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Cerebral ischemic injury is enhanced in a model of oculodentodigital dysplasia

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(1)Neuropharmacology 75 (2013) 549e556. Contents lists available at SciVerse ScienceDirect. Neuropharmacology journal homepage: www.elsevier.com/locate/neuropharm. Cerebral ischemic injury is enhanced in a model of oculodentodigital dysplasia Michael G. Kozoriz a, Simon Lai a, José L. Vega b, c, Juan C. Sáez c, d, Wun Chey Sin a, John F. Bechberger a, Christian C. Naus a, * a. Department of Cellular and Physiological Sciences, Life Sciences Institute, University of British Columbia, Vancouver, BC, Canada Experimental Physiology Laboratory (EPhyL), Instituto Antofagasta, Antofagasta, Chile Departamento de Fisiología, Pontificia Universidad Católica de Chile, Santiago, Chile d Instituto Milenio, Centro Interdiciplinario de Neurociencias de Valparaíso, Valparaíso, Chile b c. a r t i c l e i n f o. a b s t r a c t. Article history: Received 2 January 2013 Received in revised form 1 May 2013 Accepted 4 May 2013. Oculodentodigital dysplasia (ODDD) is a rare autosomal dominant disease that results in visible developmental anomalies of the limbs, face, eyes and teeth. Recently analysis of human connexin43 (Cx43) DNA sequences has revealed a number of different missense, duplication and frame shift mutations resulting in this phenotype. A mouse model of this disorder has been created with a missense point mutation of the glycine amino acid at position 60 to serine (G60S). Heterozygote þ/G60S mice exhibit a similar ODDD phenotype as observed in humans. In addition to the malformations listed above, ODDD patients often have neurological findings. In the brain, Cx43 is highly expressed in astrocytes and has been shown to play a role in neuroprotection. We were interested in determining the effect of the þ/ G60S mutation following stroke. Four days after middle cerebral artery occlusion the volume of infarct was larger in mice with the þ/G60S mutation. In astrocyte-neuron co-cultures, exposure to glutamate also resulted in greater cellular death in the þ/G60S mutants. Protein levels of Cx43 in the mutant mouse were found to be reduced when compared to the normal tissue. Cx43 protein was observed as a continual line of small punctate aggregates in the plasma membrane with increased intracellular localization, which is distinct from the larger plaques seen in the normal mouse astrocytes. Functionally, primary þ/G60S astrocytes exhibited reduced gap junctional coupling and increased hemichannel activity, which may underlie the mechanism of increased damage during stroke. This article is part of the Special Issue Section entitled ‘Current Pharmacology of Gap Junction Channels and Hemichannels’. Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved.. Keywords: Astrocytes Stroke Rare genetic disease Connexin43 Hemichannels Gap junctional communication. 1. Introduction Oculodentodigital dysplasia (ODDD) was first described by Meyer-Schwickerath et al. (1957) and further defined by Gorlin. Abbreviations: BSA, bovine serum albumin; Cx43, connexin43; DAPI, 40 ,6diamidino-2-phenylindole; DMEM, Dulbecco’s modified Eagle’s medium; EBSS, Earle’s balanced salt solution; Etd, ethidium; PI, propidium iodide; La3þ, lanthanum ion; G60S, missense point mutation of the glycine amino acid at position 60 conversion to serine; GFAP, glial fibrillary acidic protein; HBSS, Hank’s buffered salt solution; IBA-1, ionized calcium-binding adaptor molecule 1; LDH, lactate dehydrogenase; MCAO, middle cerebral artery occlusion; ODDD, oculodentodigital dysplasia; RIPA, radioimmune precipitation lysis buffer; TBS-T, Tris-buffered saline with tween. * Corresponding author. Department of Cellular and Physiological Sciences, Life Sciences Institute, University of British Columbia, 2350 Health Sciences Mall, Rm. 1352, Vancouver, BC V6T 1Z3, Canada. Tel.: þ1 604 827 4383; fax: þ1 604 827 3922. E-mail address: cnaus@interchange.ubc.ca (C.C. Naus).. et al. (1963). Common external features in this syndrome include ocular, nasal, dental and digital abnormalities (Paznekas et al., 2009). It is now known that this syndrome is caused by mutation of the gene encoding the gap junction protein connexin43 (Cx43). Currently, 62 different Cx43 mutations have been identified in ODDD patients, and in the majority of cases the mutation is dominant (Paznekas et al., 2009). Cx43 is expressed in numerous tissues and plays an important role in cellular communication. Cx43 proteins oligomerize into hexamers to form connexons, which are inserted into the plasma membrane of single cells to form hemichannels, or coupled to the connexons in neighboring cells to form gap junction channels that provide cytoplasmic continuity between cells (Sáez et al., 2003). The effect of ODDD Cx43 mutations has been studied by several groups and in general these mutants show reduced gap junction formation and increased hemichannel activity (Dobrowolski et al., 2007; McLachlan et al., 2005). Also, mutated Cx43 often exerts a. 0028-3908/$ e see front matter Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuropharm.2013.05.003.

(2) 550. M.G. Kozoriz et al. / Neuropharmacology 75 (2013) 549e556. dominant-negative effect by reducing normal Cx43 function (McLachlan et al., 2005). However, recent studies have suggested that gap junction coupling and hemichannel activity are not affected in G60S ODDD astrocytes (Wasseff et al., 2011) and C6 glioma cells expressing some ODDD mutations (Gutmann et al., 1991; Lai et al., 2006). In the brain, Cx43 is highly expressed in astrocytes, and understanding the effect of the ODDD Cx43 mutations on astrocytes remains to be clarified. There is reason to believe that mutations of Cx43 have an effect on the nervous system as ODDD patients often have neurological symptoms including ataxia, dysarthria, neurogenic bladder, seizures and spastic paraparesis (Flenniken et al., 2005; Gutmann et al., 1991; Loddenkemper et al., 2002). We have previously shown that disruption of Cx43 leads to increased infarct volume following middle cerebral artery occlusion (MCAO) (Kozoriz et al., 2010; Nakase et al., 2004; Siushansian et al., 2001), however the effect of an ODDD mutation in ischemia is not known. In this study we were interested in determining if cellular susceptibility to stroke is altered in a mouse with a þ/ G60S point mutation that exhibits an ODDD phenotype (Flenniken et al., 2005). We examined infarct volume following MCAO and neuronal death in a neuron-astrocyte co-culture preparation. We also assessed the pattern of astrocytic Cx43 expression, gap junction coupling and hemichannel activity. Taken together, the results of our study indicate that the þ/G60S Cx43 mutation results in reduced astrocytic gap junctional communication and enhanced hemichannel activity; both of these could contribute to impaired neuroprotection.. 2.3. Astrocyte cultures Astrocyte cultures from both wild-type and ODDD mice were prepared from 0 to 1 day old pups (Kozoriz et al., 2010; Ozog et al., 2002). Neocortices were dissected in PBS and placed in culture medium consisting of high glucose Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen Corp., Burlington, Canada) with 10% fetal bovine serum (FBS; HyClone, Logan, UT), 10 U/mL penicillin, and 10 U/mL streptomycin (Invitrogen Corp.). Tissue was triturated through a serological pipette and passed through a 70 mm cell strainer (BD Falcon, Bedford, MA). Cells were re-suspended in culture medium and plated on either plastic culture dishes or laminin coated coverslips and stored in a humidified incubator in 95% air/5% CO2 at 37  C. Medium changes were performed every 4 days and astrocytes were used between 14 and 21 days in vitro or when 30% confluent for the hemichannel experiments described below (w5 days). 2.4. Neuron-astrocyte co-culture cytotoxicity assay Neuron-astrocyte co-cultures were generated as previously described (Ozog et al., 2002). In brief, cultured astrocytes, prepared as described above, from each genotype were grown until confluent (10e13 days) in 35 mm plastic culture dishes. Wild-type neurons (1  106 cells/well) isolated from gestational age day 16 mice were seeded on top of the astrocyte monolayer. The co-culture media contained 3/5 neurobasal medium, 2/5 DMEM/F12, supplemented with 10 U/mL penicillin, 10 U/ mL streptomycin and B27 supplements (Invitrogen Corp.). Media changes were performed every 3 days by replacing 2/3 of the solution with fresh co-culture media. On day 8, co-cultured cells was rinsed with Earle’s balanced salt solution (EBSS) and exposed to 1 mM glutamate (SigmaeAldrich, St. Louis, MO) or vehicle in EBSS for 3 h. Cells were then maintained in co-culture media for 24 h after glutamate/ vehicle exposure. Next, samples of media were collected and frozen for subsequent analysis of lactate dehydrogenase (LDH) release using an LDH detection kit (Sigmae Aldrich). Cell cultures were also exposed to propidium iodide (30 mM in PBS) for 5 min and subsequently fixed in 4% formaldehyde solution. The tissue was mounted with ProLong Gold antifade reagent with 40 ,6-diamidino-2-phenylindole (DAPI; Molecular Probes Inc., Eugene, OR).. 2. Material and methods. 2.5. Immunohistochemistry. All experiments were performed in accordance with the guidelines established by the Canadian Council on Animal Care and were approved by The University of British Columbia Animal Care Committee. All efforts were made to minimize animal suffering, to reduce the number of animals used, and to utilize alternatives to in vivo techniques.. Immunohistochemistry was performed on cortical tissue or on cultured astrocytes from each genotype as previously described (Kozoriz et al., 2010; Nakase et al., 2004; Ozog et al., 2002). Ten mm thick glass mounted sections were rinsed in PBS and then exposed to 0.3% Triton-X-100 (Fisher Scientific) in PBS for 2 min and then rinsed in PBS. Sections were blocked in 7.5% bovine serum albumin (BSA; SigmaeAldrich) for 30 min, incubated overnight in 1% BSAePBS and primary antibody, then washed with PBS (3  10 min) and subsequently incubated for 1 h with secondary antibody in 1% BSAePBS. Slides were then rinsed 3 times for 10 min with PBS, dipped in water, and mounted with ProLong Gold antifade reagent with DAPI. Primary antibodies were used at the concentrations indicated: anti-Cx43 Cterminal (amino acid residues 363e382) antibody raised in rabbit (1:2000 dilution; SigmaeAldrich; catalog #C6219), anti-glial fibrillary acidic protein (GFAP) antibody raised in mouse (1:1000; SigmaeAldrich; catalog #G3893) and anti-ionized calcium-binding adaptor molecule 1 (IBA-1) antibody produced in rabbit (1:500; Wako Pure Chemical Industries, Richmond, VA; catalog #019-19741). Secondary antibodies consisted of a 1:500 dilution of highly cross-adsorbed goat anti-rabbit or anti-mouse IgG antibodies conjugated to Alexa Fluor 488 (catalog #A-11029 and #A-11034) or Alexa Fluor 568 (catalog #A-11031 and #A-11036) and were used as appropriate (Molecular Probes Inc.). Images were obtained using the same exposure times on a Leica TCS SP5 II Basic VIS system (Leica Microsystems Canada Inc., Concord, Ontario, Canada). Identical conditions were used for acquisition of immunofluorescence images. Immunocytochemistry on confluent astrocyte cultures was performed using the same concentration of Cx43 antibodies and secondary antibodies as used on tissue sections noted above. Astrocytes were rinsed in PBS, fixed in 4% paraformaldehyde, rinsed in PBS and then exposed to 0.1% Triton-X-100 PBS (Fisher Scientific, Ottawa, Canada) for 2 min. After a brief PBS rinse, cells were blocked in 7.5% BSA for 30 min. Astrocytes were then incubated for 1 h in PBS containing 1% BSA and primary antibody, then washed with PBS 3 times for 10 min and incubated for 1 h with a secondary antibody in PBS containing 1% BSA. Astrocytes were then rinsed 3 times for 10 min with PBS and mounted with ProLong Gold with DAPI. Images were obtained using the same exposure times on a Zeiss Axioplan2 fluorescence microscope (Carl Zeiss Ltd, Toronto, Canada). Identical conditions were used for acquisition of immunofluorescence images.. 2.1. ODDD mice and MCAO Mice from a C57BL/6 genetic background were bred with heterozygote mice harboring a þ/G60S point mutation (GjaJrt/þ strain) (Flenniken et al., 2005). The mice used in this study were obtained from the Centre for Modeling Human Disease, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto Centre for Phenogenomics, Toronto, ON Canada. They were on a FVB/NJ (25%), C3H/HEJ (25%) and C57BL/6 (50%) genetic background. In this manuscript, wildtype refers to Cx43þ/þ mice while þ/G60S refers to the autosomal dominant Cx43Jrt/þ mutant. Genotyping for endogenous and þ/G60S Cx43 has been previously described (Flenniken et al., 2005; Naus et al., 1997). The MCAO surgery was performed as previously described by our lab (Nakase et al., 2003, 2004; Siushansian et al., 2001). In brief, mice aged 8e10 weeks were anaesthetized with sodium pentobarbital (65 mg/kg intraperitoneally; MTC Pharmaceuticals, Cambridge, Canada). The mice were secured to a 37  C heating pad in a sterotaxic frame, a skin incision was made and the squamosal bone was exposed by retracting the temporalis muscle. A burr hole was made over the middle cerebral artery and the dura mater was pierced with a needle. The MCA was then occluded above and below the rhinal fissure by electrocautery. The skin incision was closed with sutures and the mice were kept in a cage warmed on a 37  C heating pad until regaining consciousness. 2.2. Infarct volume For this study a total of four mice for each genotype were used. Infarct volume was assessed as described in our previous studies (Kozoriz et al., 2010; Nakase et al., 2004; Siushansian et al., 2001). Four days after MCAO mice were deeply anesthetized with sodium pentobarbital (70 mg/kg intraperitoneally) and were transcardially perfused with phosphate-buffered saline (PBS) followed by perfusion with 10% formalin (SigmaeAldrich, Oakville, Canada). Brains were removed and stored in 10% formalin and the next day were cryoprotected in a 10% formalin/30% sucrose solution. Brain sections, 20 mm thick, were collected every 100 mm, and mounted sequentially on glass microscope slides. To quantify infarct size sections were stained with 0.125% thionin (Fisher Scientific, Ottawa, Canada) and the volume of infarct was measured by adding together the lesion area in each of the serial sections.. 2.6. Western blot Adult cortical brain tissue and astrocyte cultures were prepared and probed using standard Western blot protocols (Siushansian et al., 2001). In brief, tissue was lysed in radioimmune precipitation lysis buffer (RIPA) supplemented with protease inhibitors (Roche Laval, QC, Canada) and phosphatase inhibitors (Sigmae Aldrich). Samples were extracted with a syringe through a 26 gauge needle,.

(3) M.G. Kozoriz et al. / Neuropharmacology 75 (2013) 549e556. 551. Fig. 1. The þ/G60S mutation increases cerebral infarct volume. A, Sections obtained 4 days following MCAO indicate a larger infarct size in þ/G60S mice in comparison to Cx43þ/þ mice. B, The volume of the infarct in þ/G60S mice is significantly larger than the wild-type (*P < 0.05).. sonicated, and centrifuged at 10,000 g for 10 min. Protein concentrations were determined using the BCA protein kit quantification (Pierce, Rockford, IL). Twenty mg protein samples were boiled for 1 min in RIPA, with the exception of samples for the Cx26 blot, which were not boiled, and loaded onto a 12% polyacrylamide gel for separation. Proteins were then transferred to a polyvinylidene fluoride membrane. The membrane was blocked in 5% milk in TBS-T (Tris-buffered saline with 0.1% Tween-20) for 1 h followed by incubation in a primary antibody (see below) in 5% milk and TBS-T overnight. The blot was then washed with TBS-T and was then incubated in secondary antibody (see below) and washed. Proteins were detected by incubation with Super-Signal chemiluminescent solution (Pierce). To ensure equal loading of samples, blots were stripped and re-probed for antiglyceraldehyde 3-phosphate dehydrogenase (GAPDH). Primary antibodies were used at the following concentrations: anti-Cx43 C-terminal (1:5000), anti-Cx26 (1:500; Zymed. San Francisco, CA; catalog #51-2800), anti-Cx30 (1:250; Zymed, San Francisco, CA; catalog #71-2200) and anti-GAPDH (1:10,000; HyTest, Turku, Finland; catalog #5G4 MAb 6C5). Secondary antibodies, goat anti-mouse or antirabbit horseradish peroxidase-tagged, were used at a 1:3000 dilution (Sigma; catalog #A6154 and #A8924).. 3. Results 3.1. Cerebral infarct volume in þ/G60S mice Given the importance of Cx43 in neuroprotection following stroke (Kozoriz et al., 2010; Lin et al., 2008; Nakase et al., 2004; Siushansian et al., 2001) we were interested in determining if a þ/ G60S mutation alters the extent of ischemic injury following MCAO. The þ/G60S mutation has been reported to affect gap junction and hemichannel activity (Manias et al., 2008), therefore we anticipated there should be an increase in neuronal ischemic injury in a stroke model. Both wild-type and þ/G60S mice were subjected to MCAO and infarct volume was assessed 4 days later. þ/G60S mice had. 2.7. Gap junction coupling To assess gap junction coupling, cells were incubated with 50 mL of the gap junction permeable dye carboxyfluorescein (0.1%) and gap junction impermeable dye dextran-rhodamine (0.1%; 10 kDa) (Molecular Probes Inc., Burlington, ON, Canada) in PBS. Dye entry was induced by scraping a confluent monolayer of astrocytes with a scalpel blade and allowing the dyes to incubate for 2 min. Excess dye was washed off with several rinses of PBS. Carbenoxolone (20 mM) in PBS was added to inhibit further coupling. The extent of coupling was determined by measuring the average distance traveled by carboxyfluorescein from the dextran-rhodamine labeled cells at the scape edge. 2.8. Hemichannel assay Hemichannel activity was examined using two methods. First, using a propidium iodide uptake assay (Kozoriz et al., 2010), cells were washed with Hank’s buffered salt solution (HBSS; Invitrogen Corp.) with Ca2þ and then incubated in HBSS with or without Ca2þ containing 1.5 mM propidium iodide for 15 min. Astrocytes were then washed with HBSS, fixed and mounted with ProLong Gold antifade reagent containing DAPI for subsequent analysis. A second assay examined hemichannel activity by dye uptake and time-lapse fluorescence imaging (Orellana et al., 2010). Cells plated onto glass coverslips were washed twice with Locke’s solution containing (in mM) 154 NaCl, 5.4 KCl, 2.3 CaCl2, and 5 HEPES, at pH 7.4 and incubated in 5 mM ethidium bromide (Etd). In some experiments Ca2þ/Mg2þ free medium was used. Phase-contrast and fluorescence microscopy with time-lapse imaging were used to record cell appearance and fluorescence intensity changes in each condition. Images of Etd uptake were analyzed with the Image J software (NIH software). To test for changes in slope, regression lines were fitted to points before and after various treatments and mean values of slopes were compared using GraphPad Prism software. Lanthanum ion (La3þ; 200 mM) and carbenoxolone (25 mM) were also applied in some experiments to test the specificity of hemichannel openings versus cellular death. 2.9. Data analysis Results are reported as means  S.E.M. Unless otherwise indicated statistical comparisons were performed using a Student’s t-test with a P value of <0.05 considered statistically significant.. Fig. 2. Excitotoxic neuronal cell death is increased in þ/G60S neuron-astrocyte cocultures. A, Cellular death was evaluated by measuring LDH released into the extracellular media and comparing it to LDH within intact cells. The fraction of LDH release was greater in co-cultures from both genotypes exposed to glutamate in comparison to vehicle. Following glutamate exposure, the extent of cell death was greater in þ/G60S co-cultures in comparison to Cx43þ/þ cells (*P < 0.05; n ¼ 6 in all groups). B, Cell viability was also assessed by measuring the ability to extrude propidium iodide (PI). þ/G60S co-cultures had a larger increase in PI iodide labeled cells following exposure to glutamate in comparison to wild-type (*P < 0.05; n ¼ 6 in all groups)..

(4) 552. M.G. Kozoriz et al. / Neuropharmacology 75 (2013) 549e556. significantly larger infarct volumes in comparison to wild-type indicating there was greater damage in þ/G60S mutants (Fig. 1). We next examined the possible mechanisms for this enhanced injury in mice expressing the þ/G60S Cx43 mutation. 3.2. Excitotoxic neuronal cell death in neuron-astrocyte co-cultures To further assess the effect of a þ/G60S mutation on neuronal injury, we examined the ability of astrocytes to protect neurons. under excitotoxic conditions. Wild-type neurons were plated on a confluent layer of either wild-type astrocytes or astrocytes carrying the þ/G60S mutation. These co-cultures were subjected to glutamate at a concentration and time interval that causes death in a portion of the neurons. Cell death was determined by measuring LDH release and neuronal propidium iodide (PI) uptake 24 h after exposure to glutamate or vehicle. Glutamate exposure increased the cell death markers compared to vehicle in both genotypes (Fig. 2). In comparison to wild-type, cell death was increased following. Fig. 3. Cx43 immunolocalization in wild-type and þ/G60S brain sections and cultured astrocytes. AeC, Wild-type brain section stained for GFAP (A) displays typical punctuate Cx43 immunolocalization (B) in a cortical region similar to the infarcted region served by the middle cerebral artery. The inset in the merged image (C) shows Cx43 associated with astrocyte processes. DeF, Brain section from þ/G60S mutant mouse showing similar area of cortex stained for GFAP (D) and Cx43 (E). The merged image (F) shows extensive Cx43 immunolocalized in GFAP-positive astrocytic cell bodies and processes, most evident in the inset. GeI, Astrocytes (GFAP-positive, G) in cultures from wild-type mice also show well defined regions of Cx43 immunolocalization (H, I) between adjacent cells. JeL, Astrocyte cultures from þ/G60S mice exhibit a different pattern of immunoreactivity with a continual ring of small spots of reactivity in the membrane. Increased immunolocalization in the endoplasmic reticulum is also observed. Nuclei are labeled with DAPI (blue)..

(5) M.G. Kozoriz et al. / Neuropharmacology 75 (2013) 549e556. glutamate in þ/G60S astrocyte co-cultures indicating the þ/G60S mutation again significantly enhanced cell death (Fig. 2). 3.3. Expression pattern of Cx43 in brain tissue and cultured astrocytes from ODDD mice We next examined the cellular localization of wild-type and mutant Cx43, as well as the overall level of Cx43 protein. Immunostaining of brain sections was carried out for Cx43 and GFAP (Fig. 3A). For cerebral cortical areas, all sections showed localization of Cx43 in GFAP-positive cells. However, there was an overall reduction in the level of Cx43 immunoreactivity in sections from þ/ G60S mutant mice, as well as reduced gap junction plaque formation in the astrocytes expressing the þ/G60S mutation. It is also apparent that considerable Cx43 was localized within the cell bodies and processes of the þ/G60S mutant astrocytes. We also examined Cx43 and GFAP in astrocyte cultures prepared from þ/ G60S mutant mice (Fig. 3B). Here, we also observed reduced plaque formation with an increase in Cx43 localization in cytoplasmic compartments of þ/G60S mutant astrocytes. In addition, we observed more diffuse localization of Cx43 along the plasma membrane of þ/G60S astrocytes. Western blots were performed on samples obtained from astrocyte cultures and cortical brain tissue from both genotypes (Fig. 4). Cx43 bands were observed in both genotypes, however a reduction in the upper band (phosphorylated Cx43) was observed both in cortical and astrocyte culture samples (Fig. 4). Because mutated Cx43 may alter the expression of other connexins we examined the expression of the other major astrocyte connexin, Cx30. No difference in Cx30 expression was observed between the genotypes in adult cortical tissue (Fig. 4). As another group has shown no decrease in coupling in þ/G60S astrocyte cultures (Wasseff et al., 2011), we also probed for Cx26 as a possible connexin which is upregulated in these mutants. However, Cx26 was not detected in astrocyte cultures from either genotype, but was clearly detected in samples of mouse mammary tissue (Fig. 4). 3.4. Gap junction coupling and hemichannels activity in cultured þ/ G60S astrocytes Because gap junctional communication is thought to play a role in stroke (Kozoriz et al., 2010; Nakase et al., 2004; Rossi et al., 2007; Siushansian et al., 2001) we sought to determine if coupling was altered in þ/G60S astrocytes. A confluent monolayer of astrocytes from both genotypes was scraped in the presence of gap junction permeable and impermeable dyes. In comparison to wild-type cultures, þ/G60S cultures had reduced coupling (Fig. 5), indicating. 553. gap junctional communication is reduced in these mutant astrocytes. In addition, the hemichannel activity was increased in þ/G60S cultured astrocytes under Ca2þ and Mg2þ free conditions. This was demonstrated using propidium iodide uptake (Fig. 6), as well as in real time with Etd uptake (Fig. 7A). This effect was blocked by La3þ (200 mM) and carbenoxolone (25 mM) applied during the dye uptake assay (Fig. 7A), and reduced by preincubation with SB203580 (10 mM), a specific inhibitor of p38 MAPK (Fig. 7B), suggesting the increased hemichannel activity is dependent upon this kinase. 4. Discussion The neurological manifestations of ODDD in humans are broad (Gutmann et al., 1991; Joss et al., 2008; Loddenkemper et al., 2002). As astrocytes are the primary cell type that expresses Cx43 in the brain we examined their properties in a þ/G60S mouse model of ODDD (Flenniken et al., 2005). In astrocytes, Cx43 is known to form gap junctions and hemichannels which allow for the passage of ions and small molecules (Chen et al., 2012; Orellana et al., 2011). The effect of a þ/G60S ODDD mutation has been studied in other tissues. In ovary granulosa cells gap junction coupling is reduced. In addition gap junction plaque formation appears to be reduced in heart, ovary and teeth (Flenniken et al., 2005; Manias et al., 2008; Toth et al., 2010). Also, in rat epidermal keratinocytes transfected with þ/G60S Cx43 encoding plasmid, gap junction plaque formation was sparse and Cx43 was increased in cytoplasmic compartments (Churko et al., 2010). Our study of þ/G60S brain tissue largely echoes these findings of intracellular compartmentalization, reduced plaque formation and reduced coupling. Interestingly, another group has shown that coupling is present, but reduced, in glioma cells expressing several other ODDD mutations (Lai et al., 2006). We also examined hemichannel activity in these mutants and found that it was higher compared to wild-type. This phenomenon is associated with reduced coupling, consistent with other studies showing the activity of hemichannels and gap junction are oppositely regulated (Contreras et al., 2002; De Vuyst et al., 2007; Retamal et al., 2007). Although cultured cells, including Cx43 transfected HeLa cells and cortical astrocytes, under resting conditions show low Cx43 hemichannel activity (Contreras et al., 2002; Retamal et al., 2007; De Vuyst et al., 2009), basal dye uptake of þ/ G60S astrocytes was lower with respect to wild-type astrocytes (Fig. 7B). One explanation could be that these astrocytes have low levels of Cx43 hemichannels on the surface. Studies in mammary gland have described that Cx43 mutations related to ODDD induced a reduction in the levels of Cx43 (Plante and Laird, 2008). Although. Fig. 4. Western blot of protein from adult cortical tissue and astrocyte cultures. A, Protein samples from 4 different adult cortices indicate that the quantity of Cx43 protein in þ/ G60S mice is reduced in comparison to the wild-type, while Cx30 levels remain unchanged. GAPDH served as a loading control. B1, A reduction in expression was also found in astrocyte cultures (6 different culture preparations shown). The level of phosphorylation of Cx43 is greatly reduced in the þ/G60S mutant mouse as shown by a reduction in the intensity of the upper bands. B2, The same astrocyte protein samples as in B1 were run on a separate blot to stain for Cx26. No Cx26 was observed in either wild-type or þ/G60S cultures. Protein from mouse mammary (MAM) tissue loaded at 20 and 60 mg, respectively, served as a positive control for Cx26..

(6) 554. M.G. Kozoriz et al. / Neuropharmacology 75 (2013) 549e556. Fig. 5. Gap junction coupling is reduced in þ/G60S cultured astrocytes. A, Gap junctional communication was assessed by measuring the distance of carboxyfluorescein (green) dye passage through scraped astrocyte cultures. Dextran-rhodamine (red), which is impermeable to gap junctions, labels cells at the scrape site (yellow due to calcein and rhodamine colocalization). Examples from wild-type (A1) and þ/G60S (A2) cultures are shown. B, A graph quantifying the distance of dye passage demonstrates a reduction in gap junction coupling in þ/G60S astrocytes (*P < 0.05; n ¼ 6 in both groups). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.). Cx43 levels in the plasma membrane were not determined, we did observe the lowest levels of total Cx43 in þ/G60S astrocytes (Fig. 4). Our results showed that þ/G60S astrocytes exhibit an increased susceptibility to divalent cation removal, which induces hemichannel opening (Contreras et al., 2002; De Vuyst et al., 2009; Quist et al., 2000). These results are consistent with studies in transfected HeLa cells, with Cx43-mutations related with ODDD (G138R, G143S and I31M) that showed increased hemichannel activity (Dobrowolski et al., 2007). Moreover, calcium ion-dependent open/ closed conformations in Cx43 hemichannels require the extramembranous parts of the channel (Thimm et al., 2005). Another. Fig. 6. Hemichannel activity is increased in þ/G60S cultured astrocyte. A, Cultured astrocytes from both genotypes were exposed to propidium iodide (PI) in HBSS or Ca2þ and Mg2þ-free HBSS. In the presence of Ca2þ and Mg2þ the portion of cells taking up PI was minimal, however in the Ca2þ and Mg2þ-free media PI uptake was increased in the þ/ G60S cells in comparison to wild-type (*P < 0.05; n ¼ 6 in all groups). Examples of PI (red) uptake in Ca2þ and Mg2þ-free media for wild type (B1) and þ/G60S (B2) astrocytes is shown. Nuclei are labeled with DAPI (blue). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.). Fig. 7. Hemichannel activity is increased in þ/G60S cultured astrocytes. A. Time-lapse measurements of Etd uptake in astrocytes from wild-type (þ/þ) or þ/G60S astrocytes exposed to Locke solution containing Etd (5 mM). At 5 min, the solution was change to Ca2þ/Mg2þ-free solution. Lanthanum ion (La3þ, 200 mM) was applied after 20 min of Etd uptake measurement. B. Ratio of Etd uptake rate of wild-type (white) or þ/G60S (black) astrocytes expressed as % of maximal response under control conditions in the presence of physiological concentration of divalent cations (dashed line). As in (A) Etd uptake was then evaluated in Ca2þ/Mg2þ-free solution followed by the application of La3þ (200 mM) or carbenoxolone (CBX, 25 mM). In independent experiments cells were pretreated for 30 min with SB203580 (10 mM) and then exposed to Ca2þ/Mg2þ-free solution in the presence of SB203580 (*P < 0.05 versus control; #P < 0.05 versus without Ca2þ/Mg2þ; n ¼ 3 or more in all groups)..

(7) M.G. Kozoriz et al. / Neuropharmacology 75 (2013) 549e556. possibility is that the þ/G60S mutation (extracellular loop) causes an altered conformation and changes the intrinsic properties of the Cx43 hemichannels, changing their sensitivity to calcium ions. Interestingly, the increase in hemichannel activity was dependent on p38 MAPK kinase. This kinase is involved in the cerebral ischemia-reperfusion injury (Kovalska et al., 2012) and also modulates the activity of hemichannels (Kim et al., 1999). The hemichannel activity in ODDD mutants is variable, either shown to increase, decrease or not change, depending on the particular mutant under study (Dobrowolski et al., 2007; Lai et al., 2006; Wasseff et al., 2011). Differences in results may be due to different genetic backgrounds or dyes used to assess coupling. For example, there are some differences between our results and those reported by Wasseff et al. (2011), who also examined the þ/G60S Cx43 mutant in astrocytes. They found no changes in Lucifer yellow dye passage in þ/G60S vs wild-type astrocytes in brain slices from GjaJrt/þ mice, or in astrocytes cultured from these mice. However, when the þ/G60S Cx43 mutant was expressed in N2A cells, which do not express other connexins, there was no dye passage. Their immunocytochemistry for Cx43 in þ/G60S mutant astrocytes also shows increased localization in the membranes, while they show reduced expression of this mutant Cx43 in spinal cord and cerebellum. Some of these differences compared to our results are difficult to explain, but may arise due to strain differences. Wasseff and collaborators used mice on a C57BL/6J and C3H/HeJ genetic background, while our studies used mice from a different source with a background of FVB/NJ (25%), C3H/HEJ (25%) and C57BL/6 (50%). With respect to Cx43, there are variable effects following conditional knockout of Gja1 targeted to the CNS in mice, which depends on the genetic background (Wiencken-Barger et al., 2007). In addition to the localization of Cx43 described above, there were alterations in the phosphorylation state of Cx43 in these mutants. As discussed by Manias et al. (2008), in þ/G60S cardiac tissue there was a 60e80% reduction in Cx43 protein and mostly the highly phosphorylated form of Cx43 was disrupted. It was determined in this study that Cx43 was retained in the Golgi apparatus and it was reasoned that Cx43G60S co-oligomerizes with wild-type Cx43, ultimately affecting wild-type Cx43 trafficking and keeping it from reaching its phosphorylated state. Our findings are consistent with a decrease in phosphorylation of Cx43G60S in both cultured astrocytes as well as cerebral cortex. In this study, we found that þ/G60S astrocytes provide less neuroprotection in co-cultures with neurons compared to wild type astrocytes. More importantly, we determined that infarct volume was increased following MCAO in mice harboring a þ/G60S mutation. During focal ischemia a necrotic core develops, which is surrounded by a penumbral region and normal brain tissue. One theory of how gap junctions can be protective is that astrocytes can buffer cytotoxic substances in the penumbral region (Contreras et al., 2004; Rossi et al., 2007). Since coupling was reduced in þ/ G60S astrocytes it is possible that such buffering mechanisms were compromised during stroke leading to increased infarct volume. This effect is similar to studies from our lab where a reduction in Cx43 expression and gap junction coupling led to increased infarct damage (Kozoriz et al., 2010; Nakase et al., 2004; Siushansian et al., 2001). Since hemichannel activity promotes astroglial death by hypoxia-reoxygenation (Orellana et al., 2010), they may also contribute to increased infarct volume in these mice. The in vivo results might also be explained, in part, by an increase in hemichannel activity in other cell types such as microglia that express Cx43 hemichannels; activated microglia release glutamate and ATP, causing neurotoxicity (Orellana et al., 2010). These findings may provide insight into the neurological deficits reported for ODDD patients (Abrams and Scherer, 2012; Gutmann et al., 1991; Loddenkemper et al., 2002). To date, due to the rare nature of this. 555. disorder, there is no report regarding stroke incidence and severity in ODDD patients. Acknowledgments This work was funded by the Heart and Stroke Foundation of BC and Yukon, Canada to C.C.N. M.G.K. was supported by a CIHR e Vancouver Coastal Health Research Institute e UBC MD/PhD Studentship Award. C.C.N. is a Canada Research Chair. J.L.V. was supported by a FONDECYT grant (N 3120006). Research assistance was provided by Shannon Lozinsky. References Abrams, C.K., Scherer, S.S., 2012. Gap junctions in inherited human disorders of the central nervous system. Biochim. Biophys. Acta 1818, 2030e2047. Chen, M.J., Kress, B., Han, X., Moll, K., Peng, W., Ji, R.R., Nedergaard, M., 2012. Astrocytic CX43 hemichannels and gap junctions play a crucial role in development of chronic neuropathic pain following spinal cord injury. Glia 60, 1660e 1670. Churko, J.M., Langlois, S., Pan, X., Shao, Q., Laird, D.W., 2010. The potency of the fs260 connexin43 mutant to impair keratinocyte differentiation is distinct from other disease-linked connexin43 mutants. Biochem. J. 429, 473e483. Contreras, J.E., Sánchez, H.A., Eugenín, E.A., Speidel, D., Theis, M., Willecke, K., Bukauskas, F.F., Bennett, M.V., Sáez, J.C., 2002. Metabolic inhibition induces opening of unapposed connexin43 gap junction hemichannels and reduces gap junctional communication in cortical astrocytes in culture. Proc. Natl. Acad. Sci. U. S. A. 99, 495e500. Contreras, J.E., Sánchez, H.A., Véliz, L.P., Bukauskas, F.F., Bennett, M.V., Sáez, J.C., 2004. Role of connexin-based gap junction channels and hemichannels in ischemia-induced cell death in nervous tissue. Brain Res. Brain Res. Rev. 47, 290e303. De Vuyst, E., Decrock, E., De Bock, M., Yamasaki, H., Naus, C.C., Evans, W.H., Leybaert, L., 2007. Connexin hemichannels and gap junction channels are differentially influenced by lipopolysaccharide and basic fibroblast growth factor. Mol. Biol. Cell. 18, 34e46. De Vuyst, E., Wang, N., Decrock, E., De Bock, M., Vinken, M., Van Moorhem, M., Lai, C., Culot, M., Rogiers, V., Cecchelli, R., Naus, C.C., Evans, W.H., Leybaert, L., 2009. Ca2þ regulation of connexin43 hemichannels in C6 glioma and glial cells. Cell Calcium 46, 176e187. Dobrowolski, R., Sommershof, A., Willecke, K., 2007. Some oculodentodigital dysplasia-associated Cx43 mutations cause increased hemichannel activity in addition to deficient gap junction channels. J. Membr. Biol. 219, 9e17. Flenniken, A.M., Osborne, L.R., Anderson, N., Ciliberti, N., Fleming, C., Gittens, J.E., Gong, X.Q., Kelsey, L.B., Lounsbury, C., Moreno, L., Nieman, B.J., Peterson, K., Qu, D., Roscoe, W., Shao, Q., Tong, D., Veitch, G.I., Voronina, I., Vukobradovic, I., Wood, G.A., Zhu, Y., Zirngibl, R.A., Aubin, J.E., Bai, D., Bruneau, B.G., Grynpas, M., Henderson, J.E., Henkelman, R.M., McKerlie, C., Sled, J.G., Stanford, W.L., Laird, D.W., Kidder, G.M., Adamson, S.L., Rossant, J., 2005. A Gja1 missense mutation in a mouse model of oculodentodigital dysplasia. Development 132, 4375e4386. Gorlin, R.J., Miskin, L.H., St, G.J., 1963. Oculodentodigital dysplasia. J. Pediatr. 63, 69e 75. Gutmann, D.H., Zackai, E.H., McDonald-McGinn, D.M., Fischbeck, K.H., Kamholz, J., 1991. Oculodentodigital dysplasia syndrome associated with abnormal cerebral white matter. Am. J. Med. Genet. 41, 18e20. Joss, S.K., Ghazawy, S., Tomkins, S., Ahmed, M., Bradbury, J., Sheridan, E., 2008. Variable expression of neurological phenotype in autosomal recessive oculodentodigital dysplasia of two sibs and review of the literature. Eur. J. Pediatr. 167, 341e345. Kim, D.Y., Kam, Y., Koo, S.K., Joe, C.O., 1999. Gating connexin43 channels reconstituted in lipid vesicles by mitogen-activated protein kinase phosphorylation. J. Biol. Chem. 274, 5581e5587. Kovalska, M., Kovalska, L., Pavlikova, M., Janickova, M., Mikuskova, K., Adamkov, M., Kaplan, P., Tatarkova, Z., Lehotsky, J., 2012. Intracellular signaling MAPK pathway after cerebral ischemia-reperfusion injury. Neurochem. Res. 37, 1568e 1577. Kozoriz, M.G., Bechberger, J.F., Bechberger, G.R., Suen, M.W., Moreno, A.P., Maass, K., Willecke, K., Naus, C.C., 2010. The connexin43 C-terminal region mediates neuroprotection during stroke. J. Neuropathol. Exp. Neurol. 69, 196e206. Lai, A., Le, D.N., Paznekas, W.A., Gifford, W.D., Jabs, E.W., Charles, A.C., 2006. Oculodentodigital dysplasia connexin43 mutations result in non-functional connexin hemichannels and gap junctions in C6 glioma cells. J. Cell. Sci. 119, 532e 541. Lin, J.H., Lou, N., Kang, N., Takano, T., Hu, F., Han, X., Xu, Q., Lovatt, D., Torres, A., Willecke, K., Yang, J., Kang, J., Nedergaard, M., 2008. A central role of connexin43 in hypoxic preconditioning. J. Neurosci. 28, 681e695. Loddenkemper, T., Grote, K., Evers, S., Oelerich, M., Stogbauer, F., 2002. Neurological manifestations of the oculodentodigital dysplasia syndrome. J. Neurol. 249, 584e595..

(8) 556. M.G. Kozoriz et al. / Neuropharmacology 75 (2013) 549e556. Manias, J.L., Plante, I., Gong, X.Q., Shao, Q., Churko, J., Bai, D., Laird, D.W., 2008. Fate of connexin43 in cardiac tissue harbouring a disease-linked connexin43 mutant. Cardiovasc. Res. 80, 385e395. McLachlan, E., Manias, J.L., Gong, X.Q., Lounsbury, C.S., Shao, Q., Bernier, S.M., Bai, D., Laird, D.W., 2005. Functional characterization of oculodentodigital dysplasiaassociated Cx43 mutants. Cell. Commun. Adhes. 12, 279e292. Meyer-Schwickerath, G., Gruterich, E., Weyers, H., 1957. The microphthalmos syndrome. Klin Monbl Augenheilkd Augenarztl Fortbild 131, 18e30. Nakase, T., Fushiki, S., Naus, C.C., 2003. Astrocytic gap junctions composed of connexin43 reduce apoptotic neuronal damage in cerebral ischemia. Stroke 34, 1987e1993. Nakase, T., Sohl, G., Theis, M., Willecke, K., Naus, C.C., 2004. Increased apoptosis and inflammation after focal brain ischemia in mice lacking connexin43 in astrocytes. Am. J. Pathol. 164, 2067e2075. Naus, C.C., Bechberger, J.F., Zhang, Y., Venance, L., Yamasaki, H., Juneja, S.C., Kidder, G.M., Giaume, C., 1997. Altered gap junctional communication, intercellular signaling, and growth in cultured astrocytes deficient in connexin43. J. Neurosci. Res. 49, 528e540. Orellana, J.A., Froger, N., Ezan, P., Jiang, J.X., Bennett, M.V., Naus, C.C., Giaume, C., Sáez, J.C., 2011. ATP and glutamate released via astroglial connexin43 hemichannels mediate neuronal death through activation of pannexin 1 hemichannels. J. Neurochem. 118, 826e840. Orellana, J.A., Hernández, D.E., Ezan, P., Velarde, V., Bennett, M.V., Giaume, C., Sáez, J.C., 2010. Hypoxia in high glucose followed by reoxygenation in normal glucose reduces the viability of cortical astrocytes through increased permeability of connexin43 hemichannels. Glia 58, 329e343. Ozog, M.A., Siushansian, R., Naus, C.C., 2002. Blocked gap junctional coupling increases glutamate-induced neurotoxicity in neuron-astrocyte co-cultures. J. Neuropathol. Exp. Neurol. 61, 132e141. Paznekas, W.A., Karczeski, B., Vermeer, S., Lowry, R.B., Delatycki, M., Laurence, F., Koivisto, P.A., Van Maldergem, L., Boyadjiev, S.A., Bodurtha, J.N., Jabs, E.W., 2009.. GJA1 mutations, variants, and connexin43 dysfunction as it relates to the oculodentodigital dysplasia phenotype. Hum. Mutat. 30, 724e733. Plante, I., Laird, D.W., 2008. Decreased levels of connexin43 result in impaired development of the mammary gland in a mouse model of oculodentodigital dysplasia. Dev. Biol. 318, 312e322. Quist, A.P., Rhee, S.K., Lin, H., Lal, R., 2000. Physiological role of gap-junctional hemichannels. Extracellular calcium-dependent isosmotic volume regulation. J. Cell. Biol. 148, 1063e1074. Retamal, M.A., Froger, N., Palacios-Prado, N., Ezan, P., Sáez, P.J., Sáez, J.C., Giaume, C., 2007. Cx43 hemichannels and gap junction channels in astrocytes are regulated oppositely by proinflammatory cytokines released from activated microglia. J. Neurosci. 27, 13781e13792. Rossi, D.J., Brady, J.D., Mohr, C., 2007. Astrocyte metabolism and signaling during brain ischemia. Nat. Neurosci. 10, 1377e1386. Sáez, J.C., Berthoud, V.M., Brañes, M.C., Martínez, A.D., Beyer, E.C., 2003. Plasma membrane channels formed by connexins: their regulation and functions. Physiol. Rev. 83, 1359e1400. Siushansian, R., Bechberger, J.F., Cechetto, D.F., Hachinski, V.C., Naus, C.C., 2001. Connexin43 null mutation increases infarct size after stroke. J. Comp. Neurol. 440, 387e394. Thimm, J., Mechler, A., Lin, H., Rhee, S., Lal, R., 2005. Calcium-dependent open/ closed conformations and interfacial energy maps of reconstituted hemichannels. J. Biol. Chem. 280, 10646e10654. Toth, K., Shao, Q., Lorentz, R., Laird, D.W., 2010. Decreased levels of Cx43 gap junctions result in ameloblast dysregulation and enamel hypoplasia in Gja1Jrt/ þ mice. J. Cell. Physiol. 223, 601e609. Wasseff, S., Abrams, C.K., Scherer, S.S., 2011. A dominant connexin43 mutant does not have dominant effects on gap junction coupling in astrocytes. Neuron Glia Biol. 6, 213e223. Wiencken-Barger, A.E., Djukic, B., Casper, K.B., McCarthy, K.D., 2007. A role for connexin43 during neurodevelopment. Glia 55, 675e686..

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Figure

Fig. 2. Excitotoxic neuronal cell death is increased in þ/G60S neuron-astrocyte co- co-cultures
Fig. 3. Cx43 immunolocalization in wild-type and þ/G60S brain sections and cultured astrocytes
Fig. 4. Western blot of protein from adult cortical tissue and astrocyte cultures. A, Protein samples from 4 different adult cortices indicate that the quantity of Cx43 protein in þ/
Fig. 7. Hemichannel activity is increased in þ/G60S cultured astrocytes. A. Time-lapse measurements of Etd uptake in astrocytes from wild-type (þ/þ) or þ/G60S astrocytes exposed to Locke solution containing Etd (5 m M)

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