The connexin43 mimetic peptide Gap19 inhibits hemichannels without altering gap junctional communication in astrocytes

In the brain, astrocytes represent the cellular population that expresses the highest amount of connexins (Cxs). This family of membrane proteins is the molecular constituent of gap junction channels and hemichannels that provide pathways for direct cytoplasm-to-cytoplasm and inside-out exchange, respectively. Both types of Cx channels are permeable to ions and small signaling molecules allowing astrocytes to establish dynamic interactions with neurons. So far, most pharmacological approaches currently available do not distinguish between these two channel functions, stressing the need to develop new specific molecular tools. In astrocytes two major Cxs are expressed, Cx43 and Cx30, and there is now evidence indicating that at least Cx43 operates as a gap junction channel as well as a hemichannel in these cells. Based on studies in primary cultures as well as in acute hippocampal slices, we report here that Gap 19, a nonapeptide derived from the cytoplasmic loop of Cx43, inhibits astroglial Cx43 hemichannels in a dose-dependent manner, without affecting gap junction channels. This peptide, which not only selectively inhibits hemichannels but is also specific for Cx43, can be delivered in vivo in mice as TAT-Gap19, and displays penetration into the brain parenchyma. As a result, Gap 19 combined with other tools opens up new avenues to decipher the role of Cx43 hemichannels in interactions between astrocytes and neurons in physiological as well as pathological situations

Targeting MAPK phosphorylation of Connexin43 provides neuroprotection in stroke

Connexin43 (Cx43) function is influenced by kinases that phosphorylate specific serine sites located near its C-terminus. Stroke is a powerful inducer of kinase activity, but its effect on Cx43 is unknown. We investigated the impact of wild-type (WT) and knock-in Cx43 with serine to alanine mutations at the protein kinase C (PKC) site Cx43(S368A), the casein kinase 1 (CK1) sites Cx43(S325A/328Y/330A), and the mitogen-activated protein kinase (MAPK) sites Cx43(S255/262/279/282A) (MK4) on a permanent middle cerebral artery occlusion (pMCAO) stroke model. We demonstrate that MK4 transgenic animals exhibit a significant decrease in infarct volume that was associated with improvement in behavioral performance. An increase in astrocyte reactivity with a concomitant decrease in microglial reactivity was observed in MK4 mice. In contrast to WT, MK4 astrocytes displayed reduced Cx43 hemichannel activity. Pharmacological blockade of Cx43 hemichannels with TAT-Gap19 also significantly decreased infarct volume in WT animals. This study provides novel molecular insights and charts new avenues for therapeutic intervention associated with Cx43 function

Role of gap junction protein connexin43 in astrogliosis induced by brain injury.

Astrogliosis is a process that involves morphological and biochemical changes associated with astrocyte activation in response to cell damage in the brain. The upregulation of intermediate filament proteins including glial fibrillary acidic protein (GFAP), nestin and vimentin are often used as indicators for astrogliosis. Although connexin43 (Cx43), a channel protein widely expressed in adult astrocytes, exhibits enhanced immunoreactivity in the peri-lesion region, its role in astrogliosis is still unclear. Here, we correlated the temporal and spatial expression of Cx43 to the activation of astrocytes and microglia in response to an acute needle stab wound in vivo. We found large numbers of microglia devoid of Cx43 in the needle wound at 3 days post injury (dpi) while reactive astrocytes expressing Cx43 were present in the peripheral zone surrounding the injury site. A redistribution of Cx43 to the needle site, corresponding to the increased presence of GFAP-positive reactive astrocytes in the region, was only apparent from 6 dpi and sustained until at least 15 dpi. Interestingly, the extent of microglial activation and subsequent astrogliosis in the brain of Cx43 knockout mice was significantly larger than those of wild type, suggesting that Cx43 expression limits the degree of microgliosis. Although Cx43 is not essential for astrogliosis and microglial activation induced by a needle injury, our results demonstrate that Cx43 is a useful marker for injury induced astrogliosis due to its enhanced expression specifically within a small region of the lesion for an extended period. As a channel protein, Cx43 is a potential in vivo diagnostic tool of asymptomatic brain injury

The carboxy-terminal tail of connexin43 gap junction protein is sufficient to mediate cytoskeleton changes in human glioma cells

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Transient upregulation of NG2-glia in response to needle injury. A

<p>) Immunohistochemical staining of NG2-glia or oligodendrocyte precursor cells by NG2 antibody. Increased NG2 immunoreactivity at and around the lesion was observed at 6 day post injury (dpi) in wild type mice. There was no increased NG2 expression at 3 hour post injury (hpi). NG2-glia was back to near basal level (when compared to contra-lateral hemisphere) by 15 dpi. Cytoplasmic auto-fluorescence was observed in the needle track at 15 dpi. Red line denotes position of needle track. <b>B</b>) Co-staining of Ki67 proliferative marker with NG2 antibody at 3 dpi, indicating some NG2 cells were proliferating. <b>C)</b> Co-staining of Cx43 with surface proteoglycan NG2 showed minimal localization of Cx43 with NG2-positive cells. Blue, DAPI nuclei staining.</p

Mammary gland specific knockdown of the physiological surge in Cx26 during lactation retains normal mammary gland development and function.

International audienceConnexin26 (Cx26) is the major Cx protein expressed in the human mammary gland and is up-regulated during pregnancy while remaining elevated throughout lactation. It is currently unknown if patients with loss-of-function Cx26 mutations that result in hearing loss and skin diseases have a greater susceptibility to impaired breast development. To investigate if Cx26 plays a critical role in mammary gland development and differentiation, a novel Cx26 conditional knockout mouse model was generated by crossing Cx26fl/fl mice with mice expressing Cre under the β-Lactoglobulin promoter. Conditional knockdown of Cx26 from the mammary gland resulted in a dramatic reduction in detectable gap junction plaques confirmed by a significant ∼65-70% reduction in Cx26 mRNA and protein throughout parturition and lactation. Interestingly, this reduction was accompanied by a decrease in mammary gland Cx30 gap junction plaques at parturition, while no change was observed for Cx32 or Cx43. Whole mount, histological and immunofluorescent assessment of breast tissue revealed comparatively normal lobuloalveolar development following pregnancy in the conditionally knockdown mice compared to control mice. In addition, glands from genetically-modified mice were capable of producing milk proteins that were evident in the lumen of alveoli and ducts at similar levels as controls, suggesting normal gland function. Together, our results suggest that low levels of Cx26 expression throughout pregnancy and lactation, and not the physiological surge in Cx26, is sufficient for normal gland development and function

Expression of Cx43 in nestin-expressing cells.

<p><b>A</b>) Co-staining of nestin and GFAP markers at the injury site of wild type (WT) brains showed a transient increase in the number of cells expressing both proteins at 6 day post injury (dpi). There was no nestin expression at the wound vicinity at 3 hour post injury (hpi). At 15 dpi, nestin staining was no longer observed while GFAP immunoreactivity was still present at the needle track. Anti-GFAP was used at a concentration to detect only reactive astrocytes with upregulated GFAP expression. White dotted line denotes location of needle wound. <b>B</b>) WT and Cx43 knockdown brain of GFAP-Cre:Cx43 fl/fl mice (Cx43cKO) showed similar upregulation of nestin at 6 dpi but no nestin expression at 3 hpi, indicating that nestin dynamics is not dependent on Cx43 expression in astrocytes. Cytoplasmic auto-fluorescence associated with the wound lesion was observed in some sections. <b>C</b>) Confocal images showing colocalization of Cx43 puncta with nestin intermediate filaments (arrowheads) in WT brain at 6 dpi. Blue, DAPI nuclei staining.</p

Absence of astrocytic Cx43 does not affect number of microglia at the lesion site.

<p><b>A</b>) (<i>left panel)</i> A high magnification image showing the extension of processes perpendicular to the wound by IBA1-positive microglia in wild type (WT) and Cx43 deleted (Cx43cKO) brain of GFAP-Cre, Cx43 fl/fl mice at 3 hour post injury (hpi). <i>(right panel)</i> Graphical representation showing no difference in the number of DAPI-positive nuclei in the lesion site that co-stained with IBA1 marker in WT and Cx43cKO brain at 3 hpi. <b>B</b>) Lack of GFAP-expressing reactive astrocytes and Ki67-positive proliferating cells in response to a needle stab lesion in at 3 hpi in Cx43-expressing WT and Cx43-deficient Cx43cKO brains. Anti-GFAP antibody was used at a concentration to detect only reactive astrocytes with enhanced GFAP expression. <b>C</b>) Co-staining of Cx43 with GFAP showed limited localization of Cx43 puncta to GFAP-positive astrocytes (white arrowheads) at 3 hpi surrounding the needle hole (*) in WT mice. Cytoplasmic Cx43 was observed in some IBA1-expressing microglia (white arrows) at 3 hpi. Blue, DAPI nuclei staining.</p

Spatial and temporal distribution of Cx43 protein in response to a needle stab wound injury.

<p><b>A</b>) Wild type mouse coronal sections were stained with anti-Cx43 antibodies at 3, 6, 9, and 15 day post injury (dpi). Increased punctate staining typical of Cx43 gap junction plaques <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047311#pone.0047311-Hossain1" target="_blank">[27]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047311#pone.0047311-Haupt1" target="_blank">[29]</a> directly at the injury site was observed from 6 to 15 dpi, but not at 3 dpi. Some areas of low fluorescence signals in the wound periphery correspond to the myelinated fibers of Pencils of Wilson. Red line denotes position of needle track. Blue, DAPI nuclei staining. <b>B</b>) An enlarged image of Cx43 staining at 6 dpi from A) with outlines corresponding to the central and peripheral region of the wound lesion. <b>C</b>) The corresponding semi-quantitative analysis of Cx43 immunoreactivity was analyzed using Image J (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047311#s4" target="_blank">Materials and Methods</a>) within the central zone including the needle wound (solid black line), the peripheral zone (dotted line) and the contralateral side (dashed line). Data were pooled from 4 (3, 6, 9 dpi) and 3 (15 dpi) animals. * = p<0.05, n.s. = not significant (p>0.05).</p

Recruitment of reactive astrocytes and microglia to the needle stab wound. A

<p>) Cresyl violet staining of brain coronal sections showing the needle injury site, as identified by the aggregation of blue nuclei (white arrow). The bottom panel is a magnification of the white box in the upper panel. <b>B</b>) A low magnification overview showing the temporal and spatial distribution of GFAP- expressing reactive astrocytes and IBA1-expressing microglia in response to a needle stab lesion in adult mice at 3, 6, 9, and 15 day post injury (dpi). White dotted line indicates location of needle track. <b>C</b>) High magnification florescence images of mouse coronal sections at 3, 6, 9, and 15 dpi after needle stab wound showing the distribution of GFAP-positive astrocytes and IBA1-positive microglia in the needle wound (red dotted line) compared to the corresponding position at the contralateral hemisphere. Anti-GFAP and anti-IBA1 were used at a concentration to detect reactive astrocytes or microglia with enhanced GFAP <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047311#pone.0047311-Haupt1" target="_blank">[29]</a> or IBA1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047311#pone.0047311-Ito1" target="_blank">[52]</a> expression, respectively, as demonstrated by the lack of immunoreactivity in the contralateral region. Blue, DAPI nuclei staining. Semi-quantitative analysis of GFAP and IBA1 immunoreactivity was analyzed using Image J (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047311#s4" target="_blank">Materials and Methods</a>) within the needle wound (solid black line), the peripheral zone (dotted line) and the corresponding contralateral region (dashed line). Data were pooled from 4 (3, 6, 9 dpi) and 3 (15 dpi) animals. * = p<0.05.</p