Inhibition of Cx43 mediates protective effects on hypoxic/reoxygenated human neuroblastoma cells (original) (raw)
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Connexins in the Central Nervous System: Physiological Traits and Neuroprotective Targets
Frontiers in physiology, 2017
Cell-to-cell interaction and cell-to-extracellular environment communication are emerging as new therapeutic targets in neurodegenerative disorders. Dynamic expression of connexins leads to distinctive hemichannels and gap junctions, characterized by cell-specific conduction, exchange of stimuli or metabolites, and particular channel functions. Herein, we briefly reviewed classical physiological traits and functions of connexins, hemichannels, and gap junctions, in order to discuss the controversial role of these proteins and their mediated interactions during neuroprotection, with a particular focus on Cx43-based channels. We pointed out the contribution of connexins in neural cells populations during neurodegenerative processes to explore potential neuroprotective therapeutic applications.
Role of connexin43 in central nervous system injury
Experimental Neurology, 2010
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.
A central role of connexin 43 in hypoxic preconditioning
The Journal of …, 2008
Preconditioning is an endogenous mechanism in which a nonlethal exposure increases cellular resistance to subsequent additional severe injury. Here we show that connexin 43 (Cx43) plays a key role in protection afforded by preconditioning. Cx43 null mice were insensitive to hypoxic preconditioning, whereas wild-type littermate mice exhibited a significant reduction in infarct volume after occlusion of the middle cerebral artery. In cultures, Cx43-deficient cells responded to preconditioning only after exogenous expression of Cx43, and protection was attenuated by small interference RNA or by channel blockers. Our observations indicate that preconditioning reduced degradation of Cx43, resulting in a marked increase in the number of plasma membrane Cx43 hemichannels. Consequently, efflux of ATP through hemichannels led to accumulation of its catabolic product adenosine, a potent neuroprotective agent. Thus, adaptive modulation of Cx43 can offset environmental stress by adenosine-mediated elevation of cellular resistance.
Journal of Biological Chemistry, 2014
Background: The gap junction protein Cx43 is implicated in maintaining anti-oxidative defense in astrocytes. Results: In contrast to hypoxia/reoxygenation, oxidative stress induced by H 2 O 2 triggers more astrocytic death in the absence of Cx43 channels. Conclusion: Gap junction intercellular communication is required for Cx43-mediated resistance to H 2 O 2. Significance: An altered Cx43 phosphorylation state in response to cellular stress may be critical for Cx43-mediated cell death or recovery.
Connexin 43 confers resistance to hydrogen peroxide-mediated apoptosis
Biochemical and Biophysical Research Communications, 2007
The current study aimed to understand the anti-apoptotic effect of overexpressed gap junction forming protein connexin (Cx) 43 in C6 glioma cells. C6 cells exposed to hydrogen peroxide (H 2 O 2 ) or staurosporine demonstrated morphological and biochemical changes consistent with apoptosis, whereas C6 cells expressing Cx43 demonstrated relative resistance to H 2 O 2 , but not to staurosporine. This selective protection against H 2 O 2 was due to inhibition of caspase 3 activation in Cx43 expressing cells. siRNA knockdown experiments in rat primary astrocytes confirmed the presence of endogenous Cx43-mediated anti-apoptotic effect. Cx43 interacts with the upstream apoptosis signal-regulating kinase 1 known to mediate H 2 O 2 -induced apoptosis providing a possible mechanism for protection. These findings provided new evidence for regulation of the mitogen activated protein kinase pathway and apoptosis by Cx43 implicating this protein in intracellular signaling beyond its role as a gap junction forming protein on the plasma membrane.
The gap junction protein connexin43 (Cx43) has been reported to exist as several phosphorylated forms migrating at ~43 kDa on sodium dodecyl sulfate- polyacrylamide gel electrophoresis as well as an unphosphorylated 41-kDa form. In brain, Cx43 is expressed predominantly in astrocytes and is also expressed in several other cell types. Whereas the phosphorylated forms of Cx43 predominate in heart, several studies have indicated that high levels of the unphosphorylated form of Cx43 are present in brain. Various experiments in this report indicate that the 41-kDa molecular form in brain is a postmortem dephosphorylation product of phosphorylated Cx43. In rats killed by cranial high-energy microwave irradiation leading to rapid inactivation of brain metabolism, Cx43 in cerebral cortex was present almost exclusively as the 43-kDa phosphorylated form. Rapid dissection of brain followed by heat treatment or inclusion of phosphatase inhibitors during tissue homogenization also largely prevented the conversion of the 43- to the 41-kDa form. The 41- kDa species was generated after alkaline phosphatase digestion of the 43-kDa material obtained by immunoprecipitation from microwave-irradiated brain. Immunolabeling patterns and relative regional levels of Cx43 as seen by immunohistochemical and western blot detection were the same whether or not metabolism to the 41-kDa species was prevented. In developing rat brain, Cx43 levels in frontal cortex and brainstem increased with age, but the degree of dephosphorylation of the 43- to the 41-kDa form was greater at earlier ages in the brainstem. It appears that brain contains a phosphatase that may be involved in modulating the phosphorylation state of Cx43 and thus may regulate intercellular communication via astrocytic gap junctions.
Olfactory Ensheathing Cells Protect Cortical Neuron Cultures Exposed to Hypoxia
CNS & Neurological Disorders - Drug Targets (Formerly Current Drug Targets - CNS & Neurological Disorders)
A peculiar population of glial cells, Olfactory Ensheathing Cells (OECs), are able to support the continuous neuronal turn-over and sheathe olfactory axons. In vitro, they stimulate axonal growth, as produce several neurotrophic factors (GFs); in vivo they promote remyelination of damaged axons. In this in vitro study, OEC effects on survival of cortical neurons exposed to hypoxia were examined. Rat co-cultures of OECs and cortical neurons were placed both in normal and hypoxic conditions; subsequently cells were analyzed by immunocytochemistry. Furthermore, some neuronal cultures were grown with Glial cell Derived Neurotrophic Factor (GDNF) or basic Fibroblast Growth Factor (bFGF) to tentatively rescue cells from oxygen deprivation. Some cortical neurons grown in both conditions were considered as control cells. Some neuronal cultures were feed with conditioned medium from OECs. We show that both in co-cultures and with GFs-treatment there was an increase of the number of neurons i...
Reversion of the neoplastic phenotype of human glioblastoma cells by connexin 43 (cx43)
Cancer research, 1998
Connexins (cx), structural components of gap junction, are believed to play a role in the regulation of cell proliferation and suppression of the neoplastic phenotype. We used human brain glioblastoma tumor cells as a model system to test this hypothesis. Western blot and reverse transcription-PCR analysis indicate that the expression levels of the gap junction protein connexin 43 (cx43) are profoundly decreased in several human brain tumor cell lines examined. Transfection of human cx43 into human glioblastoma cell lines U251 and T98G profoundly reduces cell proliferation in monolayer culture, in soft agar, and in athymic nude mice. Surprisingly, these effects are not associated with the establishment of gap junction communication in cx43 transfected cells. We conclude that the loss of cx43 expression may play a role in the development of human gliomas and that cx43 acts as a tumor suppressor gene to human glioblastoma.
Proceedings of the National Academy of Sciences, 2002
Rat cortical astrocytes in pure culture are functionally coupled to neighboring cells via connexin (Cx) 43 gap junctions under ordinary conditions. Small fluorescent molecules such as Lucifer yellow (LY) pass between cell interiors via gap junctions, but do not enter the cells when externally applied. Subjecting rat and mouse cortical astrocytes to ''chemical ischemia'' by inhibition of glycolytic and oxidative metabolism induced permeabilization of cells to Lucifer yellow and ethidium bromide before loss of membrane integrity determined by dextran uptake and lactate dehydrogenase release. The gap junction blockers octanol and 18␣-glycyrrhetinic acid markedly reduced dye uptake, suggesting that uptake was mediated by opening of unapposed hemichannels. Extracellular La 3؉ also reduced dye uptake and delayed cell death. The purinergic blocker, oxidized ATP, was ineffective. Astrocytes isolated from mice with targeted deletion of the Cx43 coding DNA exhibited greatly reduced dye coupling and ischemia-induced dye uptake, evidence that dye uptake is mediated by Cx43 hemichannels. Dye coupling was reduced but not blocked by metabolic inhibition. Blockade of lipoxygenases or treatment with free radical scavengers reduced dye uptake by rat astrocytes, suggesting a role for arachidonic acid byproducts in hemichannel opening. Furthermore, permeabilization was accompanied by reduction in ATP levels and dephosphorylation of Cx43. Although hemichannel opening would tend to collapse electrochemical and metabolic gradients across the plasma membrane of dying cells, healthy cells might rescue dying cells by transfer of ions and essential metabolites via Cx43 gap junctions. Alternatively, dying astrocytes might compromise the health of neighboring cells via Cx43 gap junctions, thereby promoting the propagation of cell death.
Inhibition of connexin43 improves functional recovery after ischemic brain injury in neonatal rats
Glia, 2015
Connexin43 (Cx43) is one of the most abundant gap junction proteins in the central nervous system. Abnormal opening of Cx43 hemichannels after ischemic insults causes apoptotic cell death. In this study, we found persistently increased expression of Cx43 8 h to 7 d after hypoxia/ischemia (HI) injury in neonatal rats. Pre-treatment with Gap26 and Gap27, two Cx43 mimetic peptides, significantly reduced cerebral infarct volume. Gap26 treatment at 24 h after ischemia improved functional recovery on muscle strength, motor coordination, and spatial memory abilities. Further, Gap26 inhibited Cx43 expression and reduced active astrogliosis. Gap26 interacted and co-localized with Cx43 together in brain tissues and cultured astrocytes. After oxygen glucose deprivation, Gap26 treatment reduced the total Cx43 level in cultured astrocytes; but Cx43 level in the plasma membrane was increased. Degradation of Cx43 in the cytoplasm was mainly via the ubiquitin proteasome pathway. Concurrently, phosp...