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Acute connexin43 temporal and spatial expression in response to ischemic stroke

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Journal of Cell Communication and Signaling Aims and scope

Abstract

Connexin43 (Cx43) gap junctions expressed in astrocytes can significantly impact neuronal survival in stroke. However, little is known regarding Cx43 spatial and temporal expression during the initial stages of brain ischemia. Using immunohistochemistry and Western blot analysis, we examined Cx43 spatial and temporal expression as a function of neuronal injury within the first 24 h after permanent middle cerebral artery occlusion (pMCAO). Western blot analysis showed a significant increase in Cx43 protein expression in the core ischemic area at 2 and 3 h after pMCAO. However, after 6 h of pMCAO Cx43 levels were significantly reduced. This reduction was due to cell death and concomitant Cx43 degradation in the expanding focal ischemic region, while the peri-infarct zone revealed intense Cx43 staining. The neuronal cell-death marker Fluoro-Jade C labeled injured neurons faintly at 1 h post-pMCAO with a time-dependent increase in both intensity and size of punctate staining. In addition, decreased microtubule-associated protein 2 (MAP2) immunoreactivity and thionin staining similarly indicated cell damage beginning at 1 h after pMCAO. Taken together, Cx43 expression is sensitive to neuronal injury and can be detected as early as 2 h post-pMCAO. These findings underscore Cx43 gap junction as a potential early target for therapeutic intervention in ischemic stroke.

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Abbreviations

Cx26:

Connexin26

Cx30:

Connexin30

Cx43:

Connexin43

DAPI:

4′,6-diamidino-2-phenylindole

GFAP:

Glial Fibrillary Acidic Protein

MCA:

Middle Cerebral Artery

pMCAO:

Permanent Middle Cerebral Artery Occlusion

MAP2:

Microtubule-associated protein 2

OCT:

Optimal Cutting Temperature Compound

PBS:

Phosphate Buffered Saline

RIPA:

Radioimmunoprecipitation Assay

TBS-T:

Tris-buffered saline with Tween 20

References

  • Abudara V, Bechberger J, Freitas-Andrade M, De Bock M, Wang N, Bultynck G, Naus CC, Leybaert L, Giaume C (2014) The connexin43 mimetic peptide Gap19 inhibits hemichannels without altering gap junctional communication in astrocytes. Front Cell Neurosci 8:306

    Article  PubMed  PubMed Central  Google Scholar 

  • Anderson MF, Blomstrand F, Blomstrand C, Eriksson PS, Nilsson M (2003) Astrocytes and stroke: networking for survival? Neurochem Res 28:293–305

    Article  CAS  PubMed  Google Scholar 

  • Barreto G, White RE, Ouyang Y, Xu L, Giffard RG (2011) Astrocytes: targets for neuroprotection in stroke. Cent Nerv Syst Agents Med Chem 11:164–173

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Broussalis E, Killer M, McCoy M, Harrer A, Trinka E, Kraus J (2012) Current therapies in ischemic stroke. Part a. Recent developments in acute stroke treatment and in stroke prevention. Drug Discov Today 17:296–309

    Article  CAS  PubMed  Google Scholar 

  • Caceres A, Banker GA, Binder L (1986) Immunocytochemical localization of tubulin and microtubule-associated protein 2 during the development of hippocampal neurons in culture. J Neurosci 6:714–722

    CAS  PubMed  Google Scholar 

  • Cechetto DF, Wilson JX, Smith KE, Wolski D, Silver MD, Hachinski VC (1989) Autonomic and myocardial changes in middle cerebral artery occlusion: stroke models in the rat. Brain Res 502:296–305

    Article  CAS  PubMed  Google Scholar 

  • Chen Q, Boire A, Jin X, Valiente M, Er EE, Lopez-Soto A, Jacob LS, Patwa R, Shah H, Xu K, Cross JR, Massague J (2016) Carcinoma-astrocyte gap junctions promote brain metastasis by cGAMP transfer. Nature 533:493–498

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Chever O, Lee CY, Rouach N (2014) Astroglial connexin43 hemichannels tune basal excitatory synaptic transmission. J Neurosci 34:11228–11232

    Article  PubMed  Google Scholar 

  • Chiang T, Messing RO, Chou WH (2011) Mouse model of middle cerebral artery occlusion. J Vis Exp 13(48):e2761

  • Cronin M, Anderson PN, Cook JE, Green CR, Becker DL (2008) Blocking connexin43 expression reduces inflammation and improves functional recovery after spinal cord injury. Mol Cell Neurosci 39:152–160

    Article  CAS  PubMed  Google Scholar 

  • Damjanac M, Rioux Bilan A, Barrier L, Pontcharraud R, Anne C, Hugon J, Page G (2007) Fluoro-jade B staining as useful tool to identify activated microglia and astrocytes in a mouse transgenic model of Alzheimer's disease. Brain Res 1128:40–49

    Article  CAS  PubMed  Google Scholar 

  • Davidson JO, Green CR, Nicholson LF, O'Carroll SJ, Fraser M, Bennet L, Gunn AJ (2012) Connexin hemichannel blockade improves outcomes in a model of fetal ischemia. Ann Neurol 71:121–132

    Article  CAS  PubMed  Google Scholar 

  • Dawson DA, Hallenbeck JM (1996) Acute focal ischemia-induced alterations in MAP2 immunostaining: description of temporal changes and utilization as a marker for volumetric assessment of acute brain injury. J Cereb Blood Flow Metab 16:170–174

    Article  CAS  PubMed  Google Scholar 

  • del Zoppo GJ, Sharp FR, Heiss WD, Albers GW (2011) Heterogeneity in the penumbra. J Cereb Blood Flow Metab 31:1836–1851

    Article  PubMed  PubMed Central  Google Scholar 

  • Desplantez T, Verma V, Leybaert L, Evans WH, Weingart R (2012) Gap26, a connexin mimetic peptide, inhibits currents carried by connexin43 hemichannels and gap junction channels. Pharmacol Res 65:546–552

    Article  CAS  PubMed  Google Scholar 

  • D'Hondt C, Iyyathurai J, Himpens B, Leybaert L, Bultynck G (2014) Cx43-hemichannel function and regulation in physiology and pathophysiology: insights from the bovine corneal endothelial cell system and beyond. Front Physiol 5:348

    Article  PubMed  PubMed Central  Google Scholar 

  • Enkvist MO, McCarthy KD (1994) Astroglial gap junction communication is increased by treatment with either glutamate or high K+ concentration. J Neurochem 62:489–495

    Article  CAS  PubMed  Google Scholar 

  • Fonseca CG, Green CR, Nicholson LF (2002) Upregulation in astrocytic connexin 43 gap junction levels may exacerbate generalized seizures in mesial temporal lobe epilepsy. Brain Res 929:105–116

    Article  CAS  PubMed  Google Scholar 

  • Freitas-Andrade M, Naus CC (2016) Astrocytes in neuroprotection and neurodegeneration: the role of connexin43 and pannexin1. Neuroscience 323:207–221

    Article  CAS  PubMed  Google Scholar 

  • Freitas-Andrade M, Carmeliet P, Stanimirovic DB, Moreno M (2008) VEGFR-2-mediated increased proliferation and survival in response to oxygen and glucose deprivation in PlGF knockout astrocytes. J Neurochem 107:756–767

    Article  CAS  PubMed  Google Scholar 

  • Freitas-Andrade M, Bechberger JF, MacVicar BA, Viau V, Naus CC (2017) Pannexin1 knockout and blockade reduces ischemic stroke injury in female, but not in male mice. Oncotarget 8:36973–36983

    Article  PubMed  PubMed Central  Google Scholar 

  • Froger N, Orellana JA, Calvo CF, Amigou E, Kozoriz MG, Naus CC, Saez JC, Giaume C (2010) Inhibition of cytokine-induced connexin43 hemichannel activity in astrocytes is neuroprotective. Mol Cell Neurosci 45:37–46

    Article  CAS  PubMed  Google Scholar 

  • George PM, Steinberg GK (2015) Novel stroke therapeutics: unraveling stroke pathophysiology and its impact on clinical treatments. Neuron 87:297–309

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Giaume C, Theis M (2010) Pharmacological and genetic approaches to study connexin-mediated channels in glial cells of the central nervous system. Brain Res Rev 63:160–176

    Article  CAS  PubMed  Google Scholar 

  • Hossmann KA (2012) The two pathophysiologies of focal brain ischemia: implications for translational stroke research. J Cereb Blood Flow Metab 32:1310–1316

    Article  PubMed  PubMed Central  Google Scholar 

  • Kozoriz MG, Bechberger JF, Bechberger GR, Suen MW, Moreno AP, Maass K, Willecke K, Naus CC (2010) The connexin43 C-terminal region mediates neuroprotection during stroke. J Neuropathol Exp Neurol 69:196–206

    Article  CAS  PubMed  Google Scholar 

  • Kozoriz MG, Lai S, Vega JL, Saez JC, Sin WC, Bechberger JF, Naus CC (2013) Cerebral ischemic injury is enhanced in a model of oculodentodigital dysplasia. Neuropharmacology 75:549–556

    Article  CAS  PubMed  Google Scholar 

  • Lansberg MG, Bluhmki E, Thijs VN (2009) Efficacy and safety of tissue plasminogen activator 3 to 4.5 hours after acute ischemic stroke: a metaanalysis. Stroke 40:2438–2441

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Le HT, Sin WC, Lozinsky S, Bechberger J, Vega JL, Guo XQ, Saez JC, Naus CC (2014) Gap junction intercellular communication mediated by connexin43 in astrocytes is essential for their resistance to oxidative stress. J Biol Chem 289:1345–1354

  • Li WE, Ochalski PA, Hertzberg EL, Nagy JI (1998) Immunorecognition, ultrastructure and phosphorylation status of astrocytic gap junctions and connexin43 in rat brain after cerebral focal ischaemia. Eur J Neurosci 10:2444–2463

    Article  CAS  PubMed  Google Scholar 

  • Lo EH, Dalkara T, Moskowitz MA (2003) Mechanisms, challenges and opportunities in stroke. Nat Rev Neurosci 4:399–415

    Article  CAS  PubMed  Google Scholar 

  • Maes M, Crespo Yanguas S, Willebrords J, Weemhoff JL, da Silva TC, Decrock E, Lebofsky M, Pereira IVA, Leybaert L, Farhood A, Jaeschke H, Cogliati B, Vinken M (2017) Connexin hemichannel inhibition reduces acetaminophen-induced liver injury in mice. Toxicol Lett 278:30–37

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Meier C, Rosenkranz K (2014) Cx43 expression and function in the nervous system-implications for stem cell mediated regeneration. Front Physiol 5:106

    Article  PubMed  PubMed Central  Google Scholar 

  • Nakase T, Naus CC (2004) Gap junctions and neurological disorders of the central nervous system. Biochim Biophys Acta 1662:149–158

    Article  CAS  PubMed  Google Scholar 

  • Nakase T, Fushiki S, Naus CC (2003a) Astrocytic gap junctions composed of connexin 43 reduce apoptotic neuronal damage in cerebral ischemia. Stroke 34:1987–1993

    Article  PubMed  Google Scholar 

  • Nakase T, Fushiki S, Sohl G, Theis M, Willecke K, Naus CC (2003b) Neuroprotective role of astrocytic gap junctions in ischemic stroke. Cell Commun Adhes 10:413–417

    Article  CAS  PubMed  Google Scholar 

  • Nakase T, Sohl G, Theis M, Willecke K, Naus CC (2004) Increased apoptosis and inflammation after focal brain ischemia in mice lacking connexin43 in astrocytes. Am J Pathol 164:2067–2075

  • Nakase T, Yoshida Y, Nagata K (2006) Enhanced connexin 43 immunoreactivity in penumbral areas in the human brain following ischemia. Glia 54:369–375

    Article  PubMed  Google Scholar 

  • Orellana JA, Saez PJ, Shoji KF, Schalper KA, Palacios-Prado N, Velarde V, Giaume C, Bennett MV, Saez JC (2009) Modulation of brain hemichannels and gap junction channels by pro-inflammatory agents and their possible role in neurodegeneration. Antioxid Redox Signal 11:369–399

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Orellana JA, Hernandez DE, Ezan P, Velarde V, Bennett MV, Giaume C, Saez JC (2010) Hypoxia in high glucose followed by reoxygenation in normal glucose reduces the viability of cortical astrocytes through increased permeability of connexin 43 hemichannels. Glia 58:329–343

    PubMed  PubMed Central  Google Scholar 

  • Ponsaerts R, De Vuyst E, Retamal M, D'Hondt C, Vermeire D, Wang N, De Smedt H, Zimmermann P, Himpens B, Vereecke J, Leybaert L, Bultynck G (2010) Intramolecular loop/tail interactions are essential for connexin 43-hemichannel activity. FASEB J 24:4378–4395

    Article  CAS  PubMed  Google Scholar 

  • Ramos-Cabrer P, Campos F, Sobrino T, Castillo J (2011) Targeting the ischemic penumbra. Stroke 42:S7–11

    Article  PubMed  Google Scholar 

  • Retamal MA, Schalper KA, Shoji KF, Orellana JA, Bennett MV, Saez JC (2007a) Possible involvement of different connexin43 domains in plasma membrane permeabilization induced by ischemia-reperfusion. J Membr Biol 218:49–63

    Article  CAS  PubMed  Google Scholar 

  • Retamal MA, Froger N, Palacios-Prado N, Ezan P, Saez PJ, Saez JC, Giaume C (2007b) Cx43 hemichannels and gap junction channels in astrocytes are regulated oppositely by proinflammatory cytokines released from activated microglia. J Neurosci 27:13781–13792

    Article  CAS  PubMed  Google Scholar 

  • Rovegno M, Soto PA, Saez PJ, Naus CC, Saez JC, von Bernhardi R (2015) Connexin43 hemichannels mediate secondary cellular damage spread from the trauma zone to distal zones in astrocyte monolayers. Glia 63:1185–1199

    Article  PubMed  Google Scholar 

  • Sadler TR, Khodavirdi AC, Hinton DR, Holschneider DP (2009) Snap-frozen brain tissue sections stored with desiccant at ambient laboratory conditions without chemical fixation are resistant to degradation for a minimum of 6 months. Appl Immunohistochem Mol Morphol 17:165–171

    Article  PubMed  PubMed Central  Google Scholar 

  • Schmued LC, Stowers CC, Scallet AC, Xu L (2005) Fluoro-jade C results in ultra high resolution and contrast labeling of degenerating neurons. Brain Res 1035:24–31

    Article  CAS  PubMed  Google Scholar 

  • Sena E, van der Worp HB, Howells D, Macleod M (2007) How can we improve the pre-clinical development of drugs for stroke? Trends Neurosci 30:433–439

    Article  CAS  PubMed  Google Scholar 

  • Shinotsuka T, Yasui M, Nuriya M (2014) Astrocytic gap junctional networks suppress cellular damage in an in vitro model of ischemia. Biochem Biophys Res Commun 444:171–176

    Article  CAS  PubMed  Google Scholar 

  • Siushansian R, Bechberger JF, Cechetto DF, Hachinski VC, Naus CC (2001) Connexin43 null mutation increases infarct size after stroke. J Comp Neurol 440:387–394

    Article  CAS  PubMed  Google Scholar 

  • Skyschally A, Walter B, Schultz Hansen R, Heusch G (2013) The antiarrhythmic dipeptide ZP1609 (danegaptide) when given at reperfusion reduces myocardial infarct size in pigs. Naunyn-Schmiedeberg's. Arch Pharm 386:383–391

    Article  CAS  Google Scholar 

  • Solan JL, Lampe PD (2005) Connexin phosphorylation as a regulatory event linked to gap junction channel assembly. Biochim Biophys Acta 1711:154–163

    Article  CAS  PubMed  Google Scholar 

  • Solan JL, Lampe PD (2009) Connexin43 phosphorylation: structural changes and biological effects. Biochem J 419:261–272

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tamura K, Alessandri B, Heimann A, Kempski O (2011) The effect of a gap-junction blocker, carbenoxolone, on ischemic brain injury and cortical spreading depression. Neuroscience 194:262–271

    Article  CAS  PubMed  Google Scholar 

  • Theodoric N, Bechberger JF, Naus CC, Sin WC (2012) Role of gap junction protein Connexin43 in astrogliosis induced by brain injury. PLoS One 7:e47311

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wang N et al (2013) Selective inhibition of Cx43 hemichannels by Gap19 and its impact on myocardial ischemia/reperfusion injury. Basic Res Cardiol 108:309

    Article  PubMed  Google Scholar 

  • Willebrords J, Cogliati B, Pereira IVA, da Silva TC, Crespo Yanguas S, Maes M, Govoni VM, Lima A, Felisbino DA, Decrock E, Nogueira MS, de Castro IA, Leclercq I, Leybaert L, Rodrigues RM, Vinken M (2017) Inhibition of connexin hemichannels alleviates non-alcoholic steatohepatitis in mice. Sci Rep 7:8268

    Article  PubMed  PubMed Central  Google Scholar 

  • Yoon JJ, Green CR, O'Carroll SJ, Nicholson LF (2010a) Dose-dependent protective effect of connexin43 mimetic peptide against neurodegeneration in an ex vivo model of epileptiform lesion. Epilepsy Res 92:153–162

    Article  CAS  PubMed  Google Scholar 

  • Yoon JJ, Nicholson LF, Feng SX, Vis JC, Green CR (2010b) A novel method of organotypic brain slice culture using connexin-specific antisense oligodeoxynucleotides to improve neuronal survival. Brain Res 1353:194–203

    Article  CAS  PubMed  Google Scholar 

  • Yoshimoto T, Siesjo BK (1999) Posttreatment with the immunosuppressant cyclosporin a in transient focal ischemia. Brain Res 839:283–291

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This study was funded through a grant from the Canadian Institutes of Health Research (CIHR) to CCN and WCS, and a CIHR Team Grant (R1478A47) on “Vascular Cognitive Impairment: Animal Models of Co-morbidity.” MFA was supported by a fellowship from the Heart and Stroke Foundation of Canada. CCN holds a Canada Research Chair.

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  1. Moises Freitas-Andrade and Jennifer She contributed equally to this work.

Authors and Affiliations

  1. Department of Cellular and Physiological Sciences, Life Sciences Institute, The University of British Columbia, 2350 Health Sciences Mall, Vancouver, BC, V6T 1Z3, Canada

    Moises Freitas-Andrade, Jennifer She, John Bechberger, Christian C. Naus & Wun Chey Sin

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  1. Moises Freitas-Andrade
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  2. Jennifer She
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  3. John Bechberger
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  4. Christian C. Naus
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Correspondence to Wun Chey Sin.

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Freitas-Andrade, M., She, J., Bechberger, J. et al. Acute connexin43 temporal and spatial expression in response to ischemic stroke. J. Cell Commun. Signal. 12, 193–204 (2018). https://doi.org/10.1007/s12079-017-0430-6

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