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[This corrects the article DOI: 10.3389/fimmu.2022.870679.].

To explore the pathophysiological mechanisms of antiseizure and adverse behavioural/psychiatric effects of brivaracetam and levetiracetam, in the present study, we determined the effects of brivaracetam and levetiracetam on astroglial L-glutamate release induced by artificial high-frequency oscillation (HFO) bursts using ultra-high-performance liquid chromatography. Additionally, the effects of brivaracetam and levetiracetam on protein expressions of connexin43 (Cx43) and synaptic vesicle protein 2A (SV2A) in the plasma membrane of primary cultured rat astrocytes were determined using a capillary immunoblotting system. Acutely artificial fast-ripple HFO (500 Hz) burst stimulation use-dependently increased L-glutamate release through Cx43-containing hemichannels without affecting the expression of Cx43 or SV2A in the plasma membrane, whereas acute physiological ripple HFO (200 Hz) stimulation did not affect astroglial L-glutamate release or expression of Cx43 or SV2A. Contrarily, subchronic ripple HFO and acute pathological fast-ripple HFO (500 Hz) stimulations use-dependently increased L-glutamate release through Cx43-containing hemichannels and Cx43 expression in the plasma membrane. Subchronic fast-ripple HFO-evoked stimulation produced ectopic expression of SV2A in the plasma membrane, but subchronic ripple HFO stimulation did not generate ectopic SV2A. Subchronic administration of brivaracetam and levetiracetam concentration-dependently suppressed fast-ripple HFO-induced astroglial L-glutamate release and expression of Cx43 and SV2A in the plasma membrane. In contrast, subchronic ripple HFO-evoked stimulation induced astroglial L-glutamate release, and Cx43 expression in the plasma membrane was inhibited by subchronic levetiracetam administration, but was not affected by brivaracetam. These results suggest that brivaracetam and levetiracetam inhibit epileptogenic fast-ripple HFO-induced activated astroglial transmission associated with hemichannels. In contrast, the inhibitory effect of therapeutic-relevant concentrations of levetiracetam on physiological ripple HFO-induced astroglial responses probably contributes to the adverse behavioural/psychiatric effects of levetiracetam.

Introduction: Gap junctions can transmit signals between cells, including miRNAs, leading to the amplification of adjacent cell damage. No previous study has addressed gap junctions and miRNAs in sepsis because the internal mechanism of sepsis-induced intestinal injury is complex. Therefore, we studied the relationship between connexin43 (Cx43) and miR-181b and provided a research direction for further study of sepsis. Methods: A mouse caecal ligation and puncture method was used to construct a mouse sepsis model. Firstly, damage to intestinal tissues at different time points was analysed. The levels of Cx43, miR-181b, Sirt1, and FOXO3a in intestinal tissues and the transcription and translation of the apoptosis-related genes Bim and puma, which are downstream of FOXO3a were analysed. Secondly, the effect of Cx43 levels on miR-181b and Sirt1/FOXO3a signalling pathway activity was explored by using the Cx43 inhibitor heptanol. Finally, luciferase assays were used to determine miR-181b binding to the predicted target sequence. Results: The results show that during sepsis, intestinal injury becomes increasingly worse with time, and the expression of Cx43 and miR-181b increase. In addition, we found that heptanol could significantly reduce intestinal injury. This finding indicates that inhibiting Cx43 regulates the transfer of miR-181b between adjacent cells, thereby reducing the activity of the Sirt1/FOXO3a signalling pathway and reducing the degree of intestinal injury during sepsis. Conclusions: In sepsis, the enhancement of Cx43 gap junctions leads to an increase in miR-181b intercellular transfer, affects the downstream SIRT1/FOXO3a signalling pathway and causes cell and tissue damage.

This study was undertaken to evaluate the connexin hemichannel blocker tonabersat for the inhibition of inflammasome activation and use as a potential treatment for diabetic retinopathy. Human retinal pigment epithelial cells (ARPE-19) were stimulated with hyperglycemia and the inflammatory cytokines IL-1β and TNFα in order to mimic diabetic retinopathy molecular signs in vitro. Immunohistochemistry was used to evaluate the effect of tonabersat treatment on NLRP3, NLRP1, and cleaved caspase-1 expression and distribution. A Luminex cytokine release assay was performed to determine whether tonabersat affected proinflammatory cytokine release. NLRP1 was not activated in ARPE-19 cells, and IL-18 was not produced under disease conditions. However, NLRP3 and cleaved caspase-1 complex formation increased with hyperglycemia and cytokine challenge but was inhibited by tonabersat treatment. It also prevented the release of proinflammatory cytokines IL-1β, VEGF, and IL-6. Tonabersat therefore has the potential to reduce inflammasome-mediated inflammation in diabetic retinopathy.

Recently, accumulating preclinical findings suggest the possibility that functional abnormalities of tripartite synaptic transmission play important roles in the pathophysiology of schizophrenia and affective disorder. Therefore, to explore the novel mechanisms of mood-stabilizing effects associated with tripartite synaptic transmission, the present study determined the effects of mood-stabilizing antipsychotics, clozapine (CLZ), quetiapine (QTP) and brexpiprazole (BPZ), on the astroglial l-glutamate release and expression of connexin43 (Cx43) in the astroglial plasma membrane using cortical primary cultured astrocytes. Neither acute (for 120 min) nor subchronic (for 7 days) administrations of CLZ, QTP and BPZ affected basal astroglial l-glutamate release, whereas both acute and subchronic administration of CLZ, QTP and BPZ concentration-dependently enhanced astroglial l-glutamate release through activated hemichannels. Subchronic administration of therapeutic-relevant concentration of valproate (VPA), a histone deacetylase inhibiting mood-stabilizing antiepileptic drug, enhanced the stimulatory effects of therapeutic-relevant concentration of CLZ, QTP and BPZ on astroglial l-glutamate release through activated hemichannel. Subchronic administration of therapeutic-relevant concentration of CLZ, QTP and BPZ did not affect Cx43 protein expression in the plasma membrane during resting stage. After subchronic administration of VPA, acute and subchronic administration of therapeutic-relevant concentrations of CLZ increased Cx43 protein expression in the plasma membrane. Both acute administrations of therapeutic-relevant concentrations of QTP and BPZ did not affect, but subchronic administrations enhanced Cx43 protein expression in the astroglial plasma membrane. Furthermore, protein kinase B (Akt) inhibitor suppressed the stimulatory effects of CLZ and QTP, but did not affect Cx43 protein expression in the astroglial plasma membrane. These results suggest that three mood-stabilizing atypical antipsychotics, CLZ, QTP and BPZ enhance tripartite synaptic glutamatergic transmission due to enhancement of astroglial Cx43 containing hemichannel activities; however, the Cx43 activating mechanisms of these three mood-stabilizing antipsychotics were not identical. The enhanced astroglial glutamatergic transmission induced by CLZ, QTP and BPZ is, at least partially, involved in the actions of these three mood-stabilizing antipsychotics.

Cardiomyocyte apoptosis is a crucial factor leading to myocardial dysfunction. Adiponectin (APN) has a cardiomyocyte-protective impact. Studies have shown that the connexin43 (Cx43) and phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) signaling pathways play an important role in the heart, but whether APN plays a protective role by regulating these pathways is unclear. Our study aimed to confirm whether APN protects against lipopolysaccharide (LPS)-induced cardiomyocyte apoptosis and to explore whether it plays an important role through regulating the Cx43 and PI3K/AKT signaling pathways. In addition, our research aimed to explore the relationship between the Cx43 and PI3K/AKT signaling pathways. In vitro experiments: Before H9c2 cells were treated with LPS for 24 h, they were pre-treated with APN for 2 h. The cytotoxic effect of APN on H9c2 cells was evaluated by a CCK-8 assay. The protein levels of Bax, Bcl2, cleaved caspase-3, cleaved caspase-9, Cx43, PI3K, p-PI3K, AKT and p-AKT were evaluated by Western blot analysis, and the apoptosis rate was evaluated by flow cytometry. APN attenuated the cytotoxicity induced by LPS. LPS upregulated Bax, cleaved caspase-3 and cleaved caspase-9 and downregulated Bcl2 in H9c2 cells; however, these effects were attenuated by APN. In addition, LPS upregulated Cx43 expression, and APN downregulated Cx43 expression and activated the PI3K/AKT signaling pathway. LPS induced apoptosis and inhibited PI3K/AKT signaling pathway in H9c2 cells, and these effects were attenuated by Gap26 (a Cx43 inhibitor). Moreover, the preservation of APN expression was reversed by LY294002 (a PI3K/AKT signaling pathway inhibitor). In vivo experiments: In C57BL/6J mice, a sepsis model was established by intraperitoneal injection of LPS, and APN was injected into enterocoelia. The protein levels of Bax, Bcl2, cleaved caspase-3, and Cx43 were evaluated by Western blot analysis, and immunohistochemistry was used to detect Cx43 expression and localization in myocardial tissue. LPS upregulated Bax and cleaved caspase-3 and downregulated Bcl2 in sepsis; however, these effects were attenuated by APN. In addition, the expression of Cx43 was upregulated in septic myocardial tissue, and APN downregulated Cx43 expression in septic myocardial tissue. In conclusion, both in vitro and in vivo , the data demonstrated that APN can protect against LPS-induced apoptosis during sepsis by modifying the Cx43 and PI3K/AKT signaling pathways.

Background In response to brain injury or inflammation, astrocytes undergo hypertrophy, proliferate, and migrate to the damaged zone. These changes, collectively known as \"astrogliosis\", initially protect the brain; however, astrogliosis can also cause neuronal dysfunction. Additionally, these astrocytes undergo intracellular changes involving alterations in the expression and localization of many proteins, including α v β 3 integrin. Our previous reports indicate that Thy-1, a neuronal glycoprotein, binds to this integrin inducing Connexin43 (Cx43) hemichannel (HC) opening, ATP release, and astrocyte migration. Despite such insight, important links and molecular events leading to astrogliosis remain to be defined. Methods Using bioinformatics approaches, we analyzed different Gene Expression Omnibus datasets to identify changes occurring in reactive astrocytes as compared to astrocytes from the normal mouse brain. In silico analysis was validated by both qRT-PCR and immunoblotting using reactive astrocyte cultures from the normal rat brain treated with TNF and from the brain of a hSOD1 G93A transgenic mouse model. We evaluated the phosphorylation of Cx43 serine residue 373 (S373) by AKT and ATP release as a functional assay for HC opening. In vivo experiments were also performed with an AKT inhibitor (AKTi). Results The bioinformatics analysis revealed that genes of the PI3K/AKT signaling pathway were among the most significantly altered in reactive astrocytes. mRNA and protein levels of PI3K, AKT, as well as Cx43, were elevated in reactive astrocytes from normal rats and from hSOD1 G93A transgenic mice, as compared to controls. In vitro, reactive astrocytes stimulated with Thy-1 responded by activating AKT, which phosphorylated S373Cx43. Increased pS373Cx43 augmented the release of ATP to the extracellular medium and AKTi inhibited these Thy-1-induced responses. Furthermore, in an in vivo model of inflammation (brain damage), AKTi decreased the levels of astrocyte reactivity markers and S373Cx43 phosphorylation. Conclusions Here, we identify changes in the PI3K/AKT molecular signaling network and show how they participate in astrogliosis by regulating the HC protein Cx43. Moreover, because HC opening and ATP release are important in astrocyte reactivity, the phosphorylation of Cx43 by AKT and the associated increase in ATP release identify a potential therapeutic window of opportunity to limit the adverse effects of astrogliosis.

[This corrects the article DOI: 10.3389/froh.2024.1496819.].

Hutchinson–Gilford progeria syndrome (HGPS) is a rare genetic disease caused by defective prelamin A processing, leading to nuclear lamina alterations, severe cardiovascular pathology, and premature death. Prelamin A alterations also occur in physiological aging. It remains unknown how defective prelamin A processing affects the cardiac rhythm. We show age-dependent cardiac repolarization abnormalities in HGPS patients that are also present in the Zmpste24 −/− mouse model of HGPS. Challenge of Zmpste24 −/− mice with the β-adrenergic agonist isoproterenol did not trigger ventricular arrhythmia but caused bradycardia-related premature ventricular complexes and slow-rate polymorphic ventricular rhythms during recovery. Patch-clamping in Zmpste24 −/− cardiomyocytes revealed prolonged calcium-transient duration and reduced sarcoplasmic reticulum calcium loading and release, consistent with the absence of isoproterenol-induced ventricular arrhythmia. Zmpste24 −/− progeroid mice also developed severe fibrosis-unrelated bradycardia and PQ interval and QRS complex prolongation. These conduction defects were accompanied by overt mislocalization of the gap junction protein connexin43 (Cx43). Remarkably, Cx43 mislocalization was also evident in autopsied left ventricle tissue from HGPS patients, suggesting intercellular connectivity alterations at late stages of the disease. The similarities between HGPS patients and progeroid mice reported here strongly suggest that defective cardiac repolarization and cardiomyocyte connectivity are important abnormalities in the HGPS pathogenesis that increase the risk of arrhythmia and premature death.

The skeleton adapts to mechanical loading to promote bone formation and remodeling. While most bone cells are involved in mechanosensing, it is well accepted that osteocytes are the principal mechanosensory cells. The osteocyte cell body and processes are surrounded by a fluid-filled space, forming an extensive lacuno-canalicular network. The flow of interstitial fluid is a major stress-related factor that transmits mechanical stimulation to bone cells. The long dendritic processes of osteocytes form a gap junction channel network connecting not only neighboring osteocytes, but also cells on the bone surface, such as osteoblasts and osteoclasts. Mechanosensitive osteocytes also form hemichannels that mediate the communication between the cytoplasmic and extracellular microenvironment. This paper will discuss recent research progress regarding connexin (Cx)-forming gap junctions and hemichannels in osteocytes, osteoblasts, and other bone cells, including those richly expressing Cx43. We will then cover the recent progress regarding the regulation of these channels by mechanical loading and the role of integrins and signals in mediating Cx43 channels, and bone cell function and viability. Finally, we will summarize the recent studies regarding bone responses to mechanical unloading in Cx43 transgenic mouse models. The osteocyte has been perceived as the center of bone remodeling, and connexin channels enriched in osteocytes are a likely major player in meditating the function of bone. Based on numerous studies, connexin channels may present as a potential new therapeutic target in the treatment of bone loss and osteoporosis. This review will primarily focus on Cx43, with some discussion in other connexins expressed in bone cells.