Acute downregulation of Cx43 alters P2Y receptor expression levels in mouse spinal cord astrocytes

Propagation of intercellular calcium waves (ICW) between astrocytes depends on the diffusion of signaling molecules through gap junction channels and diffusion through the extracellular space of neuroactive substances acting on plasmalemmal receptors. The relative contributions of these two pathways vary in different brain regions and under certain pathological conditions. We have previously shown that in wild-type spinal cord astrocytes, ICW are primarily gap junction-dependent, but that deletion of the main gap junction protein (Cx43) by homologous recombination results in a switch in mode of ICW propagation to a purinoceptor-dependent mechanism. Such a compensatory mechanism for ICW propagation was related to changes in the pharmacological profile of P2Y receptors, from an adenine-sensitive P2Y(1), in wild-type, to a uridine-sensitive P2U receptor subtype, in Cx43 knockout (KO) astrocytes. Using oligonucleotide antisense to Cx43 mRNA for acute downregulation of connexin43 expression levels, we provide evidence for the molecular nature of such compensatory mechanism. Pharmacological studies and Western blot analysis indicate that there is a reciprocal regulation of P2Y(1) and P2Y(4) expression levels, such that downregulation of Cx43 leads to decreased expression of the adenine-sensitive P2Y(1) receptor and increased expression of the uridine-sensitive P2Y(4) receptor. This change in functional expression of the P2Y receptor subtype population in acutely downregulated Cx43 was paralleled by changes in the mode of ICW propagation, similar to that previously observed for Cx43 KO spinal cord astrocytes. On the basis of these results, we propose that Cx43 regulates both modes of ICW by altering P2Y receptor subtype expression in addition to providing intercellular coupling.     

Gap junction channels coordinate the propagation of intercellular Ca2+ signals generated by P2Y receptor activation

Astrocytes express gap junction proteins and multiple types of P2Y receptors (P2YRs) that contribute to the propagation of intercellular Ca(2+) waves (ICW). To gain access to the role played by gap junctional communication in ICW propagation generated by P2YR activation, we selectively expressed P2Y(1,2,4)R subtypes and Cx43 in the human 1321N1 astrocytoma cell line, which lacks endogenous P2 receptors. Fluorescence recovery after photobleaching revealed that 1321N1 cells are poorly dye-coupled and do not propagate ICW. Forced expression of Cx43 in 1321N1 cells (which did not show functional hemichannels) increased dye coupling and allowed short-range ICW transmission that was mainly mediated by intercellular diffusion of Ca(2+) generated in the stimulated cells. Astrocytoma clones expressing each of the P2YR subtypes were also able to propagate ICWs that were likely dependent on IP(3) generation. These waves exhibited properties particular to each P2YR subtype. Co-expression of eGFP-hCx43 and P2Y(1)R modified the properties of P2Y(1)R-generated ICW to those characteristics of P2Y(2)R. Increased coupling in P2Y(4)R clones induced by expression of eGFP-hCx43 abolished the ICWs observed in uncoupled P2Y(4)R clones. No changes in the behavior of ICWs generated in P2Y(2)R clones were observed after forced expression of Cx43. These data indicate that in 1321N1 cells gap junctional communication provides intercellular integration of Ca(2+) signals generated by P2YR activation, thus coordinating the propagation of intercellular calcium waves.     

Astrocyte‐restricted disruption of connexin‐43 impairs neuronal plasticity in mouse barrel cortex

There is intensive gap‐junctional coupling between glial processes, but their significance in sensory functions remains unknown. Connexin‐43 (Cx43), a major component of astrocytic gap‐junction channels, is abundantly expressed in astrocytes. To investigate the role of Cx43‐mediated gap junctions between astrocytes in sensory functions, we generated Cx43 knockout (KO) mice with a mouse line carrying loxP sites flanking exon 2 of the Cx43 gene and the transgenic line expressing Cre recombinase under control of the glial fibrillary acidic protein promoter, which exhibited a significant loss of Cx43 in astrocytes in the barrel cortex. Although Cx43 expression between the astrocytes measured by immunohistochemistry was virtually abolished in Cx43 KO mice, they had normal architecture in the barrel cortex but the intensity of cytochrome oxide histochemistry decreased significantly. In vivo electrophysiological analysis revealed that the long‐term potentiation of the vibrissal evoked responses in the barrel cortex evoked by high‐frequency rhythmic vibrissal stimuli (100 Hz, 1 s) was abolished in Cx43 KO mice. Current source density analysis also revealed that astrocytic Cx43 was important to the flow of excitation within the laminar connections in barrel cortex. Behavioral tests showed that the ability of Cx43 KO mice to sense the environment with their whiskers decreased. Even so, the jump‐stand experiment showed that they could still discriminate rough from smooth surfaces. Our findings suggest that Cx43‐mediated gap‐junctional coupling between astrocytes is important in the neuron–glia interactions required for whisker‐related sensory functions and plasticity.     

Modulation of astrocyte P2Y1 receptors by the carboxyl terminal domain of the gap junction protein Cx43

Gap junction proteins, connexins, provide intercellular channels that allow ions and small signaling molecules to be transmitted to adjacent coupled cells. Besides this function, it is becoming apparent that connexins also exert channel-independent effects, which are likely mediated by processes involving protein-protein interactions. Although a number of connexin interacting proteins have been identified, only little is known about the functional consequences of such interactions. We have previously shown that deletion of the astrocytic gap junction protein, connexin43 (Cx43) causes a right-ward shift in the dose-response curve to P2Y1R agonists and decreased P2Y1R expression levels. To evaluate whether these changes were due to reduced gap junctional communication or to protein-protein interactions, Cx43-null astrocytes were transfected with full-length Cx43 and Cx43 domains, and P2Y1R function and expression levels evaluated. Results indicate that restoration of P2Y1R function is independent of gap junctional communication and that the Cx43 carboxyl terminus spanning the SH3 binding domain (260-280) participates in the rescue of P2Y1R pharmacological behavior (shifting to the left the P2Y1R dose-response curve) without affecting its expression levels. These results suggest that the Cx43 carboxyl-terminus domain provides a binding site for an intracellular molecule, most likely a member of the c-Src tyrosine kinase family, which affects P2Y1R-induced calcium mobilization. It is here proposed that a nonchannel function of Cx43 is to serve as a decoy for such kinases. Such modulation of P2Y1R is expected to influence several neural cell functions, especially under inflammation and neurodegenerative disorders where expression levels of Cx43 are decreased.     

Mice with astrocyte-directed inactivation of connexin43 exhibit increased exploratory behaviour, impaired motor capacities, and changes in brain acetylcholine levels

Gap junctions mediate communication between many cell types in the brain. Gap junction channels are composed of membrane-spanning connexin (Cx) proteins, allowing the cell-to-cell passage of small ions and metabolites. Cx43 is the main constituent of the brain-spanning astrocytic gap junctional network, controlling activity-related changes in ion and glutamate concentrations as well as metabolic processes. In astrocytes, deletion of Cx43-coding DNA led to attenuated gap junctional coupling and impaired propagation of calcium waves, known to influence neuronal activity. Investigation of the role of Cx43 in behaviour has been impossible so far, due to postnatal lethality of its general deletion. Recently, we have shown that deletion of Cx30, which is also expressed by astrocytes, affects exploration, emotionality, and neurochemistry in the mouse. In the present study, we investigated the effects of the astrocyte-directed inactivation of Cx43 on mouse behaviour and brain neurochemistry. Deletion of Cx43 in astrocytes increased exploratory activity without influencing habituation. In the open field, but not in the elevated plus-maze, an anxiolytic-like effect was observed. Rotarod performance was initially impaired, but reached control level after further training. In the water maze, Cx43 deficient mice showed a steeper learning course, although final performance was similar between groups. Cx43 inactivation in astrocytes increased acetylcholine content in the frontal cortex of water maze-trained animals. Results are discussed in terms of altered communication between astrocytes and neurons, possible compensation processes, and differential effects of Cx30- and astrocyte-specific Cx43 deletion.     

Enhanced connexin 43 immunoreactivity in penumbral areas in the human brain following ischemia

Astrocytes support neurons not only physically but also chemically by secreting neurotrophic factors and energy substrates. Moreover, astrocytes establish a glial network and communicate through gap junctions in the brain. Connexin 43 (Cx43) is one of major component proteins in astrocytic gap junctions. Heterozygote Cx43 KO mice and astrocyte specific Cx43 KO mice exhibited amplified brain damage after ischemic insults, suggesting a neuroprotective role for astrocytic gap junctions. However, some reports mentioned unfavorable effects of gap junctions in neuronal support. Therefore, the role of astrocytic gap junctions under ischemic condition remains controversial. Since these studies have been performed using animal models, we investigated the Cx43 expression in human brain after stroke. Brain slice sections were prepared from pathological samples in our hospital. Embolic stroke brains sectioned because of the stroke were considered as acute ischemic models. Multiple infarction brains sectioned because of pneumonia or cancer were considered as chronic models. We observed the levels of Cx43 in both lesioned and intact areas, and compared them with acute and chronic models. As the results, astrocytes were strongly activated in penumbral lesions both of acute and chronic ischemic models. The Cx43 immunoreactivity was significantly amplified in the penumbra of chronic model compared to that of the acute model. Neurons were well preserved in chronic model compared to acute model. These findings suggested that the brain may generate neuronal protection by increasing the levels of Cx43 and amplifying the astrocytic gap junctional intercellular communication under hypoxic condition.     

Chronic endothelin exposure inhibits connexin43 expression in cultured cortical astroglia

Severe brain lesions are accompanied by sustained increases in endothelin (ET) levels, which in turn profoundly affect brain microcirculation and neural cell function. A known response of astrocytes to acute increases in ET levels is the rapid and transient closure of gap junctions and the subsequent decrease of gap junction-mediated intercellular communication (GJIC). Because evidence exists that the loss of GJIC alters astrocytic gene expression, we analyzed the effects of chronic ET exposure on astrocytic gap junction coupling. We found that within 24 hr, cultured cortical astrocytes respond to low nanomolar concentrations (2-10 nM) of either ET-1 or ET-3 with a robust inhibition of connexin (Cx)43 expression, the major junctional protein in astrocytes, and a subsequent decline of GIJC. We further observed that in the continuous presence of ETs, Cx43 expression remained inhibited for at least 7 days. In addition, a similar decrease of Cx43 expression occurred in cultured spinal cord astrocytes maintained with ET-1 for 3 days. Applying ETs in combination with the highly selective ETA and ETB receptor antagonists, BQ123 and BQ788, respectively, revealed that the inhibitory influences on astrocytic Cx43 expression depend on activation of ETB receptors. We suggest that the observed ET-dependent inhibition of Cx43 expression and the resulting decline of GJIC might represent a major pathway by which ETs regulate astrocytic gene expression in the injured brain.     

Endothelins regulate astrocyte gap junctions in rat hippocampal slices

Gap junctional communication (GJC) is a typical feature of astrocytes proposed to contribute to the role played by these glial cells in brain physiology and pathology. In acutely isolated hippocampal slices from rat (P11-P19), intercellular diffusion of biocytin through gap junction channels was shown to occur between hundreds of cells immuno-positive for astrocytic markers studied in the CA1/CA2 region. Single-cell RT-PCR demonstrated astrocytic mRNA expression of several connexin (Cx) subtypes, the molecular constituent of gap junction channels, whereas immunoblotting confirmed that Cx43 and Cx30 are the main gap junction proteins in hippocampal astrocytes. In the brain, astrocytes represent a major target for endothelins (Ets), a vasoactive family of peptides. Our results demonstrate that Ets decrease the expression of phosphorylated Cx43 forms and are potent inhibitors of GJC. The Et-induced effects were investigated using specific Et receptor agonists and antagonists, including Bosentan (Tracleer trade mark ), an EtA/B receptor antagonist, and using hippocampal slices and cultures from EtB-receptor-deficient rats. Interestingly, the pharmacological profile of Ets effects did not follow the classical profile established in cardiovascular systems. The present study therefore identifies Ets as potent endogenous inhibitory regulators of astrocyte networks. As such, the action of these peptides on astrocyte GJC might be involved in the contribution of astrocytes to neuroprotective processes and have a therapeutic potential in neuropathological situations.     

Brain macrophages inhibit gap junctional communication and downregulate connexin 43 expression in cultured astrocytes

Astrocytes are typically interconnected by gap junction channels that allow, in vitro as well as in vivo, a high degree of intercellular communication between these glial cells. Using cocultures of astrocytes and neurons, we have demonstrated that gap junctional communication (GJC) and connexin 43 (Cx43) expression, the major junctional protein in astrocytes, are controlled by neuronal activity. Moreover, neuronal death downregulates these two parameters. Because in several brain pathologies neuronal loss is associated with an increase in brain macrophage (BM) density, we have now investigated whether coculture with BM affects astrocyte gap junctions. We report here that addition of BM for 24 h decreases the expression of GJC and Cx43 in astrocytes in a density-dependent manner. In contrast, Cx43 is not detected in BM and no heterotypic coupling is observed between the two cell types. A soluble factor does not seem to be involved in these inhibitions because they are not observed either in the presence of BM conditioned media or in the absence of direct contact between the two cell types by using inserts. These observations could have pathophysiological relevance as neuronal death, microglial proliferation and astrocytic reactions occur in brain injuries and pathologies. Because astrocyte interactions with BM and dying neurons both result in the downregulation of Cx43 expression and in the inhibition of GJC, a critical consequence on astrocytic phenotype in those situations could be the inhibition of gap junctions.     

Connexin26 in adult rodent central nervous system: demonstration at astrocytic gap junctions and colocalization with connexin30 and connexin43.

The connexin family of proteins (Cx) that form intercellular gap junctions in vertebrates is well represented in the mammalian central nervous system. Among these, Cx30 and Cx43 are present in gap junctions of astrocytes. Cx32 is expressed by oligodendrocytes and is present in heterologous gap junctions between oligodendrocytes and astrocytes as well as at autologous gap junctions between successive myelin layers. Cx36 mRNA has been identified in neurons, and Cx36 protein has been localized at ultrastructurally defined interneuronal gap junctions. Cx26 is also expressed in the CNS, primarily in the leptomeningeal linings, but is also reported in astrocytes and in neurons of developing brain and spinal cord. To establish further the regional, cellular, and subcellular localization of Cx26 in neural tissue, we investigated this connexin in adult mouse brain and in rat brain and spinal cord using biochemical and immunocytochemical methods. Northern blotting, western blotting, and immunofluorescence studies indicated widespread and heterogeneous Cx26 expression in numerous subcortical areas of both species. By confocal microscopy, Cx26 was colocalized with both Cx30 and Cx43 in leptomeninges as well as along blood vessels in cortical and subcortical structures. It was also localized at the surface of oligodendrocyte cell bodies, where it was coassociated with Cx32. Freeze-fracture replica immunogold labeling (FRIL) demonstrated Cx26 in most gap junctions between cells of the pia mater by postnatal day 4. By postnatal day 18 and thereafter, Cx26 was present at gap junctions between astrocytes and in the astrocyte side of most gap junctions between astrocytes and oligodendrocytes. In perinatal spinal cord and in five regions of adult brain and spinal cord examined by FRIL, no evidence was obtained for the presence of Cx26 in neuronal gap junctions. In addition to its established localization in leptomeningeal gap junctions, these results identify Cx26 as a third connexin (together with Cx30 and Cx43) within astrocytic gap junctions and suggest a further level of complexity to the heterotypic connexin channel combinations formed at these junctions.     

Role of gap junction and connexin-43 in hypoxic-ischemic brain damage

INTRODUCTION Gap junction (GJ) is a special channel, which, at present, is thought to be the only one channel that can directly perform energy, sub- stance and information exchanges between two adjacent cells. GJ channel, widely existing in nervous system…     

Connexin43 phosphorylation state and intercellular communication in cultured astrocytes following hypoxia and protein phosphatase inhibition

The effects of hypoxia and phosphatase inhibitors on connexin43 (Cx43) phosphorylation state, gap junctional intercellular communication (GJIC) and immunolabelling with anti-Cx43 antibodies were investigated in cultured astrocytes. Astrocytes contained predominantly phosphorylated forms of Cx43 and these underwent dephosphorylation 30 min after hypoxia. This was preceded by a 77% reduction in GJIC 15 min after hypoxia, indicating that reduced GJIC occurs prior to Cx43 dephosphorylation. Hypoxia caused a reduction in punctate immunostaining (epitope masking) at cell-cell contacts with one anti-Cx43 antibody, and increased labelling with another antibody (13-8300) that detects only a dephosphorylated form of Cx43. Inhibition of protein phosphatase (PP)-1 and PP-2A with okadaic acid or calyculin A had little effect on hypoxia-induced Cx43 dephosphorylation. Inhibition of PP-2B (calcineurin) with cyclosporin A or FK506 reduced Cx43 dephosphorylation and junctional uncoupling seen after hypoxia. These results demonstrate that responses of astrocytic Cx43 to hypoxia in vitro are similar to those seen after ischaemia in vivo, and that inhibition of protein phosphatase protects astrocytes from hypoxia-induced Cx43 dephosphorylation and junctional uncoupling. In addition, calcineurin may play a direct role in the regulation of astrocytic GJIC and Cx43 phosphorylation state.     

Role of connexin43 in central nervous system injury

Gap junctions are specialized cell-to-cell contacts that provide direct intercellular communication. In the central nervous system (CNS), gap junction coupling occurs between both neurons and glial cells. One of the most abundant gap junction proteins in the CNS is connexin43 (Cx43). The functional syncytium formed by astrocytes via Cx43 gap junction intercellular communication has, for example, been implicated in maintaining the homeostasis of the extracellular milieu of neurons. In particular, astrocytes are involved in the spatial buffering of many ions, signalling molecules and energy sources. In this review, the role of Cx43 following CNS injury is examined by combining evidence surrounding the response of Cx43 to CNS injury and the effects of Cx43 gap junction blockade on neuronal survival in various models of injury. Combined evidence suggests that transient blockade targeting the window of initial Cx43 upregulation observed following injury is potentially therapeutic.     

Connexin43 and astrocytic gap junctions in the rat spinal cord after acute compression injury

To examine the possible role of interastrocytic gap junctions in the maintenance of tissue homeostasis after spinal cord damage, we initiated studies of the astrocytic gap junctional protein connexin43 (Cx43) in relation to temporal and spatial parameters of neuronal loss, reactive gliosis, and white matter survival in a rat model of traumatic spinal cord injury (SCI). Cx43 immunolocalization in normal and compression-injured spinal cord was compared by using two different sequence-specific anti-Cx43 antibodies that have previously exhibited different immunorecognition properties at lesion sites in brain. At 1- and 3-day survival times, gray matter areas with mild to moderate neuronal depletion exhibited a loss of immunolabeling with one of the two antibodies. At the lesion epicenter, these areas consisted of a zone that separated normal staining distal to the lesion from intensified labeling seen with both antibodies immediately adjacent to the lesion. Loss of immunoreactivity with only one of the two antibodies suggested masking of the corresponding Cx43 epitope. By 7 days post-SCI, Cx43 labeling was absent with both antibodies in all regions extending up to 1 mm from the lesion site. Reactive astrocytes displaying glial fibrillary acidic protein (GFAP) appeared by 1 day and were prominent by 3 days post-SCI. Their distribution in white and gray matter corresponded closely to that of Cx43 staining at 1 day, but less so at 3 days when GFAP-positive profiles were present at sites where Cx43 labeling was absent. By 7 days post-SCI, Cx43 again co-localized with GFAP-positive cells in the surviving subpial rim, and with astrocytic processes on radially oriented vascular profiles investing the central borders of the lesion. The results indicate that alterations in Cx43 cellular localization and Cx43 molecular modifications reflected by epitope masking, which were previously correlated with gap junction remodeling following excitotoxin-induced lesions in brain, are not responses limited to exogenously applied excitotoxins; they also occur in damaged spinal cord and are evoked by endogenous mechanisms after traumatic SCI. The GFAP/Cx43 co-localization results suggest that during their transformation to a reactive state, spinal cord astrocytes undergo a transitional phase marked by altered Cx43 localization or expression. J. Comp. Neurol. 382:199-214, 1997. © 1997 Wiley-Liss, Inc.     

The effects of bone morphogenetic protein 2 and 4 (BMP2 and BMP4) on gap junctions during neurodevelopment

Nervous system deficits account for the third largest group of fatal birth defects (after heart and respiratory problems) in North America. Although considerable advance has been made in neuroscience research, the early events involved in neurogenesis remain to be elucidated. More specifically, the effects of signaling molecules on intercellular communication during neurodevelopment have not yet been studied. The development of the central nervous system is regulated, at least in part, by signaling molecules such as bone morphogenetic proteins (BMPs). In this study, we have used the embryonal mouse P19 cell line to examine the effects of BMP2 and BMP4 on gap junctional communication as well as neuronal and astrocytic differentiation. The undifferentiated P19 cells show high levels of the gap junction protein, connexin43 (Cx43), and functional intercellular coupling. However, Cx43 expression and dye coupling decrease as these cells differentiate into neurons and astrocytes. In contrast, cells treated with BMP2 or BMP4 lose their capacity to differentiate into neurons but not astrocytes, while they maintain extensive gap junctional communication. The very few neurons that remain in the BMP-treated cultures are coupled (a characteristic not seen in the control neurons). Together, our data suggest that BMPs may play a critical role in morphogenesis of P19 cells while they affect gap junctions.     

Connexin29 and connexin32 at oligodendrocyte and astrocyte gap junctions and in myelin of the mouse central nervous system

The cellular localization, relation to other glial connexins (Cx30, Cx32, and Cx43), and developmental expression of Cx29 were investigated in the mouse central nervous system (CNS) with an anti-Cx29 antibody. Cx29 was enriched in subcellular fractions of myelin, and immunofluorescence for Cx29 was localized to oligodendrocytes and myelinated fibers throughout the brain and spinal cord. Oligodendrocyte somata displayed minute Cx29-immunopositive puncta around their periphery and intracellularly. In developing brain, Cx29 levels increased during the first few postnatal weeks and were highest in the adult brain. Immunofluorescence labeling for Cx29 in oligodendrocyte somata was intense at young ages and was dramatically shifted in localization primarily to myelinated fibers in mature CNS. Labeling for Cx32 also was localized to oligodendrocyte somata and myelin and absent in Cx32 knockout mice. Cx29 and Cx32 were minimally colocalized on oligodendrocytes somata and partly colocalized along myelinated fibers. At gap junctions on oligodendrocyte somata, Cx43/Cx32 and Cx30/Cx32 were strongly associated, but there was minimal association of Cx29 and Cx43. Cx32 was very sparsely associated with astrocytic connexins along myelinated fibers. With Cx26, Cx30, and Cx43 expressed in astrocytes and Cx29, Cx32, and Cx47 expressed in oligodendrocytes, the number of connexins localized to gap junctions of glial cells is increased to six. The results suggested that Cx29 in mature CNS contributes minimally to gap junctional intercellular communication in oligodendrocyte cell bodies but rather is targeted to myelin, where it, with Cx32, may contribute to connexin-mediated communication between adjacent layers of uncompacted myelin.     

Connexin43 null mutation increases infarct size after stroke.

Glial-neuronal interactions have been implicated in both normal information processing and neuroprotection. One pathway of cellular interactions involves gap junctional intercellular communication (GJIC). In astrocytes, gap junctions are composed primarily of the channel protein connexin43 (Cx43) and provide a substrate for formation of a functional syncytium implicated in the spatial buffering capacity of astrocytes. To study the function of gap junctions in the brain, we used heterozygous Cx43 null mice, which exhibit reduced Cx43 expression. Western blot analysis showed a reduction in the level of Cx43 protein and GJIC in astrocytes cultured from heterozygote mice. The level of Cx43 is reduced in the adult heterozygote cerebrum to 40% of that present in the wild-type. To assess the effect of reduced Cx43 and GJIC on neuroprotection, we examined brain infarct volume in wild-type and heterozygote mice after focal ischemia. In our model of focal stroke, the middle cerebral artery was occluded at two points, above and below the rhinal fissure. Four days after surgery, mice were killed, the brains were sectioned and analyzed. Cx43 heterozygous null mice exhibited a significantly larger infarct volume compared with wild-type (14.4 +/- 1.4 mm(3) vs. 7.7 +/- 0.82 mm(3), P < 0.002). These results suggest that augmentation of GJIC in astrocytes may contribute to neuroprotection after ischemic injury.     

Connexin43 enhances glioma invasion by a mechanism involving the carboxy terminus

Gliomas are particularly difficult to cure owing largely to their invasive nature. The neoplastic changes of astrocytes which give rise to these tumors frequently include a reduction of connexin43 (Cx43), the most abundant connexin isoform expressed in astrocytes. Cx43 is a subunit of gap junctions (GJ), intercellular channels which directly link the cytosol of adjacent cells and allow the regulated passage of ions and small molecules. To examine the role of Cx43 in glioma motility, we identified two variant C6 cell lines which endogenously express high (C6-H) or low (C6-L) levels of Cx43. In wound healing and transwell assays, C6-H cells were more motile than C6-L cells. To deduce whether Cx43 mediated these differences, assays were conducted on C6-H cells retrovirally transduced with Cx43 shRNA. Coincident with the stable knockdown of endogenous Cx43, a decrease in motility and invasion was observed. Gap junctional intercellular communication was also decreased, however motility assays conducted in the presence of GJ inhibitors did not reveal significant differences in cell motility. C6 cells transfected with full length or C-terminal truncated Cx43 (Cx43DeltaCT) were subjected to the aforementioned motility assays to expose alternate mechanisms of Cx43-mediated motility. Cells expressing full length Cx43 exhibited increased motility while cells expressing Cx43DeltaCT did not. This report, the first in which RNAi has been employed to reduce Cx43 expression in gliomas, indicates that the downregulation of Cx43 decreases motility of C6 cells. Furthermore, it is the first report to suggest that the Cx43 CT plays an important role in glioma motility.     

Neurons control the expression of connexin 30 and connexin 43 in mouse cortical astrocytes

A characteristic feature of astrocytes is their high level of intercellular communication mediated by gap junctions. The two main connexins, Cx30 and Cx43, that form these junctions in astrocytes of adult brain display different developmental and regional expression, with a delayed onset of appearance for Cx30. In primary cultures of astrocytes from newborn cerebral cortex, while Cx43 is abundantly expressed, Cx30 is not detectable. In the present report, Western blot and confocal immunofluorescence analysis performed in astrocyte/neuron cocultures demonstrate that neurons upregulate the expression of Cx43 and induce that of Cx30 in subsets of astrocytes preferentially located in close proximity to neuronal soma. In Cx43 lacking astrocytes cocultured with neurons, the induction of Cx30 allows the restoration of dye coupling within islets of Cx30-positive astrocytes, indicating that intercellular channels formed by Cx30 are functional. The upregulating effect of neurons on the expression of connexins in cortical astrocytes is independent of their electrical activity and requires tight interactions between both cell types. This effect is reversed after neuronal death induced by neurotoxic treatments. Furthermore, excitotoxic treatments triggering neuronal death in vivo lead to a downregulation of both connexins in reactive astrocytes located within the area depleted in neurons. Altogether these observations indicate that the expression of the two main astrocyte connexins is tightly regulated by neurons.