Please cite this article in press as: M. Nuriya, et al., Norepinephrine induces rapid and long-lasting phosphorylation and redistribution of connexin 43 in cortical astrocytes, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.09.021
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Fig. 4. Characterization of NE-dependent Cx43 phosphorylation in vivo. (A). Cx43 phosphorylation in vivo. Cortical brain lysate from juvenile (J: 16-day-old) and adolescent (A: 8week-old) animals were prepared and Western blotting was performed on the membrane processed with or without l protein phosphatase (lPPase) in vitro phosphatase reaction. The samples were probed for phospho-Cx43, total Cx43 and b-actin.(B). a1AR antagonist treatment reduces Cx43 phosphorylation in vivo. Cortical brain lysates prepared from animals treated with prazosin or vehicle, were probed for phospho-Cx43. The band intensities were normalized to those of b-actin. (C). a2AR antagonist treatment reduces Cx43 phosphorylation in vivo. Cortical brain lysates prepared from animals treated with xylazine or vehicle, were probed for phospho-Cx43. The band intensities were normalized to those of b-actin.
and further suggest that the basal phosphorylation level of Cx43 is maintained by the ambient concentration of NE in the brain in vivo. To examine this possibility, pharmacological manipulations of endogenous NE levels by activation of presynaptic inhibitory a2adrenoceptors using xylazine was performed [18]. Indeed, xylazine significantly reduced phosphorylation of Cx43 compared to vehicle-treated animals (Fig. 4C, p ¼ 3.0 102, n ¼ 7 for each group). These results support the conclusions from brain slice experiments and suggest that phosphorylation of Cx43 is subject to modifications via NE tone in vivo. 3. Discussion In this study, we demonstrate that NE induces rapid and longlasting modifications of Cx43 in the murine cerebral cortex. A pioneering study has indicated that astrocytes can be the target of NE in cultured striatal astrocytes [24], but the molecular mechanisms as well as the nature of these modifications remained unknown. Our study extends these studies to brain tissue in vitro and in vivo and reveals a gliomodulatory action of NE; NE-induces long-
lasting biochemical modification of Cx43 through its direct action on astrocytes. Our biochemical analyses revealed that NE-induced phosphorylation of Cx43 is accompanied by dissociation from gap junctional plaques to non-gap junctional pools, which occurs rapidly (<5 min) and return to the original state over a longer time course (~20 min) (Fig. 3, schematized in Supplementary Fig. 7). In addition to translocation of Cx43 proteins at the cellular level, phosphorylation of Cx43 at Ser368 has been shown to reduce gap junctional permeability [14e16]. While it is beyond the scope of this study, it is tempting to deduce the consequences of this NE-induced phosphorylation of Cx43 on astrocyte physiology. Functional down regulation of astrocyte gap junctions would result in reduced buffering capacity of local changes in ions and messengers across the extracellular space [4,5]. Therefore, NE-induced biochemical changes in astrocytes may affect synaptic transmissions and thereby information processing in the brain, resulting in higher sensitivity. Importantly, xylazine and prazosin, both of which reduce the phosphorylation of Cx43 at Ser368 in vivo (Fig. 4), are known to have sedative effects. While clearly having direct actions
Please cite this article in press as: M. Nuriya, et al., Norepinephrine induces rapid and long-lasting phosphorylation and redistribution of connexin 43 in cortical astrocytes, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.09.021
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on neurons, our results raise the possibility that some psychoactive compounds exert their actions in part through their gliomodulatory actions on astrocytes. Two technical points are worth further discussion. First, while we stimulated brain slices with NE for 5 min, as it was the shortest reliable stimulation time that could be performed in our system, it should be noted that the phosphorylation may occur in a much faster timescale than 5 min. Second, although we focused on the regulation of Ser368 of Cx43, since it is the best characterized site with proven analytical tools available, it does not mean that this is the only site being regulated by NE. Although it is beyond the scope of this study, other known potential phosphorylation sites may be simultaneously regulated by NE and may contribute to the functional regulation of astrocytic gap junctions. In conclusion, our study demonstrates that NE exerts gliomodulatory actions by directly acting on astrocytes to biochemically modulate Cx43 in a plastic manner. 4. Experimental procedures
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Sepharose Beads (Thermo Fisher Scientific) and compared to total protein. Anti-phospho-connexin 43 rabbit polyclonal antibody (Cell Signaling Technologies AB_2110169 and Abcam AB_731707), anticonnexin 43 rabbit polyclonal antibody (Thermo Fisher Scientific AB_2533973) and anti-b-actin mouse monoclonal antibody (Sigma Aldrich AB_476743) were purchased from the indicated companies. For phospho-connexin 43 immunodetection, the antibody from Cell Signaling Technologies was used for all the Western blotting except for that in Fig. 1D, where the one from Abcam was used to confirm the result. Band intensities of Cx43 were normalized to those of b-actin, which were obtained by re-probing the same membrane after stripping with stripping buffer (Nacalai Tesque). 4.4. Two-photon calcium imaging Time-lapse imaging of astrocytic intracellular calcium dynamics were performed as previously reported, using a FV1000MPE microscopy system (Olympus) equipped with MaiTaiHP femtosecond laser tuned to 840 nm (Newport) [9,21].
4.1. Slice preparation 4.5. Data analysis All procedures related to the care and the treatment of animals were approved by the committee of the School of Medicine, Keio University. Acute cortical slices were prepared essentially as described previously [11,25]. 4.2. Pharmacology Pharmacological manipulations were performed in a custommade chamber filled with ACSF containing 126 mM NaCl, 26 mM NaHCO3, 1.1 mM NaH2PO4, 10 mM dextrose, 3.0 mM KCl, 1.0 mM MgCl2, and 3.0 mM CaCl2 (pH 7.3) bubbled with 95% O2/5% CO2 at 37 C. Tetrodotoxin (Abcam, 1 mM), prazosin (Sigma Aldrich, 10 mM), and bisindolylmaleimide I (Merck, 200 nM) were applied 10 min before NE (Sigma Aldrich) stimulation for 5 min. Control samples were treated with vehicle in the same manner. For pharmacological manipulations in vivo, animals (~3-week-old) were injected with drugs by intraperitoneal injection (i.p.) at a dose of 10 mg/kg and returned to home cage for 1 h before being sacrificed for analysis. 4.3. Biochemistry Preparations of protein samples from cortical brain slices for biochemical analyses and Western blotting detections were performed as described previously [25]. In in vivo experiments, mice were anesthetized with isoflurane and the brains were immediately removed after decapitation. The cerebrum was dissected in HEPES ACSF and homogenized in lysis buffer followed by sample preparation as for brain slices. In vitro phosphatase reactions were performed with lambda protein phosphatase (New England Biolabs) in the reaction buffer supplied by the manufacturer at R.T. overnight for the Western blot membrane (Figs. 4A), and 30 C for 30 min for brain lysate (Supplementary Fig. 1). Fractionation experiments to characterize protein localization in DRM (detergent resistant membrane) and Non-DRM were performed as reported previously [11]. Biotinylation assays were performed essentially as described previously with some modifications [26]. Briefly, brain slices were transferred to micro centrifuge tubes and washed with HEPES ACSF on ice. Then, slices were incubated in 1 mg/mL Sulfo-NHS-SS-Biotin (Thermo Fisher Scientific) in HEPES ACSF for 30 min at 4 C with gentle rotation. The slices were washed with TBS three times to quench excess biotinylation reagents and lysates were prepared as above. Biotinylated proteins were then isolated using NeutrAvidin
Quantitative analyses of immunoblot data were performed using ImageJ software (National Institute of Health, USA). Plot profiles of the Western blotting were performed essentially as previously described [11]. Statistical analyses were performed using OriginPro (Origin Lab) and significance was judged by Mann-Whitney test for two groups and one-way ANOVA followed by Fisher's test for comparison among three groups or more. All data are shown as mean ± standard error of the mean and statistical significance of <0.05 and < 0.01 are indicated by * and **, respectively. Acknowledgements We dedicate this paper to our beloved friend and colleague the late Ms. Ayaka Morita. This work was supported by the JSPS KAKENHI (16K07065), the Sumitomo Foundation, the Takeda Science Foundation, the Kowa Foundation, the SUNBOR Grant, and the Ayaka Foundation. Appendix A. Supplementary data Supplementary data related to this chapter can be found at https://doi.org/10.1016/j.bbrc.2018.09.021. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.09.021. References [1] A. Verkhratsky, M. Nedergaard, Physiology of Astroglia, Physiol. Rev. 98 (2018) 239e389. [2] M.M. Halassa, T. Fellin, H. Takano, et al., Synaptic islands defined by the territory of a single astrocyte, J. Neurosci. 27 (2007) 6473e6477. [3] G.C. Petzold, V.N. Murthy, Role of astrocytes in neurovascular coupling, Neuron 71 (2011) 782e797. [4] A. Verkhratsky, M. Nedergaard, Astroglial cradle in the life of the synapse, Philos. Trans. R. Soc. Lond. B Biol. Sci. 369 (2014) 20130595. [5] G. Dallerac, O. Chever, N. Rouach, How do astrocytes shape synaptic transmission? Insights from electrophysiology, Front. Cell. Neurosci. 7 (2013) 159. [6] C. Giaume, X. Liu, From a glial syncytium to a more restricted and specific glial networking, J. Physiol. Paris 106 (1-2) (2012) 34e39. [7] N. Rouach, A. Koulakoff, V. Abudara, et al., Astroglial metabolic networks sustain hippocampal synaptic transmission, Science 322 (2008) 1551e1555. [8] U. Pannasch, L. Vargova, J. Reingruber, et al., Astroglial networks scale synaptic
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Norepinephrine induces rapid and long-lasting phosphorylation and redistribution of connexin 43 in cortical astrocytes Mutsuo Nuriya a, b, c, *, 1, Ayaka Morita a, d, 1, 2, Takanori Shinotsuka a, Tomoko Yamada a, Masato Yasui a a
Department of Pharmacology, School of Medicine, Keio University, Shinanomachi, Shinjuku, 160-8582, Japan Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency (JST), Kawaguchi, Saitama, 332-0012, Japan Graduate School of Environment and Information Sciences, Yokohama National University, Japan d College of Engineering, Yokohama National University, Kanagawa, 240-8501, Japan b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 August 2018 Accepted 5 September 2018 Available online xxx
Norepinephrine (NE) modulates brain functions depending on both the internal and external environment. While the neuromodulatory actions of NE have been well characterized, the response and involvement of cortical astrocytes to physiological noradrenergic systems remain largely unknown, especially at the molecular level. In this study, we biochemically characterize the action of NE on astrocytes of the murine neocortex. NE stimulation of acute brain slices rapidly increase phosphorylation of connexin 43 (Cx43) at Serine (Ser) 368, in slices from both juvenile and adolescent animals. The phosphorylation is mediated by the protein kinase C (PKC) pathway under the a1-adrenergic receptor and remains elevated for tens of minutes following brief exposure to NE, well after the intracellular calcium level returns to normal level, suggesting the plastic nature of this phosphorylation event. Importantly, this phosphorylation event persists in the absence of neuronal transmissions, suggesting that the effect of NE on Cx43 phosphorylation is induced directly on astrocytes. Furthermore, these NE-induced phosphorylations are associated with biochemical dissociation of Cx43 from gap-junctional plaques to non-junctional compartments. Finally, we show that pharmacological manipulation of the noradrenergic system using psychoactive drugs modulates phosphorylation of Cx43 in the cerebral cortex in vivo. These data suggest that NE acts directly on astrocytes in parallel with neurons and modulates functionally critical connexin channel proteins in a plastic manner. Thus, plasticity of astrocytes induced by the “gliomodulatory” actions of NE may play important roles in their physiological as well as pharmacological actions in the brain. © 2018 Elsevier Inc. All rights reserved.
Keywords: Connexin Phosphorylation Astrocyte Gap junction Norepinephrine
1. Introduction Astrocytes play pivotal roles across broad aspects of brain function [1]. Astrocytes exist in a tiled fashion, each occupying a non-overlapping area but covering practically the entire cerebral cortex as a whole [2]. Using this extensive coverage, astrocytes appear to monitor and maintain the metabolic state of neurons [3]. In addition, accumulating evidence suggests that astrocytes regulate extracellular ionic conditions and thereby modulate neuronal
* Corresponding author. Department of Pharmacology, School of Medicine, Keio University, Shinanomachi, Shinjuku, 160-8582, Japan. E-mail address: [email protected] (M. Nuriya). 1 These authors contributed equally to this work. 2 Deceased February 11, 2013.
activities [4,5]. Besides the various transporters and ion channels expressed on astrocytes, gap junctions are critical to their function in extracellular environment regulation. Gap junctions allow neighboring cells to communicate with each other in an analogue manner by transmitting small bioactive molecules with the size of less than ~1000 Da, based on the concentration gradient. Since astrocytes in the cerebral cortex are connected to neighboring cells via gap junctions, each occupying non-overlapping areas, this gap junctional network of astrocytes, called astrocytic syncytium, creates a long-range analogue cellular network [6]. Given these unique features, astrocyte gap junctions are critically involved in their physiological and pathophysiological functions [1,7e9]. At the molecular level, connexin 43 (Cx43) is the predominant constituent of functional gap junctions in astrocytes, especially in the juvenile animals [10]. Phosphorylation of Cx43 in cortical
https://doi.org/10.1016/j.bbrc.2018.09.021 0006-291X/© 2018 Elsevier Inc. All rights reserved.
Please cite this article in press as: M. Nuriya, et al., Norepinephrine induces rapid and long-lasting phosphorylation and redistribution of connexin 43 in cortical astrocytes, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.09.021
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Fig. 1. NE induces phosphorylation of Cx43 at Ser368 in cortical brain slices. (A). Dose dependency of phosphorylation. Acutely prepared murine cortical slices were incubated with 1, 5, 20 and 100 mM of NE at 37 C for 5 min and the brain lysates probed for Cx43 phosphorylated on Ser368. The band intensities were normalized to those of b-actin. (B) NE induces phosphorylation of Cx43 at Ser368 without changes in total protein level. Brain slices were treated with 20 mM NE for 5 min at 37 C and probed for phosphorylated Cx43 (left) and total Cx43 (right). The band intensities were normalized to those of b-actin. (C). Plot profile of the phospho- and total-Cx43 band. Intensity profiles of phosphoCx43 (left) and total Cx43 (right) were measured and plotted along the migration axis. Those under control conditions and NE-treated groups are shown in black and red, respectively. (D). Confirmation of the phosphorylation using another antibody. NE-induced change of the Cx43 S368 phosphorylation was confirmed by using another phosphospecific antibody from a different source. The antibody used in this panel was raised against the phosphopeptide corresponding to amino acid residues surrounding the phospho-
Please cite this article in press as: M. Nuriya, et al., Norepinephrine induces rapid and long-lasting phosphorylation and redistribution of connexin 43 in cortical astrocytes, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.09.021
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astrocytes is highly sensitive to various stimuli, including pathological conditions, such as ischemia, and pharmacological manipulations, including the general anesthetic propofol [11,12]. Cx43 is phosphorylated at multiple sites and some of these phosphorylation events are known to regulate the functions of Cx43 at gap junctions [13]. Among them, phosphorylation at Serine (Ser) 368 is known to be the key for functional modifications via changes in channel kinetics [14e16]. However, physiological regulation of Cx43 in cortical astrocytes remains to be understood. Norepinephrine (NE) plays essential roles in brain regulation [17]. In addition to its well-characterized actions as a neuromodulator, previous studies have shown that NE induces robust calcium signaling in cortical astrocytes [18,19]. In fact, NE appears to be the dominant stimuli in vivo [19] and has a modulatory action on astrocyte physiology [20,21], according to calcium imaging studies. However, the molecular details of these modulatory actions remain largely unknown. In this study, we employed biochemical analyses to characterize the potential effect of NE on astrocyte Cx43, and demonstrate that NE directly acts on astrocytes and regulates Cx43 by specific phosphorylation and redistribution at the membrane in a plastic manner. 2. Results First, we asked if NE stimulation alters the phosphorylation of Cx43 in the cerebral cortex. Acutely prepared murine cortical slices were treated with NE at different concentrations for 5 min at 37 C and the lysates were probed with an antibody against Cx43 phosphorylated at Ser368. This analysis revealed that 5 min stimulation with more than 20 mM NE increases phosphorylation of Cx43 at Ser368 (Fig. 1A, n ¼ 4 for each group). NE stimulation at 20 mM induced two changes; increase in the intensity of phosphorylated Cx43 (p ¼ 2.2 103, n ¼ 7 for each group) without changes in total Cx43 protein level (p ¼ 9.0 101, n ¼ 7 for each group) (Fig. 1B), and a change in migration patterns to higher molecular weight (Fig. 1C), both of which indicate increase in the phosphorylation of Cx43 at Ser368. We confirmed that this observation by several lines of investigation. First, NE-induced phosphorylation of Cx43 at Ser368 was confirmed by using an antibody from a different source that was raised against phosphopeptides from different species (human vs. rat) (Fig. 1D, p ¼ 9.4 104, n ¼ 8 for each group). Second, the migration patterns of total Cx43 changed from a broad range extending to higher molecular weights to a sharp range at lower molecular weight upon in vitro dephosphorylation reaction (Supplementary Fig. 1). Finally, the change in the migration pattern of Cx43 upon NE stimulation is abolished when the proteins were dephosphorylated in vitro (Supplementary Fig. 1). Examples of the full range of Western blotting results are shown in Supplementary Fig. 2. All of these results strongly suggest that NE stimulation induces Cx43 phosphorylation on Ser368. As astrocytic responses to neurotransmitters may change during developmental stages [22], we characterized astrocytic responses to NE in adolescent animals. Stimulation of brain slices prepared from adolescent mice (7-8 week-old mice) indicated that NE induces robust Cx43 phosphorylation at this stage (Fig. 1E, p ¼ 1.5 103, control: n ¼ 8, NE: n ¼ 7). Next, to characterize the kinetics, we monitored the phosphorylation level of Cx43 across various recovery periods following 5 min pulsatile stimulation by NE. This analysis revealed that the phosphorylation level of Cx43
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remains elevated for tens of minutes before returning to the basal level after one hour (Fig. 1F) (n ¼ 8, 6, 6, 6, 4 for control, 0 min, 5 min, 20 min and 60 min, respectively). In contrast, we found that the same 5 min NE stimulation increases intracellular calcium concentration, but it rapidly returns to basal levels within minutes when analyzed by two-photon microscopy (Supplementary Fig. 3). Therefore, these data combined suggest that when astrocytes receive transient NE stimulation, the information is conferred to phosphorylation of Cx43 that decays with its own kinetics, in much longer timescale than that of calcium dynamics. To further examine the molecular mechanisms underlying this event, we pharmacologically dissected the signaling pathway involved in NE-induced phosphorylation of Cx43. Since Cx43Ser368 is known to be phosphorylated by protein kinase C (PKC) in vitro [14,15], we hypothesized that NE may phosphorylate Cx43 at Ser368 via activation of the a1-adrenoceptor that is coupled to Gq-type G-proteins in the brain. Indeed, we found that specific activation of a1-adrenergic receptors using phenylephrine induced phosphorylation in the same manner as NE stimulation (Fig. 2A, p ¼ 1.2 102, control: n ¼ 8, NE: n ¼ 6). Furthermore, NEinduced phosphorylation of Cx43 at Ser368 was blocked by preincubation of brain slices with the a1-adrenoceptor antagonist prazosin (Fig. 2B, p ¼ 1.0, n ¼ 4 for each group). Finally, blockade of PKC activation by bisindolylmaleimide I (Go6850) abolished the phosphorylation (Fig. 2C, p ¼ 1.0, n ¼ 5 for each group). As was the case for normal NE stimulation (Fig. 1B), there was no difference in the total Cx43 level under any of these pharmacological manipulations (Supplementary Fig. 4). All of these results strongly suggest that NE-induced phosphorylation of Cx43 at Ser368 is mediated by activation of a1-adrenergic receptors and the following PKC pathway. As astrocytes communicate with neurons, NE's action on Cx43 in astrocytes may be mediated by modulation of neuronal activities and following changes in neuronal regulation on astrocytes. To test if the effect of NE is directly on astrocytes or indirectly mediated by neuronal responses, the same experiment was performed in the presence of tetrodotoxin, a blocker of voltage-gated sodium channel and therefore action potential-dependent neuronal activities. NE-induced phosphorylation of Cx43 on Ser368, however, persisted in the presence of tetrodotoxin, indicating that the effect is likely to be a direct result of astrocytic activation (Fig. 2D, p ¼ 3.1 102, n ¼ 6 for each group). To exclude the possibility of potential involvement of action potential-independent neuronal transmissions, the same experiment was performed in the presence of a glutamate receptor antagonist cocktail (CNQX, AP5 and MCPG) [21]. Again, even in the absence of glutamatergic transmissions, NE induced robust phosphorylation of Cx43 (Fig. 2E, p ¼ 2.2 103, n ¼ 7 for each group). Taken together, these data strongly suggest that the action of NE on the phosphorylation of Cx43 at Ser368 is due to the direct action on astrocytes but not indirectly through the modulation of neuronal transmission. We next assessed the consequences of NE-induced phosphorylation on Cx43 proteins. Two biochemical analyses were performed to examine gap junctional assembly (Supplementary Fig. 5) [23]. First, solubility of proteins in 1% triton X-100 was examined. When stimulated by NE for 5 min, a significantly higher fraction of Cx43 became triton X-100 soluble (Fig. 3A, p ¼ 4.9 102, n ¼ 6 for each group). As connexins at the functional gap junction become resistant to solubilization by triton [23], this result indicates that NE stimulation induce relocation of Cx43 from gap junction plaques to
Ser368 of rat Cx43, whereas the one used for the rest of figures was against the human sequence. Band intensities were normalized to those of b-actin. (E). Phosphorylation in adolescent animals. Brain slices prepared from adolescent animals (7-8 week-old mice) were treated with 20 mM NE for 5 min 37 C and probed for phosphorylated Cx43.Band intensities were normalized to those of b-actin. (F). Time course of NE-induced phosphorylation of Cx43 at Ser368. Brain slices were treated with 20 mM NE for 5 min and recovered in normal ACSF for 0, 5, 20 and 60 min. Band intensities were normalized to those of b-actin. NE was applied to brain slices from 5 to 0 min.
Please cite this article in press as: M. Nuriya, et al., Norepinephrine induces rapid and long-lasting phosphorylation and redistribution of connexin 43 in cortical astrocytes, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.09.021
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Fig. 2. Pharmacological characterization of NE-induced phosphorylation of Cx43. (A).a1-Adrenoceptor agonists mimic NE-induce phosphorylation of Cx43. Cortical brain slices were treated with 20 mM (R)-()-phenylephrine for 5 min at 37 C and the lysates were probed for Cx43 phosphorylated at Ser368. The band intensities were normalized to those of b-actin. (B) a1-Adrenoceptor antagonist blocks NE-induced phosphorylation of Cx43. Brain slices were stimulated by 20 mM NE for 5 min at 37 C in the presence of prazosin (10 mM). (C). Inhibition of PKC activity blocks NE-induced phosphorylation of Cx43. Brain slices were stimulated with 20 mM NE for 5 min at 37 C in the presence of bisindolylmaleimide I (200 nM). (D). Blockade of neuronal activities by tetrodotoxin does not alter NE-induced phosphorylation of Cx43. Brain slices were stimulated with 20 mM NE for 5 min at 37 C in the presence of tetrodotoxin (TTX, 1 mM). (E). Blockade of glutamatergic transmissions does not alter NE-induced phosphorylation of Cx43. Brain slices were stimulated with 20 mM NE for 5 min at 37 C in the presence of glutamate receptor antagonist cocktail (50 mM AP5, 50 mM CNQX and 1 mM MCPG).
non-gap junctional sites. This can be achieved by either lateral diffusion out from the gap junction plaques or by internalization (Supplementary Fig. 5). To distinguish these possibilities, a surface biotinylation assay was performed [23]. Interestingly, 5 min NE treatment significantly increased Cx43 biotinylation, suggesting a shift of Cx43 from gap junction plaques to freely accessible nonjunctional positions at the plasma membrane (Fig. 3B, p ¼ 8.2 103, n ¼ 6 for each group). In contrast, other cell surface proteins such as postsynaptic glutamate receptor GluA2 or the astrocytic protein b-dystroglycan are stable during this period (Fig. 3B and Supplementary Fig. 6, p ¼ 5.8 101 for GluA2, p ¼ 4.7 101 for b-dystroglycan, n ¼ 6 for each group). Importantly, when slices were incubated in normal artificial cerebrospinal fluid (ACSF) after 5 min of NE stimulation, changes in biotinylation efficacy remains at 20 min but returns to original levels at 60 min, suggesting that the translocation of Cx43 is a
transient and reversible process (Fig. 3C). Taken together, these results suggest that phosphorylation of Cx43 on Ser368 upon NE stimulation disassemble them from functional gap-junctional plaques to a non-functional pool at the plasma membrane. Finally, we examined whether the phosphorylation of Cx43 is regulated in vivo. First, the phosphorylation of Cx43 was examined using brain lysates. This revealed that Cx43 is phosphorylated at Ser368 in the basal state both in juvenile (2-week-old) as well as adolescent (8-week-old) mice (Fig. 4A). Dephosphorylation of proteins abolished the immunoreactivity of the phospho-specific antibody, confirming the specificity of both phosphorylation and detection (Fig. 4A). Next, the effect of the a1-adrenoceptor antagonist prazosin was examined. Treatment of animals with prazosin significantly reduced the phosphorylation of Cx43 at Ser368 in the cerebral cortex (Fig. 4B, p ¼ 1.1 102, n ¼ 7 for each group). This result confirms the conclusions obtained from in vitro experiments
Please cite this article in press as: M. Nuriya, et al., Norepinephrine induces rapid and long-lasting phosphorylation and redistribution of connexin 43 in cortical astrocytes, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.09.021
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Fig. 3. Biochemical characterization of NE-induced changes of Cx43 proteins. (A). NE stimulation changes the solubility of Cx43 to triton X-100. Cortical brain slices were treated with 20 mM NE for 5 min at 37 C and the lysate were fractionated into non-DRM (1% triton X-100 soluble) and DRM (1% triton X-100 insoluble) fractions. The right panel shows the normalized Non-DRM/DRM ratio. B. NE stimulation changes the efficacy of Cx43 surface biotinylation. Brain slices were treated with 20 mM NE for 5 min at 37 C and labeled with NHS-SS-biotin to label the surface proteins. Biotinylated proteins were then collected and probed for Cx43 (left) and GluA2 (right). The graph shows the normalized biotinylation ratio. (C). Time course of NE-induced changes in the Cx43 surface biotinylation. Brain slices were treated with 20 mM NE for 5 min at 37 C and incubated in the normal ACSF for 20 and 60 min. The slices were then biotinylated and biotinylated proteins were quantified as above.
Please cite this article in press as: M. Nuriya, et al., Norepinephrine induces rapid and long-lasting phosphorylation and redistribution of connexin 43 in cortical astrocytes, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.09.021
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Fig. 4. Characterization of NE-dependent Cx43 phosphorylation in vivo. (A). Cx43 phosphorylation in vivo. Cortical brain lysate from juvenile (J: 16-day-old) and adolescent (A: 8week-old) animals were prepared and Western blotting was performed on the membrane processed with or without l protein phosphatase (lPPase) in vitro phosphatase reaction. The samples were probed for phospho-Cx43, total Cx43 and b-actin.(B). a1AR antagonist treatment reduces Cx43 phosphorylation in vivo. Cortical brain lysates prepared from animals treated with prazosin or vehicle, were probed for phospho-Cx43. The band intensities were normalized to those of b-actin. (C). a2AR antagonist treatment reduces Cx43 phosphorylation in vivo. Cortical brain lysates prepared from animals treated with xylazine or vehicle, were probed for phospho-Cx43. The band intensities were normalized to those of b-actin.
and further suggest that the basal phosphorylation level of Cx43 is maintained by the ambient concentration of NE in the brain in vivo. To examine this possibility, pharmacological manipulations of endogenous NE levels by activation of presynaptic inhibitory a2adrenoceptors using xylazine was performed [18]. Indeed, xylazine significantly reduced phosphorylation of Cx43 compared to vehicle-treated animals (Fig. 4C, p ¼ 3.0 102, n ¼ 7 for each group). These results support the conclusions from brain slice experiments and suggest that phosphorylation of Cx43 is subject to modifications via NE tone in vivo. 3. Discussion In this study, we demonstrate that NE induces rapid and longlasting modifications of Cx43 in the murine cerebral cortex. A pioneering study has indicated that astrocytes can be the target of NE in cultured striatal astrocytes [24], but the molecular mechanisms as well as the nature of these modifications remained unknown. Our study extends these studies to brain tissue in vitro and in vivo and reveals a gliomodulatory action of NE; NE-induces long-
lasting biochemical modification of Cx43 through its direct action on astrocytes. Our biochemical analyses revealed that NE-induced phosphorylation of Cx43 is accompanied by dissociation from gap junctional plaques to non-gap junctional pools, which occurs rapidly (<5 min) and return to the original state over a longer time course (~20 min) (Fig. 3, schematized in Supplementary Fig. 7). In addition to translocation of Cx43 proteins at the cellular level, phosphorylation of Cx43 at Ser368 has been shown to reduce gap junctional permeability [14e16]. While it is beyond the scope of this study, it is tempting to deduce the consequences of this NE-induced phosphorylation of Cx43 on astrocyte physiology. Functional down regulation of astrocyte gap junctions would result in reduced buffering capacity of local changes in ions and messengers across the extracellular space [4,5]. Therefore, NE-induced biochemical changes in astrocytes may affect synaptic transmissions and thereby information processing in the brain, resulting in higher sensitivity. Importantly, xylazine and prazosin, both of which reduce the phosphorylation of Cx43 at Ser368 in vivo (Fig. 4), are known to have sedative effects. While clearly having direct actions
Please cite this article in press as: M. Nuriya, et al., Norepinephrine induces rapid and long-lasting phosphorylation and redistribution of connexin 43 in cortical astrocytes, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.09.021
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on neurons, our results raise the possibility that some psychoactive compounds exert their actions in part through their gliomodulatory actions on astrocytes. Two technical points are worth further discussion. First, while we stimulated brain slices with NE for 5 min, as it was the shortest reliable stimulation time that could be performed in our system, it should be noted that the phosphorylation may occur in a much faster timescale than 5 min. Second, although we focused on the regulation of Ser368 of Cx43, since it is the best characterized site with proven analytical tools available, it does not mean that this is the only site being regulated by NE. Although it is beyond the scope of this study, other known potential phosphorylation sites may be simultaneously regulated by NE and may contribute to the functional regulation of astrocytic gap junctions. In conclusion, our study demonstrates that NE exerts gliomodulatory actions by directly acting on astrocytes to biochemically modulate Cx43 in a plastic manner. 4. Experimental procedures
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Sepharose Beads (Thermo Fisher Scientific) and compared to total protein. Anti-phospho-connexin 43 rabbit polyclonal antibody (Cell Signaling Technologies AB_2110169 and Abcam AB_731707), anticonnexin 43 rabbit polyclonal antibody (Thermo Fisher Scientific AB_2533973) and anti-b-actin mouse monoclonal antibody (Sigma Aldrich AB_476743) were purchased from the indicated companies. For phospho-connexin 43 immunodetection, the antibody from Cell Signaling Technologies was used for all the Western blotting except for that in Fig. 1D, where the one from Abcam was used to confirm the result. Band intensities of Cx43 were normalized to those of b-actin, which were obtained by re-probing the same membrane after stripping with stripping buffer (Nacalai Tesque). 4.4. Two-photon calcium imaging Time-lapse imaging of astrocytic intracellular calcium dynamics were performed as previously reported, using a FV1000MPE microscopy system (Olympus) equipped with MaiTaiHP femtosecond laser tuned to 840 nm (Newport) [9,21].
4.1. Slice preparation 4.5. Data analysis All procedures related to the care and the treatment of animals were approved by the committee of the School of Medicine, Keio University. Acute cortical slices were prepared essentially as described previously [11,25]. 4.2. Pharmacology Pharmacological manipulations were performed in a custommade chamber filled with ACSF containing 126 mM NaCl, 26 mM NaHCO3, 1.1 mM NaH2PO4, 10 mM dextrose, 3.0 mM KCl, 1.0 mM MgCl2, and 3.0 mM CaCl2 (pH 7.3) bubbled with 95% O2/5% CO2 at 37 C. Tetrodotoxin (Abcam, 1 mM), prazosin (Sigma Aldrich, 10 mM), and bisindolylmaleimide I (Merck, 200 nM) were applied 10 min before NE (Sigma Aldrich) stimulation for 5 min. Control samples were treated with vehicle in the same manner. For pharmacological manipulations in vivo, animals (~3-week-old) were injected with drugs by intraperitoneal injection (i.p.) at a dose of 10 mg/kg and returned to home cage for 1 h before being sacrificed for analysis. 4.3. Biochemistry Preparations of protein samples from cortical brain slices for biochemical analyses and Western blotting detections were performed as described previously [25]. In in vivo experiments, mice were anesthetized with isoflurane and the brains were immediately removed after decapitation. The cerebrum was dissected in HEPES ACSF and homogenized in lysis buffer followed by sample preparation as for brain slices. In vitro phosphatase reactions were performed with lambda protein phosphatase (New England Biolabs) in the reaction buffer supplied by the manufacturer at R.T. overnight for the Western blot membrane (Figs. 4A), and 30 C for 30 min for brain lysate (Supplementary Fig. 1). Fractionation experiments to characterize protein localization in DRM (detergent resistant membrane) and Non-DRM were performed as reported previously [11]. Biotinylation assays were performed essentially as described previously with some modifications [26]. Briefly, brain slices were transferred to micro centrifuge tubes and washed with HEPES ACSF on ice. Then, slices were incubated in 1 mg/mL Sulfo-NHS-SS-Biotin (Thermo Fisher Scientific) in HEPES ACSF for 30 min at 4 C with gentle rotation. The slices were washed with TBS three times to quench excess biotinylation reagents and lysates were prepared as above. Biotinylated proteins were then isolated using NeutrAvidin
Quantitative analyses of immunoblot data were performed using ImageJ software (National Institute of Health, USA). Plot profiles of the Western blotting were performed essentially as previously described [11]. Statistical analyses were performed using OriginPro (Origin Lab) and significance was judged by Mann-Whitney test for two groups and one-way ANOVA followed by Fisher's test for comparison among three groups or more. All data are shown as mean ± standard error of the mean and statistical significance of <0.05 and < 0.01 are indicated by * and **, respectively. Acknowledgements We dedicate this paper to our beloved friend and colleague the late Ms. Ayaka Morita. This work was supported by the JSPS KAKENHI (16K07065), the Sumitomo Foundation, the Takeda Science Foundation, the Kowa Foundation, the SUNBOR Grant, and the Ayaka Foundation. Appendix A. Supplementary data Supplementary data related to this chapter can be found at https://doi.org/10.1016/j.bbrc.2018.09.021. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.09.021. References [1] A. Verkhratsky, M. Nedergaard, Physiology of Astroglia, Physiol. Rev. 98 (2018) 239e389. [2] M.M. Halassa, T. Fellin, H. Takano, et al., Synaptic islands defined by the territory of a single astrocyte, J. Neurosci. 27 (2007) 6473e6477. [3] G.C. Petzold, V.N. Murthy, Role of astrocytes in neurovascular coupling, Neuron 71 (2011) 782e797. [4] A. Verkhratsky, M. Nedergaard, Astroglial cradle in the life of the synapse, Philos. Trans. R. Soc. Lond. B Biol. Sci. 369 (2014) 20130595. [5] G. Dallerac, O. Chever, N. Rouach, How do astrocytes shape synaptic transmission? Insights from electrophysiology, Front. Cell. Neurosci. 7 (2013) 159. [6] C. Giaume, X. Liu, From a glial syncytium to a more restricted and specific glial networking, J. Physiol. Paris 106 (1-2) (2012) 34e39. [7] N. Rouach, A. Koulakoff, V. Abudara, et al., Astroglial metabolic networks sustain hippocampal synaptic transmission, Science 322 (2008) 1551e1555. [8] U. Pannasch, L. Vargova, J. Reingruber, et al., Astroglial networks scale synaptic
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Please cite this article in press as: M. Nuriya, et al., Norepinephrine induces rapid and long-lasting phosphorylation and redistribution of connexin 43 in cortical astrocytes, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.09.021