Ser364 of connexin43 and the upregulation of gap junction assembly by cAMP.

The assembly of gap junctions (GJs) is a process coordinated by growth factors, kinases, and other signaling molecules. GJ assembly can be enhanced via the elevation of cAMP and subsequent stimulation of connexon trafficking to the plasma membrane. To study the positive regulation of GJ assembly, fibroblasts derived from connexin (Cx)43 knockout (KO) and wild-type (WT) mice were transfected with WT Cx43 (WTCx43) or mutant Cx43. GJ assembly between untransfected WT fibroblasts or stably transfected WTCx43/KO fibroblasts was increased two- to fivefold by 8Br-cAMP, and this increase could be blocked by inhibition of cAMP-dependent protein kinase (PKA) or truncation of the Cx43 COOH terminus (CT). Although serine 364 (S364) of the Cx43 CT was determined to be a major site of phosphorylation, the molar ratio of Cx43 phosphorylation was not increased by 8Br-cAMP. Importantly, GJ assembly between either S364ECx43/KO or S364ECx43/WT fibroblasts was stimulated by 8Br-cAMP, but that between S364ACx43/KO or S364PCx43/KO fibroblasts was not stimulated, indicating that phosphorylation or a negative charge at S364 is required for enhancement of GJ assembly by cAMP. Furthermore, GJ assembly between S364ACx43/WT fibroblasts could be stimulated by 8Br-cAMP, but could not be between S364PCx43/WT fibroblasts. Thus, S364PCx43 interferes with enhanced GJ assembly when coexpressed with WTCx43.     

PKA negatively modulates the migration enhancing effect of Connexin 43

Connexin 43 (Cx43) expression is associated with an increased cell migration and related changes of the actin cytoskeleton (enhanced filopodia formation). These effects are mediated by the C-terminal cytoplasmic part of Cx43 in a channel-independent manner. Since this part has been shown to interact with a variety of proteins and has multiple phosphorylation sites we analyzed here a potential role of the protein kinase A (PKA) for the Cx43 mediated increase in cell migration. Mutation of the PKA-phosphorylation site (substitution of three serines by alanine or glycine) resulted in a further increase in cell motility compared to wild-type Cx43, but with a loss of directionality. Likewise, cell motility was enhanced by PKA inhibition only in Cx43 expressing cells, while reduced in the presence of the PKA activator forskolin. In contrast, cell motility remained unaffected by stimulation with forskolin in cells expressing Cx43 with the mutated PKA phosphorylation site (Cx43-PKA) as well as in Cx-deficient cells. Moreover, PKA activation resulted in increased binding of PKA and VASP to Cx43 associated with an enhanced phosphorylation of VASP, an important regulatory protein of cell polarity and directed migration. Functionally, we could confirm these results in endothelial cells endogenously expressing Cx43. A Tat-Cx43 peptide containing the PKA phosphorylation site abolished the PKA dependent reduction in endothelial cell migration. Our results indicate that PKA dependent phosphorylation of Cx43 modulates cell motility and plays a pivotal role in regulating directed cell migration.     

Connexin43 modulates cell polarity and directional cell migration by regulating microtubule dynamics

Knockout mice deficient in the gap junction gene connexin43 exhibit developmental anomalies associated with abnormal neural crest, primordial germ cell, and proepicardial cell migration. These migration defects are due to a loss of directional cell movement, and are associated with abnormal actin stress fiber organization and a loss of polarized cell morphology. To elucidate the mechanism by which Cx43 regulates cell polarity, we used a wound closure assays with mouse embryonic fibroblasts (MEFs) to examine polarized cell morphology and directional cell movement. Studies using embryonic fibroblasts from Cx43 knockout (Cx43KO) mice showed Cx43 deficiency caused cell polarity defects as characterized by a failure of the Golgi apparatus and the microtubule organizing center to reorient with the direction of wound closure. Actin stress fibers at the wound edge also failed to appropriately align, and stabilized microtubule (Glu-tubulin) levels were markedly reduced. Forced expression of Cx43 with deletion of its tubulin-binding domain (Cx43dT) in both wildtype MEFs and neural crest cell explants recapitulated the cell migration defects seen in Cx43KO cells. However, forced expression of Cx43 with point mutation causing gap junction channel closure had no effect on cell motility. TIRF imaging revealed increased microtubule instability in Cx43KO cells, and microtubule targeting of membrane localized Cx43 was reduced with expression of Cx43dT construct in wildtype cells. Together, these findings suggest the essential role of Cx43 gap junctions in development is mediated by regulation of the tubulin cytoskeleton and cell polarity by Cx43 via a nonchannel function.     

Effects of a murine germ cell-specific knockout of Connexin 43 on Connexin expression in testis and fertility

Connexin 43 (Cx 43)—expressed by germ cells (GC), Sertoli cells (SC) and Leydig cells—is one of at least eleven Cx in the murine testis. A general knockout (KO) of Cx 43 in mice results in perinatal death and a SC-specific KO of Cx 43 (SCCx43KO) causes infertility of male mice by preventing the initiation of spermatogenesis. To further elucidate the role of Cx 43 in the testis, a new mouse model with a GC-specific KO of Cx 43 (GCCx43KO) was created by using the Cre/loxP recombination system. A transgenic mouse line expressing the Cre gene under the tissue non-specific alkaline phosphatase promoter and a transgenic floxed Cx 43-LacZ mouse line were mated. The resulting F1-generation was backcrossed with homozygous Cx 43 floxed mice, and offspring was genotyped. Immunohistochemical analysis of testes of different aged homozygous mice revealed normal spermatogenesis and reduced Cx 43 immunoreactions. RT-qPCR and Western blots showed a downregulation of Cx 43 mRNA and protein, and a nearly unchanged mRNA expression of Cx 26, Cx 33 and Cx 45 in pubertal and adult KO mice. Western blots revealed considerable immunoreactive bands for Cx 26 and Cx 45. Male and female homozygous GCCx43KO mice were viable and fertile. Our data suggest, in contrast to inter SC and inter SC–GC cross talk in SCCx43KO mice which depends selectively on Cx 43 expression, that Cx 43 in GC seems not to be essential in GC–SC communication, when other Cx persist to be expressed.     

Primordial germ cell deficiency in the connexin 43 knockout mouse arises from apoptosis associated with abnormal p53 activation

Connexin 43 knockout (Cx43alpha1KO) mice exhibit germ cell deficiency, but the underlying cause for the germ cell defect was unknown. Using an Oct4-GFP reporter transgene, we tracked the distribution and migration of primordial germ cells (PGCs) in the Cx43alpha1KO mouse embryo. Analysis with dye injections showed PGCs are gap-junction-communication competent, with dye coupling being markedly reduced in Cx43alpha1-deficient PGCs. Time-lapse videomicroscopy and motion analysis showed that the directionality and speed of cell motility were reduced in the Cx43alpha1KO PGCs. This was observed both in E8.5 and E11.5 embryos. By contrast, PGC abundance did not differ between wild-type and heterozygous/homozygous Cx43alpha1KO embryos until E11.5, when a marked reduction in PGC abundance was detected in the homozygous Cx43alpha1KO embryos. This was accompanied by increased PGC apoptosis and increased expression of activated p53. Injection of alpha-pifithrin, a p53 antagonist, inhibited PGC apoptosis and prevented the loss of PGC. Analysis using a cell adhesion assay indicated a reduction in beta1-integrin function in the Cx43alpha1KO PGCs. Together with the abnormal activation of p53, these findings suggest the possibility of anoikis-mediated apoptosis. Overall, these findings show Cx43alpha1 is essential for PGC survival, with abnormal p53 activation playing a crucial role in the apoptotic loss of PGCs in the Cx43alpha1KO mouse embryos.     

Connexin 43 phosphorylation and degradation are required for adipogenesis

Connexin-43 (Cx43) is a membrane phosphoprotein that mediates direct inter-cellular communication by forming gap junctions. In this way Cx43 can influence gene expression, differentiation and growth. Its role in adipogenesis, however, is poorly understood. In this study, we established that Cx43 becomes highly phosphorylated in early adipocyte differentiation and translocates to the plasma membrane from the endoplasmic reticulum. As preadipocytes differentiate, Cx43 phosphorylation declines, the protein is displaced from the plasma membrane, and total cellular levels are reduced via proteosomal degradation. Notably, we show that inhibiting Cx43 degradation or constitutively over-expressing Cx43 blocks adipocyte differentiation. These data reveal that transient activation of Cx43 via phosphorylation followed by its degradation is vital for preadipocyte differentiation and maturation of functional adipocytes.     

Gap Junction Intercellular Communication Mediated by Connexin43 in Astrocytes Is Essential for Their Resistance to Oxidative Stress

Oxidative stress induced by reactive oxygen species (ROS) is associated with various neurological disorders including aging, neurodegenerative diseases, as well as traumatic and ischemic insults. Astrocytes have an important role in the anti-oxidative defense in the brain. The gap junction protein connexin43 (Cx43) forms intercellular channels as well as hemichannels in astrocytes. In the present study, we investigated the contribution of Cx43 to astrocytic death induced by the ROS hydrogen peroxide (H₂O₂) and the mechanism by which Cx43 exerts its effects. Lack of Cx43 expression or blockage of Cx43 channels resulted in increased ROS-induced astrocytic death, supporting a cell protective effect of functional Cx43 channels. H₂O₂ transiently increased hemichannel activity, but reduced gap junction intercellular communication (GJIC). GJIC in wild-type astrocytes recovered after 7 h, but was absent in Cx43 knock-out astrocytes. Blockage of Cx43 hemichannels incompletely inhibited H₂O₂-induced hemichannel activity, indicating the presence of other hemichannel proteins. Panx1, which is predicted to be a major hemichannel contributor in astrocytes, did not appear to have any cell protective effect from H₂O₂ insults. Our data suggest that GJIC is important for Cx43-mediated ROS resistance. In contrast to hypoxia/reoxygenation, H₂O₂ treatment decreased the ratio of the hypophosphorylated isoform to total Cx43 level. Cx43 has been reported to promote astrocytic death induced by hypoxia/reoxygenation. We therefore speculate the increase in Cx43 dephosphorylation may account for the facilitation of astrocytic death. Our findings suggest that the role of Cx43 in response to cellular stress is dependent on the activation of signaling pathways leading to alteration of Cx43 phosphorylation states.     

Mechanism of PKA-dependent and lipid-raft independent stimulation of Connexin43 expression by oxytoxin in mouse embryonic stem cells

Previous studies shows that connexins appear very early during murine embryo development, the gap junctional intercellular communication found in the inner cell mass of early embryo is also maintained in embryonic stem cells (ESC), and expression of oxytocin receptor (OTR) is developmentally regulated at early embryonic development. However, effect of oxytocin (OT) on the regulation of the connexin43 (Cx43) and maintenance of undifferentiation is not fully understood in stem cells. Therefore, we investigated the effect of OT on Cx43 expression and related signaling cascades in mouse ESC. OT increased Cx43 expression that was inhibited by the OTR inhibitor atosiban. In experiments to examine whether the effect of OT depends on lipid rafts, caveolin-1 (cav-1), cav-2, and flotillin-2, but not OTR, were detected in lipid raft fractions. Also, colocalization of OTR, cav-1, and cav-2 was not detected. Moreover, the lipid raft disruptor methyl-β-cyclodextrin did not attenuate OT-induced Cx43 expression. In experiments to examine related signaling pathways, OT activated cAMP/protein kinase A (PKA) which was inhibited by adenylyl cyclase inhibitor SQ 22536 and PKA inhibitor PKI. OT increased nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) phosphorylation which was inhibited by PKI. OT also increased cAMP response element-binding (CREB)/CREB-binding protein (CBP) expression in the nucleus and induced the formation of CREB1/NF-κB/CBP complexes, which was blocked by the NF-κB-specific small interfering RNA, NF-κB inhibitors, SN50, and bay11-7082. Complex disruption by NF-κB inhibitors decreased OT-induced Cx43 expression. In conclusion, OT stimulates Cx43 expression through the NF-κB/CREB/CBP complex via the lipid raft-independent OTR-mediated cAMP/PKA in mouse ESC.     

Neuroprotective role of astrocytic gap junctions in ischemic stroke

The role of astrocytic gap junctions in ischemia remains controversial. Several studies support that astrocytic gap junctions play a role in the spread of hypoxic injury, while other reports have demonstrated that blocking astrocytic gap junctions increases neuronal death. Using a stroke model on animals in which the astrocytic gap junction protein connexin43 (Cx43) was compromised, we explored the neuroprotective role of astrocytic gap junctions. A focal brain stroke was performed on heterozygous Cx43 null [Cx43(+/-)] mice, wild type [Cx43(+/+)] mice, astrocyte-directed Cx43 deficient [Cx43(fl/ fl)/hGFAP-cre] mice (here designated as Cre(+) mice), and their corresponding controls [Cx43(fl/fl)] (here designated as Cre(-) mice). Four days following stroke, ischemic lesions were measured for size and analyzed immunohistochemically. Stroke volume was significantly larger in Cx43(+/-) and Cre(+) mice compared to Cx43(+/+) and Cre(-) mice, respectively. Apoptosis as detected by TUNEL labeling and caspase-3 immunostaining was amplified in Cx43(+/-) and Cre(+) mice compared to their control groups. Furthermore, increased inflammation as characterized by the immunohistochemical staining of the microglial marker CD11b was observed in the Cre(+) mice penumbra. Astrocytic gap junctions may reduce apoptosis and inflammation in the penumbra following ischemic insult, suggesting that coupled astrocytes fulfill a neuroprotective role under ischemic stroke conditions.     

Neuroprotective Role of Astrocytic Gap Junctions in Ischemic Stroke

The role of astrocytic gap junctions in ischemia remains controversial. Several studies support that astrocytic gap junctions play a role in the spread of hypoxic injury, while other reports have demonstrated that blocking astrocytic gap junctions increases neuronal death. Using a stroke model on animals in which the astrocytic gap junction protein connexin43 (Cx43) was compromised, we explored the neuroprotective role of astrocytic gap junctions. A focal brain stroke was performed on heterozygous Cx43 null [Cx43(+/?)] mice, wild type [Cx43(+/+)] mice, astrocyte-directed Cx43 deficient [Cx43^{fl/ fl}/hGFAP-cre] mice (here designated as Cre(+) mice), and their corresponding controls [Cx43^fl/fl] (here designated as Cre(?) mice). Four days following stroke, ischemic lesions were measured for size and analyzed immunohistochemically. Stroke volume was significantly larger in Cx43(+/?) and Cre(+) mice compared to Cx43(+/+) and Cre(?) mice, respectively. Apoptosis as detected by TUNEL labeling and caspase-3 immunostaining was amplified in Cx43(+/?) and Cre(+) mice compared to their control groups. Furthermore, increased inflammation as characterized by the immunohistochemical staining of the microglial marker CD11b was observed in the Cre(+) mice penumbra. Astrocytic gap junctions may reduce apoptosis and inflammation in the penumbra following ischemic insult, suggesting that coupled astrocytes fulfill a neuroprotective role under ischemic stroke conditions.     

Reciprocal regulation between proinflammatory cytokine-induced inducible NO synthase (iNOS) and connexin43 in bladder smooth muscle cells

Gap junctions (GJs) play an important role in the control of bladder contractile response and in the regulation of various immune inflammatory processes. Here, we investigated the possible interaction between inflammation and GJs in bladder smooth muscle cells (BSMCs). Stimulation of BSMCs with IL1β and TNFα increased connexin43 (Cx43) expression and function, which was associated with increased phosphorylation of vasodilator-stimulated phosphoprotein. Inhibition of PKA with H89 or down-regulation of CREB with specific siRNAs largely abolished the Cx43-elevating effect. Further analysis revealed that IL1β/TNFα induced NFκB-dependent inducible NO synthase (iNOS) expression. Inhibition of iNOS with G-nitro-l-arginine methyl ester abrogated and an exogenous NO donor mimicked the effect of the cytokines on Cx43. Intraperitoneal injection of LPS into mice also induced bladder Cx43 expression, which was largely blocked by an iNOS inhibitor. Finally, the elevated Cx43 was found to negatively regulate iNOS expression. Dysfunction of GJs with various blockers or down-regulation of Cx43 with siRNA significantly potentiated the expression of iNOS. Fibroblasts from Cx43 knock-out (Cx43(-/-)) mice also displayed a significantly higher response to the cytokine-induced iNOS expression than cells from Cx43 wild-type (Cx43(+/+)) littermates. Collectively, our study revealed a previously unrecognized reciprocal regulation loop between cytokine-induced NO and GJs. Our findings may provide an important molecular mechanism for the symptoms of bladder infection. In addition, it may further our understanding of the roles of GJs in inflammatory diseases.     

Reversal of Rotenone-Induced Dysfunction of Astrocytic Connexin43 by Opening Mitochondrial ATP-Sensitive Potassium Channels

Growing evidence suggests that the astrocytic gap junctions (GJs), mainly formed by connexin 43 (Cx43), play an important role in physiological maintenance and various central nervous system (CNS) pathological conditions. However, little is known about the role of Cx43 in Parkinson’s disease (PD). In this article, we report that rotenone, a classic neurotoxin for PD, could inhibit expression of astrocytic Cx43 and gap junction permeability. ATP-sensitive potassium (K_ATP) channel openers, iptakalim (IPT) and diazoxide (DZ), exerted protective effect on rotenone-induced dysfunction of Cx43 and astrocyte apoptosis, which was reversed by selective mitochondrial K_ATP (mitoK_ATP) channel blocker 5-hydroxydecanoate (5-HD). Taken together, our findings reveal that rotenone-induced dysfunction of astrocytic Cx43 may be involved in the pathology of PD. Moreover, opening mitoK_ATP channels in astrocytes can reverse rotenone-induced dysfunction of astrocytic Cx43 and therefore protect against toxicity of rotenone on astrocytes.     

Protein kinase A-mediated phosphorylation of connexin36 in mouse retina results in decreased gap junctional communication between AII amacrine cells

Gap junctions in AII amacrine cells of mammalian retina participate in the coordination of the rod and cone signaling pathway involved in visual adaptation. Upon stimulation by light, released dopamine binds to D(1) receptors on AII amacrine cells leading to increased intracellular cAMP (cyclic adenosine monophosphate) levels. AII amacrine cells express the gap junctional protein connexin36 (Cx36). Phosphorylation of Cx36 has been hypothesized to regulate gap junctional activity of AII amacrine cells. However, until now in vivo phosphorylation of Cx36 has not been reported. Indeed, it had been concluded that Cx36 in bovine retina is not phosphorylated, but in vitro phosphorylation for Cx35, the bass ortholog of Cx36, had been shown. To clarify this experimental discrepancy, we examined protein kinase A (PKA)-induced phosphorylation of Cx36 in mouse retina as a possible mechanism to modulate the extent of gap junctional coupling. The cytoplasmic domains of Cx36 and the total Cx36 protein were phosphorylated in vitro by PKA. Mass spectroscopy revealed that all four possible PKA consensus motifs were phosphorylated; however, domains point mutated at the sites in question showed a prevalent usage of Ser-110 and Ser-293. Additionally, we demonstrated that Cx36 was phosphorylated in cultured mouse retina. Furthermore, activation of PKA increased the level of phosphorylation of Cx36. cAMP-stimulated, PKA-mediated phosphorylation of Cx36 protein was accompanied by a decrease of tracer coupling between AII amacrine cells. Our results link increased phosphorylation of Cx36 to down-regulation of permeability through gap junction channels mediating light adaptation in the retina.     

Connexin 43-mediated modulation of polarized cell movement and the directional migration of cardiac neural crest cells

Connexin 43 knockout (Cx43alpha1KO) mice have conotruncal heart defects that are associated with a reduction in the abundance of cardiac neural crest cells (CNCs) targeted to the heart. In this study, we show CNCs can respond to changing fibronectin matrix density by adjusting their migratory behavior, with directionality increasing and speed decreasing with increasing fibronectin density. However, compared with wild-type CNCs, Cx43alpha1KO CNCs show reduced directionality and speed, while CNCs overexpressing Cx43alpha1 from the CMV43 transgenic mice show increased directionality and speed. Altered integrin signaling was indicated by changes in the distribution of vinculin containing focal contacts, and altered temporal response of Cx43alpha1KO and CMV43 CNCs to beta1 integrin function blocking antibody treatment. High resolution motion analysis showed Cx43alpha1KO CNCs have increased cell protrusive activity accompanied by the loss of polarized cell movement. They exhibited an unusual polygonal arrangement of actin stress fibers that indicated a profound change in cytoskeletal organization. Semaphorin 3A, a chemorepellent known to inhibit integrin activation, was found to inhibit CNC motility, but in the Cx43alpha1KO and CMV43 CNCs, cell processes failed to retract with semaphorin 3A treatment. Immunohistochemical and biochemical analyses suggested close interactions between Cx43alpha1, vinculin and other actin-binding proteins. However, dye coupling analysis showed no correlation between gap junction communication level and fibronectin plating density. Overall, these findings indicate Cx43alpha1 may have a novel function in mediating crosstalk with cell signaling pathways that regulate polarized cell movement essential for the directional migration of CNCs.     

Parkin Expression Profile in Dopamine D3 Receptor Knock-Out Mice Brains

Patients affected by autosomic recessive juvenile parkinsonism (ARJP) exhibit parkin gene mutations with brain decrease in dopamine D2/D3 binding sites. To date, there are no data indicating whether the reduction in dopamine D3 receptors (DRD3) may be associated with the expression of specific parkin variants. In the present study we investigated parkin expression profile in DRD3 knock-out mice brains. RT-PCR analysis was performed to assess qualitative changes in parkin isoforms’ distribution pattern and in exons’ expression both in wild type controls and dopamine D3 receptor’s knock-out mice. Real-time PCR was performed to quantify single exons mRNA. Results demonstrated that exons 1, 2, 4, 6, 7, 8, were more expressed in wild type compared to dopamine D3 receptor KO mice brains while some other (3, 9, 10) were lower expressed. The expression levels of exons 5, 11 and 12 did not change in both animal groups. Our analysis was confirmed by western blot, which showed that parkin protein levels were influenced by the absence of DRD3.     

Identification of Akt-independent Regulation of Hepatic Lipogenesis by Mammalian Target of Rapamycin (mTOR) Complex 2

Mammalian target of rapamycin complex 2 (mTORC2) is a key activator of protein kinases that act downstream of insulin and growth factor signaling. Here we report that mice lacking the essential mTORC2 component rictor in liver (Lrictor(KO)) are unable to respond normally to insulin. In response to insulin, Lrictor(KO) mice failed to inhibit hepatic glucose output. Lrictor(KO) mice also fail to develop hepatic steatosis on a high fat diet and manifest half-normal serum cholesterol levels. This is accompanied by lower levels of expression of SREBP-1c and SREBP-2 and genes of fatty acid and cholesterol biosynthesis. Lrictor(KO) mice had defects in insulin-stimulated Akt Ser-473 and Thr-308 phosphorylation, leading to decreased phosphorylation of Akt substrates FoxO, GSK-3β, PRAS40, AS160, and Tsc2. Lrictor(KO) mice also manifest defects in insulin-activated mTORC1 activity, evidenced by decreased S6 kinase and Lipin1 phosphorylation. Glucose intolerance and insulin resistance of Lrictor(KO) mice could be fully rescued by hepatic expression of activated Akt2 or dominant negative FoxO1. However, in the absence of mTORC2, forced Akt2 activation was unable to drive hepatic lipogenesis. Thus, we have identified an Akt-independent relay from mTORC2 to hepatic lipogenesis that separates the effects of insulin on glucose and lipid metabolism.     

Spinal Astrocytic Activation Contributes to Mechanical Allodynia in a Rat Chemotherapy-Induced Neuropathic Pain Model

Chemotherapy-induced neuropathic pain (CNP) is the major dose-limiting factor in cancer chemotherapy. However, the neural mechanisms underlying CNP remain enigmatic. Accumulating evidence implicates the involvement of spinal glia in some neuropathic pain models. In this study, using a vincristine-evoked CNP rat model with obvious mechanical allodynia, we found that spinal astrocyte rather than microglia was dramatically activated. The mechanical allodynia was dose-dependently attenuated by intrathecal administratration of L-α-aminoadipate (astrocytic specific inhibitor); whereas minocycline (microglial specific inhibitor) had no such effect, indicating that spinal astrocytic activation contributes to allodynia in CNP rat. Furthermore, oxidative stress mediated the development of spinal astrocytic activation, and activated astrocytes dramatically increased interleukin-1β expression which induced N-methyl-D-aspartic acid receptor (NMDAR) phosphorylation in spinal neurons to strengthen pain transmission. Taken together, our findings suggest that spinal activated astrocytes may be a crucial component of the pathophysiology of CNP and “Astrocyte-Cytokine-NMDAR-neuron” pathway may be one detailed neural mechanisms underlying CNP. Thus, inhibiting spinal astrocytic activation may represent a novel therapeutic strategy for treating CNP.     

AGEs-RAGE mediated up-regulation of connexin43 in activated human microglial CHME-5 cells

Microglial activation is a significant contributor to the pathogenesis of many neurodegenerative diseases. Microglia respond to a range of stimuli including pathogenic protein deposits such as advanced glycation endproducts (AGEs). AGEs are prominent inflammatory stimuli that accumulate in the ageing brain. AGEs can activate microglia, leading to the production of excessive amounts of inflammatory cytokines and coupling via gap junction proteins especially connexin43 (Cx43). The literature on the expression of microglial Cx43 during inflammation is controversial. Many cellular effects of AGEs are thought to be mediated by the receptor RAGE. There is however, no evidence suggesting Cx43 is a downstream effector of AGEs-RAGE interaction in microglia. In addition, most of the AGEs-related studies have been undertaken using rodent microglia; the information on human microglia is sparse. Microglia of human and rodent origin respond differently to certain stimuli. The aims of this study were to investigate the AGEs-RAGE-mediated activation of human microglia and establish if Cx43 is one of the downstream effectors of AGEs-RAGE interaction in these cells. Human microglial CHME-5 cells were treated with different doses of AGEs for a selected time-period and microglial activation studied using specific markers. The protein expression of RAGE, Cx43 and TNF-α-receptors (RI and RII) was analysed in response to AGEs in the absence/presence of various doses of anti-RAGE Fabs. TNF-α levels in media were measured using ELISA. TNF-α-induced opening of gap junctional channels was assessed by dye uptake assays and the effect of neutralising TNFRII on Cx43 levels was also studied. CHME-5 cells showed an up-regulation of RAGE, TNF-α, TNFRs (especially TNFRII) and Cx43 upon AGEs treatment and a significant dose-dependent drop in the levels of TNF-α, TNFRII and Cx43 in the presence of anti-RAGE Fabs. TNF-α induced gap junctional/hemichannel opening whereas blocking TNFRII inhibited TNF-α-induced increase in Cx43 levels. Results suggested that TNF-α, TNFRII and Cx43 are downstream effectors of the AGEs-RAGE interaction in human microglial CHME-5 cells.     

Connexin-43 hemichannels contribute to the propagation of μ-calpain-mediated neuronal death in a cortical ablation injury model

We investigated the role of the astrocytic and neuronal hemichannels (HCs) in the spread of cortical neuronal death in a rat cortical injury model. Over time (by 6 h), propidium iodide (PI)-positive cells with labeling either with anti-neuron specific enolase or anti-parvalbumin (indicating GABAnergic interneurons) antibody spread in the deep cortical layers adjacent to the injury and co-localized with activated μ-calpain. Connexin (Cx)-43, glial fibrillary acidic protein (GFAP), activated μ-calpain and α-fodrin breakdown product (FBP) increased post-injury, peaking at 1 h, in the injury and adjacent areas. GFAP-Cx43-positive reactivated astrocytes exhibited similar distribution to the dead neurons. Cx43 and Cx36 primarily comprise HCs in the astrocyte and neuron, respectively. Ethidium bromide (EtBr) uptake was enhanced post-injury, and confirmed in the Cx43- and Cx36-positive cells. A Cx43-HC inhibitor Gap26 prevented the opening of the Cx43-HC and Cx36-HC, μ-calpain activation, α-fodrin proteolysis and death in the deep cortical neurons. Collectively, opening of the astrocytic Cx43-HC and neuronal Cx36-HC would induce the regional spread of cortical neuronal death through μ-calpain activation in the rat brain injury model.