Christian C. Naus

Intercellular Calcium Signaling in Astrocytes via ATP Release through Connexin Hemichannels

Astrocytes are capable of widespread intercellular communication via propagated increases in intracellular Ca2+ concentration. We have used patch clamp, dye flux, ATP assay, and Ca2+ imaging techniques to show that one mechanism for this intercellular Ca2+ signaling in astrocytes is the release of ATP through connexin channels (“hemichannels”) in individual cells. Astrocytes showed low Ca2+-activated whole-cell currents consistent with connexin hemichannel currents that were inhibited by the connexin channel inhibitor flufenamic acid (FFA). Astrocytes also showed molecular weight-specific influx and release of dyes, consistent with flux through connexin hemichannels. Transmembrane dye flux evoked by mechanical stimulation was potentiated by low Ca2+ and was inhibited by FFA and Gd3+. Mechanical stimulation also evoked release of ATP that was potentiated by low Ca2+ and inhibited by FFA and Gd3+. Similar whole-cell currents, transmembrane dye flux, and ATP release were observed in C6 glioma cells expressing connexin43 but were not observed in parent C6 cells. The connexin hemichannel activator quinine evoked ATP release and Ca2+ signaling in astrocytes and in C6 cells expressing connexin43. The propagation of intercellular Ca2+ waves in astrocytes was also potentiated by quinine and inhibited by FFA and Gd3+. Release of ATP through connexin hemichannels represents a novel signaling pathway for intercellular communication in astrocytes and other non-excitable cells.

Pannexin channels are not gap junction hemichannels

Pannexins, a class of membrane channels, bear significant sequence homology with the invertebrate gap junction proteins, innexins, and more distant similarities in their membrane topologies and pharmacological sensitivities with the gap junction proteins, connexins. However, the functional role for the pannexin oligomers or pannexons, is different from connexin oligomers, the connexons. Many pannexin publications have used the term “hemichannels” to describe pannexin oligomers while others use the term “channels” instead. This has led to confusion within the literature about the function of pannexins that promotes the idea that pannexons serve as gap junction hemichannels and thus, have an assembly and functional state as gap junctional intercellular channels. Here, we present the case that unlike the connexin gap junction intercellular channels, so far, pannexin oligomers have repeatedly been shown to be channels that are functional in single membranes, but not as intercellular channels in appositional membranes. Hence, they should be referred to as channels and not hemichannels. Thus, we advocate that in the absence of firm evidence that pannexins form gap junctions, the use of the term “hemichannel” be discontinued within the pannexin literature.

Intracellular calcium changes trigger connexin 32 hemichannel opening

Connexin hemichannels have been proposed as a diffusion pathway for the release of extracellular messengers like ATP and others, based on connexin expression models and inhibition by gap junction blockers. Hemichannels are opened by various experimental stimuli, but the physiological intracellular triggers are currently not known. We investigated the hypothesis that an increase of cytoplasmic calcium concentration ([Ca2+]i) triggers hemichannel opening, making use of peptides that are identical to a short amino‐acid sequence on the connexin subunit to specifically block hemichannels, but not gap junction channels. Our work performed on connexin 32 (Cx32)‐expressing cells showed that an increase in [Ca2+]i triggers ATP release and dye uptake that is dependent on Cx32 expression, blocked by Cx32 (but not Cx43) mimetic peptides and a calmodulin antagonist, and critically dependent on [Ca2+]i elevation within a window situated around 500 nM. Our results indicate that [Ca2+]i elevation triggers hemichannel opening, and suggest that these channels are under physiological control.

ATP and glutamate released via astroglial connexin 43 hemichannels mediate neuronal death through activation of pannexin 1 hemichannels

J. Neurochem. (2011) 118, 826–840.

Implications and challenges of connexin connections to cancer

The idea that the gap junction family of proteins, connexins, are tumour suppressors has been widely supported through numerous cancer models. However, the paradigm that connexins and enhanced gap junctional intercellular communication is of universal benefit by restricting tumour growth has been challenged by more recent evidence that suggests a role for connexins in facilitating tumour progression and metastasis. Therefore, connexins might be better classified as conditional tumour suppressors that modulate cell proliferation, as well as adhesion and migration.

Increased apoptosis and inflammation after focal brain ischemia in mice lacking connexin43 in astrocytes

Astrocytes secrete cytokines and neurotrophic factors to neurons, consistent with a neurosupportive role for astrocytes. However, in ischemic or metabolic insults, the function of astrocytic gap junctions composed mainly from connexin43 (Cx43) remains controversial. We have previously shown that heterozygous Cx43 null mice subjected to middle cerebral artery occlusion exhibited significantly enhanced stroke volume and apoptosis compared to wild-type mice. In this study, we used mice in which the human GFAP promoter-driven cre transgene deletes the floxed Cx43 gene in astrocytes, excluding the effects from reduced Cx43 expression in many other cell types as well as astrocytes. We induced focal brain ischemia in mice lacking Cx43 in astrocytes [Cre(+)] and control littermates [Cre(-)]. Cre(+) mice showed a significantly increased stroke volume and enhanced apoptosis, detected by terminal dUTP nick-end labeling and caspase-3 immunostaining, compared to Cre(-) mice. Inflammatory response assessed by the microglial marker CD11b was amplified in the penumbra of Cre(+) mice compared to that of Cre(-) mice. Our results suggest that astrocytic gap junctions could be important for the regulation of neuronal apoptosis and the inflammatory response after stroke. These findings support the view that astrocytes play a critical role in neuroprotection during ischemic insults.

Amyloid β-Induced Death in Neurons Involves Glial and Neuronal Hemichannels

The mechanisms involved in Alzheimers disease are not completely understood and how glial cells contribute to this neurodegenerative disease remains to be elucidated. Because inflammatory treatments and products released from activated microglia increase glial hemichannel activity, we investigated whether amyloid-β peptide (Aβ) could regulate these channels in glial cells and affect neuronal viability. Microglia, astrocytes, or neuronal cultures as well as acute hippocampal slices made from GFAP-eGFP transgenic mice were treated with the active fragment of Aβ. Hemichannel activity was monitored by single-channel recordings and by time-lapse ethidium uptake, whereas neuronal death was assessed by Fluoro-Jade C staining. We report that low concentrations of Aβ25–35 increased hemichannel activity in all three cell types and microglia initiate these effects triggered by Aβ. Finally, neuronal damage occurs by activation of neuronal hemichannels induced by ATP and glutamate released from Aβ25–35-activated glia. These responses were observed in the presence of external calcium and were differently inhibited by hemichannel blockers, whereas the Aβ25–35-induced neuronal damage was importantly reduced in acute slices made from Cx43 knock-out mice. Thus, Aβ leads to a cascade of hemichannel activation in which microglia promote the release of glutamate and ATP through glial (microglia and astrocytes) hemichannels that induces neuronal death by triggering hemichannels in neurons. Consequently, this work opens novel avenues for alternative treatments that target glial cells and neurons to maintain neuronal survival in the presence of Aβ.

The role of gap junctions in seizures

Electrotonic synaptic communication between neurons via gap junctions (gjs) is increasingly recognized as an important synchronizing mechanism in the brain. At the same time, the biology of central nervous system (CNS) gjs is being unravelled. The pathogenesis of the abnormal neuronal synchrony underlying seizures, formerly thought to be based mainly on chemical synaptic transmission, now includes a role for gap junctional communication. This concept has been strengthened by evidence from several in vitro seizure models, in which pharmacological manipulations of gap junctional communication predictably affect the generation of seizures: blockers diminishing seizures and enhancers increasing the seizures. Evidence for interneurons, coupled in part by gjs, generating synchronous neural network activity including seizures, is presented. Also neuromodelling studies, which have enhanced our ability to understand the functional role that gap junctional communication plays in the generation and maintenance of neural synchrony and seizures, are presented. Gap junctional communication appears to be a promising target for the development of future anticonvulsant therapy.

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 mm3 vs. 7.7 ± 0.82 mm3, P < 0.002). These results suggest that augmentation of GJIC in astrocytes may contribute to neuroprotection after ischemic injury. J. Comp. Neurol. 440:387–394, 2001.

Tumor-Suppressive Effects of Pannexin 1 in C6 Glioma Cells

Mammalian gap junction proteins, connexins, have long been implicated in tumor suppression. Recently, a novel family of proteins named pannexins has been identified as the mammalian counterpart of the invertebrate gap junction proteins, innexins. To date, pannexin 1 (Panx1) and pannexin 2 (Panx2) mRNAs are reported to be expressed in the brain. Most neoplastic cells, including rat C6 gliomas, exhibit reduced connexin expression, aberrant gap junctional intercellular communication (GJIC), and an increased proliferation rate. When gap junctions are up-regulated by transfecting C6 cells with connexin43, GJIC is restored and the proliferation is reduced. In this study, we examined the tumor-suppressive effects of Panx1 expression in C6 cells. Reverse transcription-PCR analysis revealed that C6 cells do not express any of the pannexin transcripts, whereas its nontumorigenic counterpart, rat primary astrocytes, exhibited mRNAs for all three pannexins. On generation of stable C6 transfectants with tagged Panx1 [myc or enhanced green fluorescent protein (EGFP)], a localization of Panx1 expression to the Golgi apparatus and plasma membrane was observed. In addition, Panx1 transfectants exhibited a flattened morphology, which differs greatly from the spindle-shaped control cells (EGFP only). Moreover, Panx1 expression increased gap junctional coupling as shown by the passage of sulforhodamine 101. Finally, we showed that stable expression of Panx1 in C6 cells significantly reduced cell proliferation in monolayers, cell motility, anchorage-independent growth, and in vivo tumor growth in athymic nude mice. Altogether, we conclude that the loss of pannexin expression may participate in the development of C6 gliomas, whereas restoration of Panx1 plays a tumor-suppressive role.