Neuroprotective gap-junction-mediated bystander transformations in the adult zebrafish spinal cord after injury.

The adult zebrafish spinal cord displays an impressive innate ability to regenerate after traumatic insults, yet the underlying adaptive cellular mechanisms remain elusive. Here, we show that while the cellular and tissue responses after injury are largely conserved among vertebrates, the large-size fast spinal zebrafish motoneurons are remarkably resilient by remaining viable and functional. We also reveal the dynamic changes in motoneuron glutamatergic input, excitability, and calcium signaling, and we underscore the critical role of calretinin (CR) in binding and buffering the intracellular calcium after injury. Importantly, we demonstrate the presence and the dynamics of a neuron-to-neuron bystander neuroprotective biochemical cooperation mediated through gap junction channels. Our findings support a model in which the intimate and dynamic interplay between glutamate signaling, calcium buffering, gap junction channels, and intercellular cooperation upholds cell survival and promotes the initiation of regeneration.

Metformin suppresses NFE2L1 pathway activation to inhibit gap junction beta protein expression in NSCLC.

OBJECTIVE: Non-small-cell lung cancer (NSCLC) is a deadly form of cancer that exhibits extensive intercellular communication which contributed to chemoradiotherapy resistance. Recent evidence suggests that arrange of key proteins are involved in lung cancer progression, including gap junction proteins (GJPs). METHODS AND RESULTS: In this study, we examined the expression patterns of GJPs in NSCLC, uncovering that both gap junction protein, beta 2 (GJB2) and gap junction protein, beta 2 (GJB3) are increased in LUAD and LUSC. We observed a correlation between the upregulation of GJB2, GJB3 in clinical samples and a worse prognosis in patients with NSCLC. By examining the mechanics, we additionally discovered that nuclear factor erythroid-2-related factor 1 (NFE2L1) had the capability to enhance the expression of connexin26 and connexin 31 in the NSCLC cell line A549. In addition, the use of metformin was discovered to cause significant downregulation of gap junction protein, betas (GJBs) by limiting the presence of NFE2L1 in the cytoplasm. CONCLUSION: This emphasizes the potential of targeting GJBs as a viable treatment approach for NSCLC patients receiving metformin.

Connexin 43 Prevents Radiation-Induced Intestinal Damage via the Ca2+-Dependent PI3K/Akt Signaling Pathway.

Radiation-induced intestinal damage (RIID) is a common side effect of radiotherapy in patients with abdominopelvic malignancies. Gap junctions are special structures consisting of connexins (Cxs). This study aimed to investigate the expression and role of connexins in RIID and underlying mechanism. In this study, a calcein-AM fluorescence probe was used to detect changes in gap junctional intercellular communication in intestinal epithelial IEC-6 cells. Our results show that gap junctional intercellular communication of IEC-6 cells was reduced at 6, 12, 24, and 48 h after irradiation, with the most pronounced effect at 24 h. Western blotting and immunofluorescence results showed that the expression of Cx43, but not other connexins, was reduced in irradiated intestinal epithelial cells. Silencing of Cx43 reduced gap junctional intercellular communication between irradiated intestinal epithelial cells with increased ROS and intracellular Ca2+ levels. Furthermore, knockdown of Cx43 reduced the number of clonal clusters, decreased cell proliferation with increased cytotoxicity and apoptosis. Western blotting results showed that silencing of Cx43 resulted in changed γ-H2AX and PI3K/AKT pathway proteins in irradiated intestinal epithelial cells. Administration of the PI3K/AKT pathway inhibitor LY294002 inhibited the radioprotective effects in Cx43-overexpressing intestinal epithelial cells. Our study demonstrated that Cx43 expression is decreased by ionizing radiation, which facilitates the radioprotection of intestinal epithelial cells.

Does BRRD mitigate the bank-to-sovereign risk channel?

We investigate the effectiveness of the Bank Recovery and Resolution Directive (BRRD) in mitigating the transmission of credit risk from banks to their sovereign, using CDS spreads to capture bank and sovereign credit risk for a sample of 43 banks in 8 Euro Area countries over the period 2009-2020. If the BRRD bail-in framework is credible, changes in bank default risk should not be transmitted to sovereign risk. In a novel approach we use banks earnings announcements to identify exogenous shocks to bank credit risk and investigate to what extent bank risk is transmitted to sovereign risk before and during the BRRD era. We find that bank-to-sovereign risk transmission has diminished after the introduction of the BRRD, suggesting that financial markets judge the BRRD framework as credible. The decline in bank-sovereign risk transmission is particularly significant in the periphery Euro Area countries, especially Italy and Spain, where the bank-sovereign nexus was most pronounced during the sovereign debt crisis. We report that the lower bank-to-sovereign credit risk transmission is associated with the parliamentary approval of the BRRD and not with the OMT program launched by the ECB to affect sovereign yield spreads, nor with specific bail-in or bailout cases which occurred during the BRRD era. Finally, we document that the reduction in risk transmission is most pronounced for banks classified as a Global Systemically Important Bank (G-SIB), stressing the importance of additional capital buffers imposed by Basel III.

The E3 ubiquitin ligase ITCH negatively regulates intercellular communication via gap junctions by targeting connexin43 for lysosomal degradation.

Intercellular communication via gap junctions has a fundamental role in regulating cell growth and tissue homeostasis, and its dysregulation may be involved in cancer development and radio- and chemotherapy resistance. Connexin43 (Cx43) is the most ubiquitously expressed gap junction channel protein in human tissues. Emerging evidence indicates that dysregulation of the sorting of Cx43 to lysosomes is important in mediating the loss of Cx43-based gap junctions in cancer cells. However, the molecular basis underlying this process is currently poorly understood. Here, we identified the E3 ubiquitin ligase ITCH as a novel regulator of intercellular communication via gap junctions. We demonstrate that ITCH promotes loss of gap junctions in cervical cancer cells, which is associated with increased degradation of Cx43 in lysosomes. The data further indicate that ITCH interacts with and regulates Cx43 ubiquitination and that the ITCH-induced loss of Cx43-based gap junctions requires its catalytic HECT (homologous to E6-AP C-terminus) domain. The data also suggest that the ability of ITCH to efficiently promote loss of Cx43-based gap junctions and degradation of Cx43 depends on a functional PY (PPXY) motif in the C-terminal tail of Cx43. Together, these data provide new insights into the molecular basis underlying the degradation of Cx43 and have implications for the understanding of how intercellular communication via gap junctions is lost during cancer development.

Gap Junctions in Ctenophora.

Gap junction proteins form specialized intercellular communication channels, including electrical synapses, that regulate cellular metabolism and signaling. We present a molecular inventory of the gap junction proteins-innexins (INX-like) in ctenophores, focusing on two reference species, Pleurobrachia bachei and Mnemiopsis leidyi. Innexins were identified in more than 15 ctenophore species, including such genera as Euplokamis, Pukia, Hormiphora, Bolinopsis, Cestum, Ocyropsis, Dryodora, Beroe, benthic ctenophores, Coeloplana and Vallicula, and undescribed species of Mertensiidae. The observed diversity of innexins resulted from the independent expansion of this family from the common ancestor of ctenophores. Innexins show the conserved topology with four transmembrane domains connected by two extracellular loops, which bridge intracellular gaps. However, INX-like genes have highly diverse exon organization and low percentage identity for their amino acid sequences within the same species and between ctenophore species. Such a broad scope of molecular diversity differs from innexins in other phyla. We predicted posttranslational modifications in innexins: 249 and 188 for M. leidyi and P. bachei, respectively. Neither their number nor their locations were conserved within or between species. When the number of posttranslational modifications is factored into the innexins' radiation, the potential for molecular and physiological diversity within gap junctions of ctenophores is almost unfathomable. RNA-seq and in situ hybridization data revealed that innexins are expressed across embryogenesis, including early cleavage stages and gastrulation. They are abundant in all adult tissues, with the highest expression level in the aboral organ (the major integrative center and the gravity sensor in ctenophores), followed by tentacles and comb plates. Nevertheless, each organ and tissue has a unique combination of innexins, suggesting their involvement in complex integrative functions and behaviors of ctenophores.

Photocatalytic manipulation of Ca2+ signaling for regulating cellular and animal behaviors via MOF-enabled H2O2 generation.

The in situ generation of H2O2 in cells in response to external stimulation has exceptional advantages in modulating intracellular Ca2+ dynamics, including high controllability and biological safety, but has been rarely explored. Here, we develop photocatalyst-based metal-organic frameworks (DCSA-MOFs) to modulate Ca2+ responses in cells, multicellular spheroids, and organs. By virtue of the efficient photocatalytic oxygen reduction to H2O2 without sacrificial agents, photoexcited DCSA-MOFs can rapidly trigger Ca2+ outflow from the endoplasmic reticulum with single-cell precision in a repeatable and controllable manner, enabling the propagation of intercellular Ca2+ waves (ICW) over long distances in two-dimensional and three-dimensional cell cultures. After photoexcitation, ICWs induced by DCSA-MOFs can activate neural activities in the optical tectum of tadpoles and thighs of spinal frogs, eliciting the corresponding motor behaviors. Our study offers a versatile optical nongenetic modulation technique that enables remote, repeatable, and controlled manipulation of cellular and animal behaviors.

Blood-testis barrier: a review on regulators in maintaining cell junction integrity between Sertoli cells.

The blood-testis barrier (BTB) is formed adjacent to the seminiferous basement membrane. It is a distinct ultrastructure, partitioning testicular seminiferous epithelium into apical (adluminal) and basal compartments. It plays a vital role in developing and maturing spermatocytes into spermatozoa via reorganizing its structure. This enables the transportation of preleptotene spermatocytes across the BTB, from basal to adluminal compartments in the seminiferous tubules. Several bioactive peptides and biomolecules secreted by testicular cells regulate the BTB function and support spermatogenesis. These peptides activate various downstream signaling proteins and can also be the target themself, which could improve the diffusion of drugs across the BTB. The gap junction (GJ) and its coexisting junctions at the BTB maintain the immunological barrier integrity and can be the "gateway" during spermatocyte transition. These junctions are the possible route for toxicant entry, causing male reproductive dysfunction. Herein, we summarize the detailed mechanism of all the regulators playing an essential role in the maintenance of the BTB, which will help researchers to understand and find targets for drug delivery inside the testis.

Expression, Purification, and Nanodisc Reconstitution of Connexin-43 Hemichannels for Structural Characterization by Cryo-Electron Microscopy.

Connexins are polytopic domain membrane proteins that form hexameric hemichannels (HCs) which can assemble into gap junction channels (GJCs) at the interface of two neighboring cells. The HCs may be involved in ion and small-molecule transport across the cellular plasma membrane in response to various stimuli. Despite their importance, relatively few structures of connexin HCs are available to date, compared to the structures of the GJCs. Here, we describe a protocol for expression, purification, and nanodisc reconstitution of connexin-43 (Cx43) HCs, which we have recently structurally characterized using cryo-EM analysis. Application of similar protocols to other connexin family members will lead to breakthroughs in the understanding of the structure and function of connexin HCs.

Spatial and Temporal Localization of Connexins in Cells Using Confocal Microscopy.

The 21-member connexin family found in humans is the building block of both single-membrane spanning channels (hemichannels) and double-membrane spanning intercellular channels. These large-pore channels are dynamic and typically have a short life span of only a few hours. Imaging connexins from the time of synthesis in the endoplasmic reticulum through to their degradation can be challenging given their distinct assembly states and transient residences in many subcellular compartments. Here, we describe how connexins can be effectively imaged on a confocal microscope in living cells when tagged with fluorescent proteins and when immunolabeled with high affinity anti-connexin antibodies in fixed cells. Temporal and spatial localization of multiple connexins and disease-linked connexin mutants at the subcellular level extensively informs on the mechanisms governing connexin regulation in health and disease.

Assessment of Connexin43 Hemichannel Functionality Based on Cytosolic Uptake of Yo-Pro1.

Connexin proteins are the building blocks of gap junctions and connexin hemichannels. Both provide a pathway for cellular communication. Gap junctions support intercellular communication mechanisms and regulate homeostasis. In contrast, open connexin hemichannels connect the intracellular compartment and the extracellular environment, and their activation fuels inflammation and cell death. The development of clinically applicable connexin hemichannel blockers for therapeutic purposes is therefore gaining momentum. This chapter describes a well-established protocol optimized for assessing connexin hemichannel activity by using the reporter dye Yo-Pro1.

Purification, Reconstitution, and Functional Analysis of Connexin Hemichannels.

Connexins are the proteins that form the gap junction channels that are essential for cell-to-cell communication. These channels are formed by head-to-head docking of hemichannels (each from one of two adjacent cells). Free "undocked" hemichannels at the plasma membrane are mostly closed, although they are still important under physiological conditions. However, abnormal and sustained increase in hemichannel activity due to connexin mutations or acquired conditions can produce or contribute to cell damage. For example, mutations of Cx26, a connexin isoform, can increase hemichannel activity and cause deafness. Studies using purified isolated systems under well-controlled conditions are essential for a full understanding of molecular mechanisms of hemichannel function under normal conditions and in disease, and here, we present methodology for the expression, purification, and functional analysis of hemichannels formed by Cx26.

Protocol for the Study of Connexin and DNA Interactions.

Connexins (Cxs) are transmembrane proteins which form hemichannels and gap junction channels at the plasma membrane. These channels allow the exchange of ions and molecules between the intra- and extracellular space and between cytoplasm of adjacent cells, respectively. The channel function of Cx assemblies has been extensively studied; however, "noncanonical" functions have emerged in the last few decades and have capture the attentions of many researchers, including the role of some Cxs as gene modulators or transcription factors. In this chapter, we describe a protocol to study the interaction of Cx46 with DNA in HeLa cells. These methods can facilitate understanding the role of Cxs in physiological processes and pathological mechanisms, including, for example, the contribution of Cx46 in maintaining stemness of glioma cancer stem cells.

Enhanced Methodologies for Investigating the Electrophysiological Characteristics of Endogenous Pannexin 1 Intercellular Cell-Cell Channels.

Gap junctions, pivotal intercellular conduits, serve as communication channels between adjacent cells, playing a critical role in modulating membrane potential distribution across cellular networks. The family of Pannexin (Panx) proteins, in particular Pannexin1 (Panx1), are widely expressed in vertebrate cells and exhibit sequence homology with innexins, the invertebrate gap junction channel constituents. Despite being ubiquitously expressed, detailed functional and pharmacological properties of Panx1 intercellular cell-cell channels require further investigation. In this chapter, we introduce optimized cell culture methodologies and electrophysiology protocols to expedite the exploration of endogenous Panx1 cell-cell channels in TC620 cells, a human oligodendroglioma cell line that naturally expresses Panx1. We anticipate these refined protocols will significantly contribute to future characterizations of Panx1-based intercellular cell-cell channels across diverse cell types and offer valuable insights into both normal cellular physiology and pathophysiology.

Phosphorylation-dependent allosteric regulation of Cx43 gap junction inhibitor potency.

Physiological and pathological processes such as homeostasis, embryogenesis, development, tumorigenesis, and cell movement depend on the intercellular communication through gap junctions (GJIC). Connexin (Cx)-based GJ channels are formed of two apposing hemichannels in the contiguous cells and provide a direct pathway for electrical and metabolic intercellular communication. The main modulators of GJ conductance are transjunctional voltage, intracellular pH, Ca2+, Mg2+, and phosphorylation. Chemical modulators of GJIC are being used in cases of various intercellular communication-dependent diseases. In this study, we used molecular docking, dual whole-cell patch-clamp, and Western blotting to investigate the impact of connexin phosphorylation on GJ chemical gating by α-pinene and other GJ inhibitors (octanol, carbenoxolone, mefloquine, intracellular pH, glycyrrhetinic acid, and sevoflurane) in HeLa cells expressing exogenous Cx43 (full length and truncated at amino acid 258) and other connexins typical of heart and/or nervous system (Cx36, Cx40, Cx45, and Cx47), and in cells expressing endogenous Cx43 (Novikoff and U-87). We found that Ca2+-regulated kinases, such as Ca2+/calmodulin-dependent kinase II, atypical protein kinase C, cyclin-dependent kinase, and Pyk2 kinase may allosterically modulate the potency of α-pinene through phosphorylation of Cx43 C-terminus. The identified new phenomenon was Cx isoform-, inhibitor-, and cell type-dependent. Overall, these results suggest that compounds, the potency of which depends on receptor phosphorylation, might be of particular interest in developing targeted therapies for diseases accompanied by high kinase activity, such as cardiac arrhythmias, epilepsy, stroke, essential tremor, inflammation, and cancer.

NGF increases Connexin-43 expression and function in pulmonary arterial smooth muscle cells to induce pulmonary artery hyperreactivity.

AIMS: Pulmonary hypertension (PH) is characterised by an increase in pulmonary arterial pressure, ultimately leading to right ventricular failure and death. We have previously shown that nerve growth factor (NGF) plays a critical role in PH. Our objectives here were to determine whether NGF controls Connexin-43 (Cx43) expression and function in the pulmonary arterial smooth muscle, and whether this mechanism contributes to NGF-induced pulmonary artery hyperreactivity. METHODS AND RESULTS: NGF activates its TrkA receptor to increase Cx43 expression, phosphorylation, and localization at the plasma membrane in human pulmonary arterial smooth muscle cells, thus leading to enhanced activity of Cx43-dependent GAP junctions as shown by Lucifer Yellow dye assay transfer and fluorescence recovery after photobleaching -FRAP- experiments. Using both in vitro pharmacological and in vivo SiRNA approaches, we demonstrate that NGF-dependent increase in Cx43 expression and activity in the rat pulmonary circulation causes pulmonary artery hyperreactivity. We also show that, in a rat model of PH induced by chronic hypoxia, in vivo blockade of NGF or of its TrkA receptor significantly reduces Cx43 increased pulmonary arterial expression induced by chronic hypoxia and displays preventive effects on pulmonary arterial pressure increase and right heart hypertrophy. CONCLUSIONS: Modulation of Cx43 by NGF in pulmonary arterial smooth muscle cells contributes to NGF-induced alterations of pulmonary artery reactivity. Since NGF and its TrkA receptor play a role in vivo in Cx43 increased expression in PH induced by chronic hypoxia, these NGF/Cx43-dependent mechanisms may therefore play a significant role in human PH pathophysiology.

Interplay between paclitaxel, gap junctions, and kinases: unraveling mechanisms of action and resistance in cancer therapy.

This comprehensive review elucidates the multifaceted roles of paclitaxel, a key chemotherapeutic agent, in cancer therapy, with a focus on its interactions with gap junctions and related kinases. Paclitaxel, with its complex diterpene structure, mediates its anticancer effects predominantly through specific interactions with ß-tubulin, instigating cell cycle arrest and triggering various cell death pathways, including apoptosis, pyroptosis, ferroptosis, and necroptosis. The paper systematically delineates the chemical attributes and action mechanisms of paclitaxel and its analogs, underscoring their capacity to disrupt microtubule dynamics, thereby leading to mitotic arrest and subsequent cell death induction. It also scrutinizes the pivotal role of gap junctions, composed of connexin proteins, in the modulation of cancer cell behavior and chemoresistance, especially in the milieu of paclitaxel administration. The review articulates how gap junctions can either suppress tumors or contribute to cancer progression, thereby influencing chemotherapy outcomes. Furthermore, the paper provides an in-depth analysis of how paclitaxel modulates gap junction-associated kinases via phosphorylation, influencing the drug's therapeutic efficacy and resistance profiles. By integrating insights from numerous key studies, the review offers a comprehensive understanding of the interplay between paclitaxel, gap junctions, and kinases, shedding light on potential approaches to augment paclitaxel's anti-tumor effectiveness and counteract chemoresistance in cancer treatment.

Unveiling the mechanisms of trichloroethylene hypersensitivity syndrome: Exploring the role of connexin 43 gap junctions in severe skin damage.

Trichloroethylene (TCE), extensively used as an organic solvent in various industrial applications, has been identified as a causative factor in inducing hypersensitivity syndrome (THS). Currently, there is no specific treatment for THS, and most patients experience serious adverse outcomes due to extensive skin damage leading to severe infection. However, the pathogenesis of THS-associated skin damage remains unclear. This study aims to elucidate the mechanism underlying skin damage from the perspective of intercellular communication and gap junctions in THS. Our results verified that hyperactivation of connexin43 gap junctions, caused by the aberrantly elevated expression of connexin43, triggers a bystander effect that promotes apoptosis and inflammation in THS via the TNF-TNFRSF1B and mitochondria-associated pathways. Additionally, we identified the gap junction inhibitor Carbenoxolone disodium (CBX) as a promising agent for the treatment of skin damage in THS. CBX protects against inflammatory cell infiltration in the skin and decreases immune cell imbalance in the peripheral blood of THS mice. Furthermore, CBX reduces connexin43 expression, apoptosis and inflammation in THS mice. The study reveals new insights into the mechanisms underlying TCE-induced skin damage, offering a potential treatment strategy for the development of effective therapies targeting severe dermatitis induced by chemical exposure.

Assembly mechanisms of the neuronal gap junction channel connexin 36 elucidated by Cryo-EM.

Electrical synapses are essential components of neural circuits. Neuronal signal transduction across electrical synapses is primarily mediated by gap junction channels composed of Connexin36 (Cx36), the lack of which causes impaired electrical coupling between certain neurons including cortical interneurons and thalamic reticular nucleus (TRN) neurons. However, the structural basis underlying Cx36 function and assembly remains elusive. Recently, Lee et al. reported cryo-EM structures of Cx36, thus provided first insights of its gating mechanism. Here, we report a consistent cryo-EM structure of Cx36 determined in parallel, and describe unique interactions underpinning its assembly mechanism in complementary to the competing work. In particular, we found non-canonical electrostatic interactions between protomers from opposing hemichannels and a steric complementary site between adjacent protomers within a hemichannel, which together provide a structural explanation for the assembly specificity in homomeric and heteromeric gap junction channels.