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Background: Pathogenic variants in Gap junction protein beta 1 (GJB1), which encodes Connexin 32, are known to cause X-linked Charcot-Marie-Tooth disease (CMTX), the second most common form of CMT. CMTX presents with the following five central nervous systems (CNS) phenotypes: subclinical electrophysiological abnormalities, mild fixed abnormalities on neurological examination and/or imaging, transient CNS dysfunction, cognitive impairment, and persistent CNS manifestations., Case Presentation: A 40-year-old Japanese male showed CNS symptoms, including nystagmus, prominent spastic paraplegia, and mild cerebellar ataxia, accompanied by subclinical peripheral neuropathy. Brain magnetic resonance imaging revealed hyperintensities in diffusion-weighted images of the white matter, particularly along the pyramidal tract, which had persisted since childhood. Nerve conduction assessment showed a mild decrease in motor conduction velocity, and auditory brainstem responses beyond wave II were absent. Peripheral and central conduction times in somatosensory evoked potentials elicited by stimulation of the median nerve were prolonged. Genetic analysis identified a hemizygous GJB1 variant, NM_000166.6:c.520C > T p.Pro174Ser., Conclusions: The patient in the case described here, with a GJB1 p.Pro174Ser variant, presented with a unique CNS-dominant phenotype, characterized by spastic paraplegia and persistent extensive leukoencephalopathy, rather than CMTX. Similar phenotypes have also been observed in patients with GJC2 and CLCN2 variants, likely because of the common function of these genes in regulating ion and water balance, which is essential for maintaining white matter function. CMTX should be considered within the spectrum of GJB1-related disorders, which can include patients with predominant CNS symptoms, some of which can potentially be classified as a new type of spastic paraplegia., (© 2024. The Author(s).)

Charcot-Marie-Tooth disease (CMT) is a genetically heterogeneous disorder. As more genes are identified and overlapping of neuropathy phenotypes, the traditional classification of CMT often proves inadequate. A young male with typical clinical features was suspected to have CMT, but family history about the inheritance pattern and distinct features suggestive of particular subtypes were lacking. He was directly evaluated by whole exome sequencing (WES). The libraries from extracted DNA were sequenced on Illumina sequencing platform. The variant detected in WES was confirmed by Sanger sequencing. WES shows a likely pathogenic hemizygous missense variation in exon 2 of the gap junction protein beta 1(GJB1) gene (chrX: 70444104; C > T; Depth: 66x) that results in the substitution of cysteine for arginine at codon 183 (p.Arg183Cys). This novel variation expands the spectrum of GJB1 mutations associated with X-linked CMT (CMTX) to establish a specific genetic cause., (Copyright © 2024 Copyright: © 2024 Neurology India, Neurological Society of India.)

Background and Aims: The genetic epidemiology of inherited neuropathies in children remains largely unknown. In this study, we specifically investigated the genetic profile of a Brazilian cohort of pediatric patients with pure or complex axonal neuropathies, a crucial knowledge in the near future for establishing treatment priorities and perspectives for this group of patients., Methods: Fifty-three pediatric patients who were assessed prior to reaching the age of 20, and who had clinical diagnoses of axonal hereditary neuropathy or presented with axonal neuropathy as the primary clinical feature, were included in the study. The recruitment of these cases took place from January 1, 2018, to December 31, 2020. The diagnosis was based on clinical and electrophysiological data. A molecular assessment was made using target-gene panel or whole-exome sequencing. Subsequently, segregation analysis was performed on available family members, and all candidate variants found were confirmed through Sanger., Results: A molecular diagnosis was reached in 68% of the patients (n = 36/53), considering only pathogenic and probably pathogenic variants. Variants in MFN2 (n = 15) and GJB1 (n = 3) accounted for half of the genetically confirmed patients (50%; n = 18/36). The other 18 genetically diagnosed patients had variants in several less common genes., Interpretation: Apart from MFN2 and GJB1 genes, universally recognized as a frequent cause of axonal neuropathies in most studied population, our Brazilian cohort of children with axonal neuropathies showed an important genetic heterogeneity, probably reflecting the multi ethnicity of the Brazilian population. Diagnostic, counseling, and future interventions should consider this characteristic., (© 2024 Peripheral Nerve Society.)

Background: X-linked Charcot-Marie-Tooth type 1 (CMT1X) disease is one of the most common forms of inherited neuropathy caused by mutations in the gap junction beta-1 protein (GJB1) gene (also known as connexin 32). This study presented the clinical and genetic features of a series of Chinese patients with GJB1 gene mutations. Methods: A total of 22 patients from unrelated families, who were referred to Department of Neurology, Peking University First Hospital from January 2005 to January 2016, were identified with GJB1 mutations. Their clinical records and laboratory findings were retrospectively collected and reviewed. Mutations in the GJB1 gene were analyzed by targeted next-generation sequencing (NGS). Nucleotide alternations were confirmed with Sanger sequencing. Results: The CMT1X patients predominantly showed distal muscle weakness of lower limbs with mild sensory disturbance. The mean age of onset was 15.6 ± 8.7 years (ranging from 1 year to 42 years). The sudden onset of cerebral symptoms appeared in four patients (18.2%); two were initial symptoms. One case had constant central nervous system (CNS) signs. There were 19 different heterozygous mutations, including 15 known mutations and four novel mutations (c.115G>T, c.380T>A, c.263C>A, and c.818_819insGGGCT). Among the 22 Chinese patients with CMT1X, the frequency of the GJB1 mutation was 4.5% in transmembrane domain 1 (TM1), 4.5% in TM2, 22.7% in TM3, 9.1% in TM4, 4.5% in extracellular 1 (EC1), 27.3% in EC2, 9.1% in intracellular loop, 13.6% in the N-terminal domain, and 4.5% in the C-terminal domain. CMT1X with CNS impairment appeared in five (22.7%) of these patients. Conclusions: This study indicated that CNS impairment was not rare in Chinese CMT1X patients. Mutations in the EC2 domain of the GJB1 gene were hotspot in Chinese CMT1X patients.

X-linked Charcot-Marie-Tooth disease type 1 (CMTX1), the most common form of CMTX, is caused by gap-junction beta 1 (GJB1) mutations. We herein report a 25-year-old Japanese man with disorientation, right hemiparesis, and dysarthria. Brain magnetic resonance imaging (MRI) showed high signal intensities in the bilateral cerebral white matter on diffusion-weighted imaging. He had experienced 2 episodes of transient central nervous system symptoms (at 7 and 13 years old). A genetic analysis identified a novel GJB1 mutation, c.169C>T, p.Gln57*. MRI abnormalities shifted from the cerebral white matter to the corpus callosum and had disappeared at the five-month follow-up. Transient changes between these lesions may indicate CMTX1.

Stable cell pools have the advantage of providing a definite, consistent, and reproducible transmission of a transgene of interest, compared to transient expression from a plasmid transfection. Stably expressing a transgene of interest in cells under induction is a powerful way to (switch on and) study a gene function in both in vitro and in vivo assays. Taking advantage of the ability of lentivirus (LV) to promote transgene delivery, and genomic integration and expression in both dividing and nondividing cells, a doxycycline-inducible transfer vector expressing a bicistronic transgene was developed to study the function of connexins in HeLa DH cells. Here, delving on connexin 32 (Cx32), we report how to use the backbone of this vector as a tool to generate stable pools to study the function of a gene of interest (GOI), especially with assays involving Ca 2+ imaging, employing the GCaMP6s indicator. We describe a step-by-step protocol to produce the LV particle by transient transfection and the direct use of the harvested LV stock to generate stable cell pools. We further present step-by-step immunolabeling protocols to characterize the transgene protein expression by confocal microscopy using an antibody that targets an extracellular domain epitope of Cx32 in living cells, and in fixed permeabilized cells using high affinity anti-Cx32 antibodies. Using common molecular biology laboratory techniques, this protocol can be adapted to generate stable pools expressing any transgene of interest, for both in vitro and in vivo functional assays, including molecular, immune, and optical assays., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Charcot-Marie-Tooth disease (CMT) due to GJB1 variants (CMTX1) is the second most common form of CMT. It is an X-linked disorder characterized by progressive sensory and motor neuropathy with males affected more severely than females. Many reported GJB1 variants remain classified as variants of uncertain significance (VUS). In this large, international, multicentre study we prospectively collected demographic, clinical and genetic data on patients with CMT associated with GJB1 variants. Pathogenicity for each variant was defined using adapted American College of Medical Genetics criteria. Baseline and longitudinal analyses were conducted to study genotype-phenotype correlations, to calculate longitudinal change using the CMT Examination Score (CMTES), to compare males versus females, and pathogenic/likely pathogenic (P/LP) variants versus VUS. We present 387 patients from 295 families harbouring 154 variants in GJB1. Of these, 319 patients (82.4%) were deemed to have P/LP variants, 65 had VUS (16.8%) and three benign variants (0.8%; excluded from analysis); an increased proportion of patients with P/LP variants compared with using ClinVar's classification (74.6%). Male patients (166/319, 52.0%, P/LP only) were more severely affected at baseline. Baseline measures in patients with P/LP variants and VUS showed no significant differences, and regression analysis suggested the disease groups were near identical at baseline. Genotype-phenotype analysis suggested c.-17G>A produces the most severe phenotype of the five most common variants, and missense variants in the intracellular domain are less severe than other domains. Progression of disease was seen with increasing CMTES over time up to 8 years follow-up. Standard response mean (SRM), a measure of outcome responsiveness, peaked at 3 years with moderate responsiveness [change in CMTES (ΔCMTES) = 1.3 ± 2.6, P = 0.00016, SRM = 0.50]. Males and females progressed similarly up to 8 years, but baseline regression analysis suggested that over a longer period, females progress more slowly. Progression was most pronounced for mild phenotypes (CMTES = 0-7; 3-year ΔCMTES = 2.3 ± 2.5, P = 0.001, SRM = 0.90). Enhanced variant interpretation has yielded an increased proportion of GJB1 variants classified as P/LP and will aid future variant interpretation in this gene. Baseline and longitudinal analysis of this large cohort of CMTX1 patients describes the natural history of the disease including the rate of progression; CMTES showed moderate responsiveness for the whole group at 3 years and higher responsiveness for the mild group at 3, 4 and 5 years. These results have implications for patient selection for upcoming clinical trials., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Guarantors of Brain.)

In myelinating Schwann cells, connection between myelin layers is mediated by gap junction channels (GJCs) formed by docked connexin 32 (Cx32) hemichannels (HCs). Mutations in Cx32 cause the X-linked Charcot-Marie-Tooth disease (CMT1X), a degenerative neuropathy without a cure. A molecular link between Cx32 dysfunction and CMT1X pathogenesis is still missing. Here, we describe the high-resolution cryo-electron cryo-myography (cryo-EM) structures of the Cx32 GJC and HC, along with two CMT1X-linked mutants, W3S and R22G. While the structures of wild-type and mutant GJCs are virtually identical, the HCs show a major difference: In the W3S and R22G mutant HCs, the amino-terminal gating helix partially occludes the pore, consistent with a diminished HC activity. Our results suggest that HC dysfunction may be involved in the pathogenesis of CMT1X.

Aims: Renal oxidative stress (OSS) is the leading cause of diabetic nephropathy (DN). The silent information regulator 1/forkhead boxo3a (Sirt1/Foxo3a) pathway plays an essential role in regulating the antioxidant enzyme system. In this study, we aimed to investigate the mechanism of connexin32 (Cx32) on the antioxidant enzyme system in DN. Results: In this study, Cx32 overexpression significantly reduced reactive oxygen species generation and effectively inhibited the excessive production of extracellular matrix such as fibronectin (FN) and intercellular adhesion molecule-1 (ICAM-1) in high-glucose (HG)-induced glomerular mesangial cells. In addition, Cx32 overexpression reversed the downregulation of Sirt1, and promoted the nuclear transcription of Foxo3a, subsequently activating the antioxidant enzymes including catalase and manganese superoxide dismutase (MnSOD), however, Cx32 knockdown showed the opposite effects. A further mechanism study showed that Cx32 promoted the autoubiquitination and degradation of Smad ubiquitylation regulatory factor-1 (Smurf1), thereby reducing the ubiquitination of Sirt1 at Lys 335 and the degradation of Sirt1. Moreover, the in vivo results showed that adenovirus-mediated Cx32 overexpression activated the Sirt1/Foxo3a pathway, and inhibited OSS in the kidney tissues, eventually improving the renal function and glomerulosclerosis in diabetic mice. Innovation: This study highlighted the antioxidant role of Cx32-Sirt1-Foxo3a axis to alleviate DN, which is a new mechanism of Cx32 alleviating DN. Conclusion: Cx32 alleviated DN via activating the Sirt1/Foxo3a antioxidant pathway. The specific mechanism was that Cx32 upregulated the Sirt1 expression through reducing the ubiquitination of Lys 335 of Sirt1 by inhibiting Smurf1. Antioxid. Redox Signal. 39, 241-261.

Glioblastoma (GBM) is the most lethal malignant primary brain tumor. Although multimodal therapy has been applied for GBM, the median survival time remains less than 16 months. Thus, better therapeutic targets in GBM are urgently needed. Herein, we first identified five new N-terminal-truncated Cx32 isoforms (GJB1-28k, GJB1-22k, GJB1-20k, GJB1-15k, and GJB1-13k) and further demonstrated that they were generated via cap-independent internal translation through internal ribosome entry sites (IRESs) in the coding sequence of GJB1 mRNA. Among these isoforms, GJB1-13k inhibited proliferation, promoted apoptosis, and limited cell cycle progression in GBM cells by inhibiting global mRNA translation. In vivo experiments further confirmed the antitumor activity of GJB1-13k against GBM cells. In addition, TSR3, a ribosomal maturation factor, was demonstrated to directly interact with GJB1-13k. Moreover, GBM cells with high TSR3 expression exhibited low sensitivity to GJB1-13k treatment, while GJB1-13k sensitivity was restored by TSR3 knockdown. Our work identifies a new IRES-mediated protein, GJB1-13k, and suggests that overexpression of GJB1-13k in GBM cells with low TSR3 expression or combined targeting of GJB1-13k and TSR3 in GBM cells with high TSR3 expression constitutes a potential therapeutic strategy for GBM., Competing Interests: Conflict of interest statement The authors declare that they have no conflicts of interest., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Background: Charcot-Marie-Tooth (CMT) disease is the most frequent hereditary motor sensory neurological disease. GJB1 gene is the second most frequent cause of CMT, accounting for approximately 10% of CMT cases worldwide. We identified a large Han family with X-linked CMT disease., Methods: In this study, the probands and his mother underwent electrophysiological examinations and other family members were assessed retrospectively. Whole-exome sequencing, Sanger sequencing, and SNP array linkage analysis were performed to find and confirm the variant. The functional effect of the identified variant was further investigated in HEK293 cells and MCF-7 cells by minigene splicing assay., Results: The affected individuals had some clinical symptoms including symmetric atrophy and progressive weakness of the distal muscles in their twenties. Electrophysiological examinations result in peripheral nerve injury of the upper and lower limbs. Whole-exome sequencing identified a novel hemizygous deletion mutation (NM_000166: c.-16-8_-14del) in the GJB1 gene. SNP array linkage analysis and co-segregation analysis confirmed this mutation. Minigene splicing assay verified that this mutation leads to the activation of cryptic splicing sites in exon 2 which results in the deletion of exon 2., Conclusion: Our study provides theoretical guidance for prenatal diagnosis and subsequent fertility of this family. This result expands the spectrum of mutations in GJB1 known to be associated with CMTX and contributes to the diagnosis of CMT and clinical genetic counseling., (© 2022 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)

The X-linked form of Charcot-Marie-Tooth disease (CMTX1) is the second most common form of CMT. In this study we used CRISPR/Cas9 to develop new "knock-in" models of CMTX1 that are more representative of the spectrum of mutations seen with CMTX1 than the Cx32 knockout (KO) mouse model used previously. We compared mice of four genotypes - wild-type, Cx32KO, p.T55I, and p.R75W. Sciatic motor conduction velocity slowing was the most robust electrophysiologic indicator of neuropathy, showing reductions in the Cx32KO by 3 months and in the p.T55I and p.R75W mice by 6 months. At both 6 and 12 months, all three mutant genotypes showed reduced four limb and hind limb grip strength compared to WT mice. Performance on 6 and 12 mm width balance beams revealed deficits that were most pronounced at on the 6 mm balance beam at 6 months of age. There were pathological changes of myelinated axons in the femoral motor nerve in all three mutant lines by 3 months of age, and these became more pronounced at 6 and 12 months of age; sensory nerves (femoral sensory and the caudal nerve of the tail) appeared normal at all ages examined. Our results demonstrate that mice can be used to show the pathogenicity of human GJB1 mutations, and these new models for CMTX1 should facilitate the preclinical work for developing treatments for CMTX1., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Rationale: Charcot-Marie-Tooth disease (CMT) is a highly heterogeneous genetic disorder. To date, more than 90 genes have been implicated in the pathogenesis of CMT. Here, we report the identification of a rare causative mutation in a Chinese family with CMT and a pregnant patient underwent prenatal diagnosis., Patient Concerns: A 33-year-old woman with 21 + 6 weeks of pregnancy presented with progressive weakness of distal extremities after 23 years of age. A total of 8 individuals in 4 generations of her family had similar muscle weakness. On proband whole-exome sequencing (WES), a rare c.121G > A variant in the GJB1 gene was identified., Diagnosis: Based on the clinical and genetic findings, this patient was finally diagnosed with CMT., Interventions: The prenatal diagnosis was performed on the proband fetus., Outcomes: The fetus did not carry this rare variant, and the pregnancy continued., Lessons: Our findings provide the first clinical evidence for the causative role of GJB1 c.121G > A variant in CMT. WES is a valuable method for diagnosing patients with CMT., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2022 the Author(s). Published by Wolters Kluwer Health, Inc.)

Liver cancer stem cells (LCSCs) are responsible for liver cancer recurrence, metastasis, and drug resistance. Previous studies by the authors demonstrated that upregulated expression of connexin 32 (Cx32) reversed doxorubicin resistance and reduced invasion and metastasis of liver cancer cells. However, the role of Cx32 in expansion of LCSCs remains unclear. A total of 85 patients were enrolled in the present study and followed‑up for 5 years. The expression of Cx32 in hepatocellular carcinoma (HCC) tissues and corresponding paracancerous tissues were detected by immunohistochemistry (IHC). Cx32 was silenced in HepG2 cells and overexpressed in HCCLM3 cells and the stemness of liver cells was examined by detecting the expression of LCSC markers (EpCAM, CD133, Nanog, Oct4, Sox9, c‑Myc), sphere formation, and xenograft tumorigenesis. Finally, the effect of the phosphoinositide 3‑kinase (PI3K)/protein kinase B (Akt) pathway on Cx32‑regulated LCSC expansion was investigated. Cx32 was downregulated in LCSCs and HCC tissues, and predicted poor prognosis in patients with HCC. Overexpression of Cx32 in HCCLM3 cells significantly inhibited LCSC expansion, tumorigenesis, and phosphoinositide 3‑kinase/protein kinase B (PI3K/Akt) pathway activity. By contrast, silencing of Cx32 in HepG2 cells upregulated expansion of LCSCs and PI3K/Akt pathway activity. Modulating the activity of the PI3K/Akt pathway by SC‑79 and LY294002 in HepG2 and HCCLM3 cells, respectively, confirmed that Cx32 could affect the expansion of LCSCs through PI3K/Akt signaling. In conclusion, the present study demonstrated that Cx32 regulated the expansion of LCSCs, and increased expression of Cx32 significantly inhibited the expansion of LCSCs, suggesting that Cx32 may be an optimal target for intervention of HCC.

Renal tubulointerstitial fibrosis (RIF), characterized by epithelial-to-mesenchymal transition (EMT) of renal tubular epithelial cells (TECs), is the main cause of diabetic renal fibrosis. Oxidative stress plays a pivotal role in the development of diabetic RIF. Connexin32 (Cx32), prominently expressed in renal TECs, has emerged as an important player in the regulation of oxidative stress. However, the role of Cx32 in diabetic RIF has not been explored yet. Here, we showed that adenovirus-mediated Cx32 overexpression suppressed EMT to ameliorate RIF and renal function in STZ-induced diabetic mice, while knockout (KO) of Cx32 exacerbated RIF in diabetic mice. Moreover, overexpression of Cx32 inhibited EMT and the production of extra cellular matrix (ECM) in high glucose (HG) induced NRK-52E cells, whereas knockdown of Cx32 showed the opposite effects. Furthermore, we showed that NOX4, the main source of ROS in renal tubular, was down-regulated by Cx32. Mechanistically, Cx32 down-regulated the expression of PKC alpha in a carboxyl-terminal-dependent manner, thereby inhibiting the phosphorylation at Thr 147 of p22phox triggered by PKC alpha, which ultimately repressed the formation of the p22phox-NOX4 complex to reduce the protein level of NOX4. Thus, we establish Cx32 as a novel target and confirm the protection mechanism in RIF., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Berbamine is a bisbenzylisoquinoline alkaloid extracted from Berberis poiretii of Berberis of Berberidaceae. It has been reported that it can significantly inhibit the proliferation of a variety of malignant tumor cells, including liver cancer. However, the effect of berbamine on the invasion and metastasis of liver cancer has not been reported. The present study demonstrated that berbamine inhibited the migration and invasion of SMMC-7721 cells in a concentration-dependent manner and obviously increased the gap junction function and the expression of Cx32 in SMMC-7721 cells compared with control group. However, after silencing Cx32, berbamine had no significant effect on cell invasion and metastasis. Before silencing Cx32, the expression of PI3K and P-AKT were decreased after berbamine treated on SMMC-7721 cells for 24 h. After silencing Cx32, the expression of PI3K and P-AKT were increased in SMMC-7721 cells. The expression of PI3K and P-AKT had no significant effect after berbamine treated on SMMC-7721 cells for 24 h with silencing Cx32. In conclusion, the results of the present study suggest that berbamine could inhibit the SMMC-7721 cell migration and invasion, and its mechanism may be related to the regulation of PI3K/AKT signaling pathway by enhancing the expression of Cx32., (Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc.)

The roles of gap junctions (GJs) and its components, connexins, in the autophagy of cervical cancer cells have been rarely investigated. Our previous study demonstrated that connexin 32 (Cx32) exerted an anti‑apoptotic effect on cervical cancer. However, as an important regulator of apoptosis, whether the autophagy is involved in the function of Cx32 on cervical cancer cells is not well defined. The present study aimed to investigate the role of Cx32 on autophagy and apoptosis inhibition in cervical cancer cells. The expression levels of Cx32 and the autophagy‑associated protein LC3‑Ⅱ in paracancerous cervical tissues (n=30) and cervical cancer (n=50) tissues were determined via western blotting. In total, 45 cervical cancer specimens were used to evaluate the clinical relevance of Cx32 and LC3‑Ⅱ. It was found that both Cx32 and LC3‑Ⅱ were upregulated in cervical cancer tissues compared with those in paracancerous cervical tissues. The effect of Cx32 on autophagy was examined by detecting the change of LC3‑Ⅱ using western blotting, transfection with enhanced green fluorescent protein‑LC3 plasmid and transmission electron microscopy analysis. Overexpression of Cx32 significantly enhanced autophagy in HeLa‑Cx32 cells, whereas knockdown of Cx32 suppressed autophagy in C‑33A cells. The flow cytometry results demonstrated that Cx32 inhibited the apoptosis of cervical cancer cells by promoting autophagy. Moreover, Cx32 triggered autophagy via the activation of the AMP‑activated protein kinase (AMPK) signalling, regardless of the presence or absence of GJs. Collectively, it was identified that Cx32 exerted its anti‑apoptotic effect by activating autophagy via the AMPK pathway in cervical cancer, which demonstrates a novel mechanism for Cx32 in human cervical cancer progression.

Non-alcoholic steatohepatitis (NASH) can cause liver cirrhosis and hepatocellular carcinoma (HCC), with cases increasing worldwide. To reduce the incidence of liver cirrhosis and HCC, NASH is targeted for the development of treatments, along with viral hepatitis and alcoholic hepatitis. Lactoferrin (LF) has antioxidant, anti-cancer, and anti-inflammatory activities. However, whether LF affects NASH and fibrosis remains unelucidated. We aimed to clarify the chemopreventive effect of LF on NASH progression. We used a NASH model with metabolic syndrome established using connexin 32 (Cx32) dominant negative transgenic (Cx32ΔTg) rats. Cx32ΔTg rats (7 weeks old) were fed a high-fat diet and intraperitoneally injected with dimethylnitrosamine (DMN). Rats were divided into three groups for LF treatment at 0, 100, or 500 mg/kg/day for 17 weeks. Lactoferrin significantly protected steatosis and lobular inflammation in Cx32ΔTg rat livers and attenuated bridging fibrosis or liver cirrhosis induced by DMN. By quantitative RT-PCR, LF significantly down-regulated inflammatory ( Tnf-α , Il-6 , Il-18 , and Il-1β ) and fibrosis-related ( Tgf-β1 , Timp2 , and Col1a1 ) cytokine mRNAs. Phosphorylated nuclear factor (NF)-κB protein decreased in response to LF, while phosphorylated JNK protein was unaffected. These results indicate that LF might act as a chemopreventive agent to prevent hepatic injury, inflammation, and fibrosis in NASH via NF-κB inactivation.

Gap junction beta 1 (GJB1) is the pathogenic gene of X-linked Charcot-Marie-Tooth type 1 (CMTX1), a rare hereditary sensorimotor neuropathy. However, different mutations of GJB1 result in heterogeneous clinical manifestations with only some mutations leading to central nervous system involvement. We previously reported two GJB1 missense mutations: one novel mutation (c.212T > G) found in a CMTX1 family that only manifested as peripheral neuropathy, and another previously reported mutation GJB1(c.311A > C) leading to involvement of the peripheral nerves and cerebral white matter. However, the mechanism by which GJB1 mutations lead to CMTX1 has not been fully characterized. Here, we generated Schwann cells and primary cultured oligodendrocytes with these two mutations, resulting in the Cx32 I71S (GJB1 c.212T > G) and Cx32 K104T (GJB1 c.311A > C) mutants, to analyze the pathogenic mechanism using cytology, molecular biology, and electrophysiological methods. Both mutants showed abnormal endoplasmic reticulum aggregation, especially the Cx32 K104T mutant, leading to an increase in endoplasmic reticulum stress, resulting in apoptosis. Furthermore, whole-cell patch clamp experiments in oligodendrocytes revealed that the Cx32 K104T mutant reduced the cell membrane potential and inwardly rectifying potassium currents, which may be a vital element for central involvement. Therefore, our results may provide a new perspective for understanding the pathogenesis of CMTX1., Competing Interests: Declaration of competing interest There is no conflict of interest in this manuscript., (Copyright © 2021 Elsevier Inc. All rights reserved.)

To optimize gene delivery to myelinating Schwann cells we compared clinically relevant AAV serotypes and injection routes. AAV9 and AAVrh10 vectors expressing either EGFP or the neuropathy-associated gene GJB1/Connexin32 (Cx32) under a myelin specific promoter were injected intrathecally or intravenously in wild type and Gjb1-null mice, respectively. Vector biodistribution in lumbar roots and sciatic nerves was higher in AAVrh10 injected mice while EGFP and Cx32 expression rates and levels were similar between the two serotypes. A gradient of biodistribution away from the injection site was seen with both intrathecal and intravenous delivery, while similar expression rates were achieved despite higher vector amounts injected intravenously. Quantified immune cells in relevant tissues were similar to non-injected littermates. Overall, AAV9 and AAVrh10 efficiently transduce Schwann cells throughout the peripheral nervous system with both clinically relevant routes of administration, although AAV9 and intrathecal injection may offer a more efficient approach for treating demyelinating neuropathies., (© 2021. The Author(s).)

Liver cancer cell lines are frequently used in vitro tools to test candidate anti-cancer agents as well as to elucidate mechanisms of liver carcinogenesis. Among such mechanisms is cellular communication mediated by connexin-based gap junctions. The present study investigated changes in connexin expression and gap junction functionality in liver cancer in vitro. For this purpose, seven human liver cancer cell lines, as well as primary human hepatocytes, were subjected to connexin and gap junction analysis at the transcriptional, translational and activity level. Real-time quantitative reverse transcription polymerase chain reaction analysis showed enhanced expression of connexin43 in the majority of liver cancer cell lines at the expense of connexin32 and connexin26. Some of these changes were paralleled at the protein level, as evidenced by immunoblot analysis and in situ immunocytochemistry. Gap junctional intercellular communication, assessed by the scrape loading/dye transfer assay, was generally low in all liver cancer cell lines. Collectively, these results provide a full scenario of modifications in hepatocyte connexin production and gap junction activity in cultured liver cancer cell lines. The findings may be valuable for the selection of neoplastic hepatocytes for future mechanistic investigation and testing of anti-cancer drugs that target connexins and their channels.

Connexin-based channels play key roles in cellular communication and can be affected by deleterious chemicals. In this study, the effects of various genotoxic carcinogenic compounds, non-genotoxic carcinogenic compounds and non-carcinogenic compounds on the expression and functionality of connexin-based channels, both gap junctions and connexin hemichannels, were investigated in human hepatoma HepaRG cell cultures. Expression of connexin26, connexin32, and connexin43 was evaluated by means of real-time reverse transcription quantitative polymerase chain reaction analysis, immunoblot analysis and in situ immunostaining. Gap junction functionality was assessed via a scrape loading/dye transfer assay. Opening of connexin hemichannels was monitored by measuring extracellular release of adenosine triphosphate. It was found that both genotoxic and non-genotoxic carcinogenic compounds negatively affect connexin32 expression. However, no specific effects related to chemical type were observed at gap junction or connexin hemichannel functionality level.

The pathogenesis of cerebral ischemia-reperfusion (I/R) is complex. Cx32 expression has been reported to be up-regulated in ischemic lesions of aged human brain. Nevertheless, the function of Cx32 during cerebral I/R is poorly understood. Autophagy is of vital importance in the pathogenesis of cerebral I/R. In the current study, we found that oxygen-glucose deprivation/reoxygenation (OGD/R) or I/R insult significantly induced the up-regulation of Cx32 and activation of autophagy. Inhibition of Cx32 alleviated OGD/R or I/R injury, and further activated autophagy. In addition, Nur77 expression was found to be up-regulated after OGD/R or I/R. After inhibiting Cx32, the expression of Nur77 was further increased and Nur77 was translocated from nucleus to mitochondrial. Inhibition of Cx32 also activated mitophagy by promoting autophagosome formation and up-regulating the expression of mitochondrial autophagy marker molecules. Of note, in the siNur77-transfected cells, the number of dysfunctional mitochondrial was increased, and mitophagy was suppressed, which aggravated OGD/R-induced neuronal injury. In conclusion, Cx32 might act as a regulatory factor of Nur77 controlling neuronal autophagy in the brains. Understanding the mechanism of this regulatory pathway will provide new insight into the role Cx32 and Nur77 in cerebral ischemia, offering new opportunities for therapeutics.

Gap junctions (GJs) are formed by the assembly of constituent transmembrane proteins called connexins (Cxs). Aberrations in this assembly of Cxs are observed in several genetic diseases as well as in cancers. Hence it becomes imperative to understand the molecular mechanisms underlying such assembly defect. The polarized cells in the epithelia express Connexin32 (Cx32). The C-terminal tail (CT) of Cx32 orchestrates several aspects of GJ dynamics, function and growth. The study here was aimed at determining if post-translational modifications, specifically, palmitoylation of cysteine residues, present in the CT of Cx32, has any effect on GJ assembly. The CT of Cx32 was found to harbor three cysteine residues, which are likely to be modified by palmitoylation. The study here has revealed for the first time that Cx32 is palmitoylated at cysteine 217 (C217) in cell line derived from prostate tumors. However, it was found that mutating C217 to alanine affected neither the trafficking nor the ability of Cx32 to assemble into GJs. Intriguingly, it was discovered that mutating cysteine 280 and 283, only in combination, blocked the trafficking of Cx32 from the trans-Golgi network to the cell surface. The mutants showed reduced stability due to enhanced lysosomal degradation. Overall, the findings reveal the importance of the two C-terminal cysteine residues of Cx32 in regulating its trafficking and stability and hence its ability to assemble into GJs., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Necroptosis is an alternative form of programmed cell death that generally occurs under apoptosis-deficient conditions. Our previous work showed that connexin32 (Cx32) promotes the malignant progress of hepatocellular carcinoma (HCC) by enhancing the ability of resisting apoptosis in vivo and in vitro. Whether triggering necroptosis is a promising strategy to eliminate the apoptosis-resistant HCC cells with high Cx32 expression remains unknown. In this study, we found that Cx32 expression was positively correlated with the expression of necroptosis protein biomarkers in human HCC specimens, cell lines, and a xenograft model. Treatment with shikonin, a well-used necroptosis inducer, markedly caused necroptosis in HCC cells. Interestingly, overexpressed Cx32 exacerbated shikonin-induced necroptosis, but downregulation of Cx32 alleviated necroptosis in vitro and in vivo. Mechanistically, Cx32 was found to bind to Src and promote Src-mediated caspase 8 phosphorylation and inactivation, which ultimately reduced the activated caspase 8-mediated proteolysis of receptor-interacting serine-threonine protein kinase 1/3, the key molecule for necroptosis activation. In conclusion, we showed that Cx32 contributed to the activation of necroptosis in HCC cells through binding to Src and then mediating the inactivation of caspase 8. The present study suggested that necroptosis inducers could be more favorable than apoptosis inducers to eliminate HCC cells with high expression of Cx32., (© 2021 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Background: Connexins (Cxs) are gap junction proteins involved in the communication between acute myeloid leukemia (AML) and stromal cells. They consist of intercellular channels termed "connexions", which can cause uncontrolled cell proliferation if dysregulated. This study aimed to evaluate the expression levels of the Cx32 and Cx43 genes and their correlations with other prognostic markers in patients with AML., Methods: This cross sectional study was performed on peripheral blood samples from 60 newly diagnosed patients with AML and 40 healthy control subjects at Kasr Alainy School of Medicine, Cairo University, from June 2016 to December 2017. The quantitative real-time polymerase chain reaction (qRT-PCR) test was used to examine the relative expression level of Cx43 and Cx32 genes in the patients and the control subjects. The Chi square test or the Fisher exact test was employed to examine the relationship between qualitative variables, while the independent t test or the Mann-Whitney test was employed for quantitative data. All the tests were two-tailed, and a P value of less than 0.05 was considered significant., Results: Among the patients with AML, 65% had a high Cx32 expression level, whereas 63.3% had a low Cx43 expression level. There was a statistically significant difference in the fold change values of Cx32 and Cx43 expression between the patient group and the control group (P=0.009 vs P=0.013, respectively). There was a remarkable association between both Cxs and CD34 and HLA-DR cells., Conclusion: Cx expression in samples may add to the diagnostic workup of AML. Although we found a negative correlation between Cx43 expression and the peripheral blood blast percentage, the response after the first induction of chemotherapy showed no significant relationship with Cx43 and Cx32 ., (Copyright: © Iranian Journal of Medical Sciences.)

Oligodendrocytes express two gap junction forming connexins, connexin 32 (Cx32) and Cx47; therefore, formation of heteromeric channels containing both Cx47 and Cx32 monomers might occur. Mutations in Cx47 cause both Pelizaeus-Merzbacher-like disease Type 1 (PMLD1) and hereditary spastic paraparesis Type 44 (SPG44) and heteromer formation between these mutants and Cx32 may contribute to the pathogenesis of these disorders. Here, we utilized electrophysiological and antibody-based techniques to examine this possibility. When cells expressing both Cx32 and Cx47 were paired with cells expressing either Cx32 or Cx47, properties were indistinguishable from those produced by cells expressing homotypic Cx32 or Cx47 channels. Similarly, pairing cells expressing both Cx32 and Cx47 with cells expressing Cx30 or Cx43 produced channels indistinguishable from heterotypic Cx32/Cx30 or Cx47/Cx43 channels, respectively. The same assessments were performed on cells expressing Cx32 and four mutant forms of Cx47 (p.I33M associated with SPG44 or p.P87S, p.Y269D or p.M283T associated with PMLD1). None of these mutants showed a functional effect on Cx32. Immunostained cells co-expressing Cx32WT (wild type) and Cx47WT showed a Pearson correlation coefficient close to zero, suggesting that any overlap was due to chance. p.Y269D showed a statistically significant negative correlation with Cx32, suggesting that Cx32 and this mutant overlap less than expected by chance. Co-immunoprecipitation of Cx32 with Cx47WT and mutants show only very low levels of co-immunoprecipitated protein. Overall, our data suggest that interactions between PMLD1 or SPG44 mutants and Cx32 gap junctions do not contribute to the pathogenesis of these disorders., (© 2021 Wiley Periodicals LLC.)

The long-term sequelae of nerve injury as well as age-related neurodegeneration have been documented in numerous studies, however the role of Cx32 in these processes is not well understood. There is a need for better understanding of the molecular mechanisms that underlie long-term suboptimal nerve function and for approaches to prevent or improve it. In this communication we describe our studies using whole animal electrophysiology to examine the long-term sequelae of sciatic nerve crush in both WT and Cx32KO mice, a model of X-linked Charcot Marie Tooth disease, a subtype of inherited peripheral neuropathies. We present results from electrical nerve recordings done 14 to 27 days and 18 to 20 months after a unilateral sciatic nerve crush performed on 35 to 37-day old mice. Contrary to expectations, we find that whereas crush injury leads to a degradation of WT nerve function relative to uninjured nerves at 18 to 20 months, previously crushed Cx32KO nerves perform at the same level as their uninjured counterparts. Thus, 18 to 20 months after injury, WT nerves perform below the level of normal (uninjured) WT nerves in both motor and sensory nerve function. In contrast, measures of nerve function in Cx32KO mice are degraded for sensory axons but exhibit no additional dysfunction in motor axons. Early nerve injury has no negative electrophysiologic effect on the Cx32 KO motor nerves. Based on our prior demonstration that the transcriptomic profile of uninjured Cx32KO and injured WT sciatic nerves are very similar, the lack of an additional effect of crush on Cx32KO motor nerve parameters suggests that Cx32 knockout may implement a form of neuroprotection that limits the effects of subsequent injury., (© 2021 Peripheral Nerve Society.)

Long non-coding RNA (lncRNA) plays a crucial role in multiple disorders, while the role of it in Parkinson's disease (PD) is still unclear. Here, the increased lncRNA NEAT1 was discovered in MPP + -induced SH-SY5Y cells. Then, we proved that NEAT1 decreasing suppressed MPP + -induced neuronal apoptosis, upregulation of α-syn and activation of NLRP3 inflammasome. Rescue experiments shown that the inhibition of NEAT1 decreasing to MPP + -induced activation of NLRP3 inflammasome and subsequent neuronal apoptosis can be reversed by overexpressed α-syn. Subsequently, we indicated the interaction between NEAT1 and miR-1301-3p, as well as between NEAT1 and miR-5047. Interesting, we found that NEAT1 decreasing repressed the expression of GJB1, a downstream target of miR-1301-3p and miR-5047, through promoting miR-1301-3p rather than miR-5047 expression. Finally, we transfected miR-1301-3p inhibitor to MPP + -induced SH-SY5Y cells following si-NEAT1, and found that downregulation of NEAT1 repressed α-syn-mediated the activation of NLRP3 inflammasome through regulating miR-1301-3p/GJB1 signaling pathway. Overall, our data demonstrated that NEAT1 decreasing effectively suppressed MPP + -induced neuronal apoptosis. Mechanismly, downregulation of NEAT1 repressed α-syn-induced activation of NLRP3 inflammasome via inhibiting the expression of GJB1 by targeting miR-1301-3p. Our study supported a new and reliable evidence for lncRNA NEAT1 as a potential target for PD treatment.

Adipose-derived stem cells (ASCs) represent a valuable tool for regenerative medicine being able to differentiate toward several cell lines, such as adipocytes, chondrocytes and osteocytes. During ASC adipogenic differentiation, changes in connexin (Cx) expression were evaluated in the present study. Three different Cxs were investigated: Cx43, Cx32 and Cx31.9. Cx43 is the most abundant in human tissues, Cx32 is prevalently found in nervous tissue and Cx31.9 is found at the myocardial level. Human ASCs undergoing adipogenic differentiation were isolated from raw lipoaspirate and characterized as mesenchymal stem cells. After multiple days of culture (1, 7, 14, 21 and 28 days), adipogenic differentiation was assessed by Oil Red O staining and Acetyl-CoA carboxylase (ACC) levels by western blotting. Cx expression was evaluated by western blotting at the same time points. In treated ASCs, lipidic vacuoles were detected from day 7 of treatment. Their number and size progressively increased over the entire period of observation. A parallel increase of ACC expression was also found. Lower levels of Cx expression were detected during adipogenic differentiation. Such decreases were particularly evident for Cx32, already after the first day of treatment. Cx31.9 and Cx43 also decreased, but starting from day 7. Our results suggest that ASCs may initially be equipped with a variety of Cxs, which is not surprising assuming their multipotential differentiation ability. Although some Cxs may be selectively enhanced depending on specific induction strategies toward different tissues, they seem markedly downregulated during adipogenic differentiation.

Connexins (Cx), the basic subunit of gap junctions, play important roles in cell homeostasis, and their abnormal expression and function are associated with human hereditary diseases and cancers. In tumorigenesis, connexins were observed to have both anti-tumorigenic and pro-tumorigenic roles in a context- and stage-dependent manner. Initially, Cx26 and Cx43 were thought to be the only connexins involved in normal breast homeostasis and breast cancer. Later on, association of Cx32 expression with lymph node metastasis of breast cancer and subsequent demonstration of its expression in normal breast tissue suggested that Cx32 contributes to breast tissue homeostasis. Here, we aimed to determine the effects of Cx32 on normal breast cells, MCF10A, and on breast cancer cells, MDA-MB-231. Cx32 overexpression had profound effects on MCF10A cells, decreasing cell proliferation by increasing the doubling time of MCF10A. Furthermore, MCF10A cells acquired mesenchymal-like appearance upon Cx32 expression and had increased migration capacity and expression of both E-cadherin and vimentin. In contrast, Cx32 overexpression altered the EMT markers of MDA-MB-231 by increasing the expression of mesenchymal markers, such as slug and vimentin, and decreasing E-cadherin expression without affecting their proliferation and morphology. Our results indicate, for the first time in the literature, that Cx32 has tumor-promoting roles in MCF10A and MDA-MB-231 cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Connexins are membrane-spanning proteins that form membrane channels and hemichannels. They are involved in the cellular communication and in the maintenance of tissue homeostasis. Recent studies in humans and animals have demonstrated that the expression and distribution of Cx43, the most studied connexin, can change during aging. However, the research on the involvement of the other connexins in cardiac and hepatic aging is, at present, still very poor. Hence, the aim of this study is to evaluate the expression of Cx43 and Cx26 in the heart as well as Cx26 and Cx32 in the liver of a rat model that aged naturally, rather than prematurely because of genetic mutations or age-related diseases. The results obtained in the present study have demonstrated that these connexins decrease in rat cardiomyocytes and in rat hepatocytes as they age. This change was revealed only at protein level, as connexin-mRNAs remained unchanged during aging. Moreover, the aged rats showed an increase in body fat, whose subcutaneous layer tended to be higher. Finally, how these changes could represent signs of physiological adaptation in successful aging was discussed., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

We previously found that polydatin could attenuate renal oxidative stress in diabetic mice and improve renal fibrosis. Recent evidence shows that NADPH oxidase 4 (Nox4)-derived reactive oxygen species (ROS) contribute to inflammatory and fibrotic processes in diabetic kidneys. In this study we investigated whether polydatin attenuated renal fibrosis by regulating Nox4 in vitro and in vivo. In high glucose-treated rat glomerular mesangial cells, polydatin significantly decreased the protein levels of Nox4 by promoting its K48-linked polyubiquitination, thus inhibited the production of ROS, and eventually decreasing the expression of fibronectin (FN) and intercellular adhesion molecule-1 (ICAM-1), the main factors that exacerbate diabetic renal fibrosis. Overexpression of Nox4 abolished the inhibitory effects of polydatin on FN and ICAM-1 expression. In addition, the expression of Connexin32 (Cx32) was significantly decreased, which was restored by polydatin treatment. Cx32 interacted with Nox4 and reduced its protein levels. Knockdown of Cx32 abolished the inhibitory effects of polydatin on the expression of FN and ICAM-1. In the kidneys of streptozocin-induced diabetic mice, administration of polydatin (100 mg·kg -1 ·d -1 , ig, 6 days a week for 12 weeks) increased Cx32 expression and reduced Nox4 expression, decreased renal oxidative stress levels and the expression of fibrotic factors, eventually attenuating renal injury and fibrosis. In conclusion, polydatin promotes K48-linked polyubiquitination and degradation of Nox4 by restoring Cx32 expression, thereby decreasing renal oxidative stress levels and ultimately ameliorating the pathological progress of diabetic renal fibrosis. Thus, polydatin reduces renal oxidative stress levels and attenuates diabetic renal fibrosis through regulating the Cx32-Nox4 signaling pathway.

Non-alcoholic steatohepatitis (NASH) is a recognized risk factor for liver fibrosis and malignancies, and is associated with features of metabolic syndrome, such as obesity and insulin resistance (IR). We previously demonstrated that the disturbance of connexin 32 (Cx32), a gap junctional protein of hepatocytes, exacerbated NASH in Cx32 dominant-negative transgenic (Cx32ΔTg) rats fed methionine choline-deficient diet (MCDD). MCDD is well-established means of inducing NASH in rodents; however, the Cx32ΔTg-MCDD NASH model does not reproduce obesity and IR. In this study, we aimed to establish an improved NASH model. Eight-week-old male Cx32ΔTg and wild-type (Wt) rats received a high-fat diet (HFD) with dimethylnitrosamine (DMN) for 12 weeks. The HFD with DMN led to gains in body, liver, and visceral fat weights in both genotypes. IR was significantly greater in Cx32ΔTg than in Wt rats. Elevation of serum hepatic enzymes (AST, ALT), inflammatory cytokine expressions (Tnfα, Il-6, Tgf-β1, Il-1β, Timp2, and Col1a1), steatohepatitis, and fibrosis were significantly greater in Cx32ΔTg as compared with Wt rats. Regarding carcinogenesis, the number and area of glutathione S-transferase placental form (GST-P)-positive preneoplastic hepatic foci were significantly increased in Cx32ΔTg versus Wt rats. Moreover, activation of NF-κB and JNK contributed to the progression of NASH in Cx32ΔTg rats. These results suggest that Cx32 dysfunction promoted the progression of NASH, metabolic syndrome, and carcinogenesis. Therefore, the novel Cx32ΔTg-HFD-DMN NASH model may be a rapid and useful tool for evaluating the progression of NASH.

Extracellular ATP (eATP) is a potent singling molecule in activation of fish innate immunity while the molecular determinants for eATP release in fish were not completely understood. Connexin32 (Cx32) is a member of gap junction protein family that plays important immunological functions in mammals. However, the immune relevance of Cx32 and its role in ATP release in fish has not been investigated. Here, we identified, characterized three Cx32 isoform genes (Cx32.2, Cx32.2x and Cx32.7) from the Japanese flounder Paralichthys olivaceus, and investigated their role in inflammation-induced ATP release in fish. Expression analysis revealed that even though all the three Cx32 genes are constitutively expressed in all examined Japanese flounder tissues, Cx32.2 and Cx32.2x are dominantly expressed in liver, and Cx32.7 is highly expressed in intestine and head kidney macrophages. In addition, we showed that gene expression of all the three Cx32 isoforms was modulated by cAMP stimulation and inflammatory challenges. Furthermore, we revealed that Cx32 expression was upregulated in TNF-alpha overexpressed Japanese flounder FG-9307 cells. Moreover, overexpression of the three Cx32 isoforms significantly reduced the gene expression level of LPS-induced pro-inflammatory cytokine IL-8 and TNF-alpha, indicating that Cx32 is involved in modulating inflammatory response in fish. Finally, we showed that inflammation-induced ATP release was significantly increased in Cx32-overexpressed Japanese flounder FG-9307 cells, and this increased ATP release could be attenuated by pre-incubation with gap junction protein blocker carbenoxolone. Taken together, we for the first time reported the involvement of Cx32 in fish immunity. Our findings suggested that in addition to Cx43 and pannexin1 channels, Cx32 also plays a role in inflammation-induced ATP release in fish., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: Hepatic ischemia reperfusion (HIR) leads to a lung inflammatory response and subsequent pulmonary barrier dysfunction. The gap junction communication protein connexin 32 (Cx32), which is widely expressed in the lungs, participates in intercellular signaling. This study determined whether the communication protein Cx32 could affect pulmonary inflammation caused by HIR., Methods: Mice were randomly allocated into four groups (n = 8/group): (i) Cx32 +/+ sham group; (ii) Cx32 +/+ HIR model group; (iii) Cx32 -/- sham group; and (iv) Cx32 -/- HIR model group. Twenty-four hours after surgery, lung tissues were collected for bright field microscopy, western blot (Cx32, JAK2, p-JAK2, STAT3, p-STAT3), and immunofluorescence (ZO-1, 8-OHDG) analyses. The collected bronchoalveolar fluid was tested for levels of interleukin-6 (IL-6), matrix metalloproteinase 12 (MMP-12), and antitrypsin (α1-AT). Lung mmu-miR-26a/b expression was detected using a PCR assay., Results: Increased expression of Cx32 mRNA and protein was noted in the lungs after HIR. Cx32 deletion significantly aggravated pulmonary function from acute lung injury induced by HIR. In addition, Cx32 deletion decreased the protein level of ZO-1 (pulmonary function) and increased the level of the oxidative stress marker 8-OHDG in the lungs. Moreover, in the Cx32 -/- HIR model group, the levels of IL-6 and MMP-12 in bronchoalveolar lavage fluid were significantly increased leading to activation of the JAK2/STAT3 pathway, and decreased α1-AT levels. Furthermore, we found mmu-miR-26a/b was significantly downregulated in the Cx32 -/- HIR model group., Conclusion: HIR leads to acute lung inflammatory injury. Cx32 deletion aggravates hepatic-derived lung inflammation, partly through blocking the transferring of mmu-miR-26a/b and leading to IL-6-related JAK2/STAT3 pathway activation.

Connexins are goal keepers of tissue homeostasis, including in the liver. As a result, they are frequently involved in disease. The current study was set up to investigate the effects of cholestatic disease on the production of connexin26, connexin32 and connexin43 in the liver. For this purpose, bile duct ligation, a well-known trigger of cholestatic liver injury, was applied to mice. In parallel, human hepatoma HepaRG cell cultures were exposed to cholestatic drugs and bile acids. Samples from both the in vivo and in vitro settings were subsequently subjected to assessment of mRNA and protein quantities as well as to in situ immunostaining. While the outcome of cholestasis on connexin26 and connexin43 varied among experimental settings, a more generalized repressing effect was seen for connexin32. This has also been observed in many other liver pathologies and could suggest a role for connexin32 as a robust biomarker of liver disease and toxicity.

Diagnostic yield of genetic studies for Charcot-Marie-Tooth disease (CMT) is little known, with a lack of epidemiological data to build better diagnostic strategies outside the United States and Europe. We aimed to evaluate the performance of two molecular diagnostic strategies for patients with CMT, and to characterize epidemiological findings of these conditions in southern Brazil. We performed a single-center cross-sectional study, in which 94 patients (55 families) with CMT suspicion were evaluated. Overall, the diagnostic yield of the combined strategy of Multiplex-ligation-dependent-probe-amplification (MLPA) of PMP22/GJB1/MPZ and GJB1/MPZ/PMP22 Sanger sequencing was 63.6% (28/44) for index cases with demyelinating/intermediate CMT suspicion (21 CMT1A-PMP22, 5 CMTX1-GJB1 and 2 with probably CMT1B-MPZ diagnosis). Five of the 11 index cases (45.4%) with axonal CMT had at least a possible diagnosis with next generation sequencing (NGS) panel of 104 inherited neuropathies-related genes (one each with CMT1A-PMP22, CMT2A-MFN2, CMT2K-GDAP1, CMT2U-MARS, CMT2W-HARS1). Detailed clinical, neurophysiological and molecular data of families are provided. Sequential molecular diagnosis strategies with MLPA plus target Sanger sequencing for demyelinating/intermediate CMT had high diagnostic yield, and almost half of axonal CMT families had at least a possible diagnosis with the comprehensive NGS panel. Most frequent subtypes of CMT in our region are CMT1A-PMP22 and CMTX1-GJB1., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background/aim: Altered connexin expression has been associated with cancer development and progression. This study evaluated connexin 26, 32, and 43 expression in association with the long-term outcomes of gastric cancer after gastrectomy., Patients and Methods: Pathological specimens were collected from 113 patients who underwent gastrectomy for gastric cancer, of whom 104 were included in the study. The expression levels of the connexins were assessed by immunohistochemistry. The patients were classified into low and high groups according to the median histoscore of connexin., Results: Cancer-specific survival (CSS) was significantly longer in the high than low connexin 43 group (p=0.030), particularly in patients >65 years old (p=0.030). A multivariate analysis identified pathological stage, differentiation type, and connexin 43 expression as possible predictors of CSS in patients (difference in the area under the curve=0.232, 0.090, and 0.094)., Conclusion: Low connexin 43 expression predicted a poor CSS in gastric cancer patients after gastrectomy., (Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Chemoresistance is a main obstacle in ovarian cancer therapy and new treatment strategies and further information regarding the mechanism of the medication cisplatin are urgently needed. Nitric oxide has a critical role in modulating the activity of chemotherapeutic drugs. Our previous work showed that connexin32 contributed to cisplatin resistance. However, whether nitric oxide is involved in connexin32-mediated cisplatin resistance remains unknown. In this study, using A2780 and A2780 cisplatin-resistant cells, we found that S-nitroso-N-acetyl-penicillamine, a nitric oxide donor, attenuated cisplatin toxicity by decreasing gap junctions in A2780 cells. Enhancement of gap junctions using retinoic acid reversed the effects of S-nitroso-N-acetyl-penicillamine on cisplatin toxicity. In A2780 cisplatin-resistant cells, however, S-nitroso-N-acetyl-penicillamine enhanced cisplatin toxicity by decreasing connexin32 expression. Downregulation of connexin32 expression by small interfering RNA exacerbated the effects of S-nitroso-N-acetyl-penicillamine on cisplatin cytotoxicity and upregulation of connexin32 expression by pcDNA transfection reversed the effects of S-nitroso-N-acetyl-penicillamine on cisplatin cytotoxicity. Our study suggests for the first time that combining cisplatin with nitric oxide in clinical therapies for ovarian cancer should be avoided before cisplatin resistance emerges. The present study provides a productive area of further study for increasing the efficacy of cisplatin by combining cisplatin with the specific inhibitors or enhancers of nitric oxide in clinical treatment., (© 2020 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

The Calmodulin-Cork gating model is based on evidence for the direct role of calmodulin (CaM) in channel gating. Indeed, chemical gating of cell-to-cell channels is sensitive to nanomolar cytosolic calcium concentrations [Ca 2+ ] i . Calmodulin inhibitors and inhibition of CaM expression prevent chemical gating. CaMCC, a CaM mutant with higher Ca 2+ -sensitivity greatly increases chemical gating sensitivity (in CaMCC the NH 2 -terminal EF-hand pair (res. 9-76) is replaced by the COOH-terminal pair (res. 82-148). Calmodulin colocalizes with connexins. Connexins have high-affinity CaM binding sites. Several connexin mutants paired to wild-type connexins have a high gating sensitivity that is eliminated by inhibition of CaM expression. Repeated transjunctional voltage (Vj) pulses slowly and progressively close a large number of channels by the chemical/slow gate (CaM lobe). At the single-channel level, the chemical/slow gate closes and opens slowly with on-off fluctuations. The model proposes two types of CaM-driven gating: "Ca-CaM-Cork" and "CaM-Cork". In the first, gating involves Ca 2+ -induced CaM-activation. In the second, gating takes place without [Ca 2+ ] i rise. The Ca-CaM-Cork gating is only reversed by a return of [Ca 2+ ] i to resting values, while the CaM-Cork gating is reversed by Vj positive at the gated side.

Hepatic ischemia/reperfusion (I/R) injury is a common complication in the clinical setting. Our previous study has shown that connexin 32 (Cx32) plays a major role in renal I/R injury; however, the role of Cx32 in hepatic I/R injury remains unknown. Liver tissue and serum samples from patients undergoing orthotopic liver transplantation (OLT) were used to evaluate the function of Cx32 in OLT post-reperfusion injury. Then, partial hepatic ischemia was established in global Cx32 knockout mice and wild-type mice followed by reperfusion. Hepatic injury markers were examined. Cx32 small interfering RNA and the p53 inhibitor, pifithrin-α, tenovin-1 were used to examine the relationship between Cx32 and the p53/puma pathways in the BRL-3A and murine primary hepatocytes hypoxia/reoxygenation (H/R) model. Corresponding to liver damage, Cx32 was significantly induced both during OLT in human patients and partial hepatic I/R in mice. Cx32 KO mice exhibited less liver injury than controls. Cx32 deficiency significantly suppressed the p53/puma pathways and hepatocyte apoptosis. Similar results were observed in the BRL-3A and murine primary hepatocytes H/R model. Propofol protected against OLT post-reperfusion injury and hepatocyte apoptosis by inhibiting Cx32. In conclusion Cx32 is a novel regulator of hepatic I/R injury through the modulation of hepatocyte apoptosis and damage, largely via the p53/puma signaling pathway., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Charcot-Marie-Tooth (CMT) hereditary polyneuropathies pose a diagnostic challenge. Our aim here is to describe CMT patients diagnosed by whole exome sequencing (WES) following years of fruitless testing., Methods/results: Three patients with polyneuropathy suspected to be genetic in origin, but not harboring PMP22 gene deletion/duplication, were offered WES. The first patient, a 66-year-old man, had been suffering from progressive weakness and atrophies in the lower and upper extremities for 20 years. Due to ambiguous electrophysiological findings, immune therapies were administered to no avail. Twelve years after PMP22 deletion/duplication testing, WES revealed two pathogenic variants in the FIG4 gene (p.Ile41Thr and p.Phe598fs, respectively), as a cause of CMT 4J. The second patient, a 19-year-old man, had been suffering from hearing and gait impairment since at least his infancy, and recently presented with weakness and dystonia of the lower extremities. In this patient, WES identified the p.Leu122Val LITAF gene variant in heterozygous state, suggesting the diagnosis of CMT 1C, several years after initial genetic analyses. The third patient, a 44-year-old man, presented with progressive weakness and atrophies of the lower and upper extremities since the age of 17 years old. In this patient, WES identified the hemizygous p.Arg164Gln pathogenic variant in the GJB1 gene, establishing the diagnosis of CMT X1, 8 years after testing for PMP22 deletion/duplication., Conclusion: Novel diagnostic techniques, such as WES, offer the possibility to decipher the cause of CMT subtypes, ending the diagnostic Odyssey of the patients and sparing them from unnecessary and potentially harmful treatments., (© 2020 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.)

Objective: X-linked Charcot-Marie-Tooth disease (CMTX) is an hereditary neuropathy caused by mutations in GJB1 coding for connexin-32, found in Schwann cells, but also expressed in oligodendrocytes. Reports have identified CNS involvement in CMTX, but no systematic study of cognitive function has been published., Methods: We assessed 24 CMTX patients (13 males; 9GJB1 mutations) with a comprehensive neuropsychological battery, including tests of memory, language, and executive functions., Results: No differences in cognitive performance were observed between males and females. A case-by-case investigation revealed selective deficits in individual patients. One subgroup (29%) demonstrated executive abnormalities; and a non-overlapping subgroup (29%), prominent reading (decoding) abnormalities., Conclusions: The present data provide evidence for cognitive deficits in CMTX. Emerging neuropsychological patterns are also discussed.

Background: Charcot-Marie-Tooth (CMT) disease is a group of hereditary neuropathies with high phenotypic and genetic heterogeneity. In this study, we report a large family with X-linked CMT (CMTX) caused by a novel GJB1 mutation., Methods: A family with the clinical diagnosis of CMTX was investigated. For mutation analysis, the coding region of GJB1 was sequenced using DNA from 15 family members. The identified GJB1 mutation was investigated by DHPLC in 120 normal controls. Mutation reanalysis was performed based on whole-exome sequencing (WES). Cell transfection studies were performed to characterize the function of the novel mutation., Results: A missense mutation (c.605T>A) in GJB1 was detected in five patients and eight female carriers but not in two unaffected members of the family. The mutation was not found in 120 healthy controls and has not been previously reported. WES excluded other pathogenic mutations in the family. The pathogenicity of the mutation was confirmed by disrupting the membrane localization of the encoded proteins., Conclusion: Our findings demonstrate that a novel mutation (c.605T>A) in GJB1 is associated with CMTX and adds to the repertoire of GJB1 mutations related to CMTX., (© 2020 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.)

Pannexin (Px) and connexin (Cx) hemichannels mediate bidirectional membrane transport in response to various stimuli and are involved in drug efficacy and toxicity. The purpose of the present study was to clarify in detail the transport characteristics of Px1 and Cx32 hemichannels by establishing transport assay systems using human Px1- and P2RX7 receptor-overexpressing HEK293 cells (Px1/P2RX7/HEK293) and Cx32-overexpressing HEK293 cells (Cx32/HEK293), in which P2RX7 and an extracellular Ca 2+ -depleted condition serve as the opening trigger, respectively. Uptake of the cationic fluorescent dye propidium iodide (PI) was significantly increased in Px1/P2RX7/HEK293 cells compared to that in mock cells, whereas there was no significant uptake of the anionic fluorescent dye sulforhodamine 101 (SR101). Uptake of [ 3 H]cholesterol by Px1/P2RX7/HEK293 cells was significantly decreased, whereas that of [ 3 H]taurine was not, compared to mock cells. On the other hand, uptakes of PI and SR-101 by Cx32/HEK293 cells were both significantly increased compared to mock cells. The PI uptake by Cx32/HEK293 cells was significantly inhibited by thioacetamide, acetaminophen, and N-acetyl-p-benzoquinoneimine. Cellular uptake of [ 3 H]cholesterol was significantly increased in Cx32/HEK293 cells and that of [ 3 H]taurine was significantly decreased. These results support the idea that Px1 and Cx32 hemichannels have distinct substrate recognition specificities and transport directions., (Copyright © 2020 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Charcot-Marie-Tooth disease (CMT) encompasses numerous genetically heterogeneous inherited neuropathies, which together are one of the commonest neurogenetic disorders. Axonal CMT types result from mutations in neuronally expressed genes, whereas demyelinating CMT forms mostly result from mutations in genes expressed by myelinating Schwann cells. The demyelinating forms are the most common, and may be caused by dominant mutations and gene dosage effects (as in CMT1), as well as by recessive mutations and loss of function mechanisms (as in CMT4). The discovery of causative genes and increasing insights into molecular mechanisms through the study of experimental disease models has provided the basis for the development of gene therapy approaches. For demyelinating CMT, gene silencing or gene replacement strategies need to be targeted to Schwann cells. Progress in gene replacement for two different CMT forms, including CMT1X caused by GJB1 gene mutations, and CMT4C, caused by SH3TC2 gene mutations, has been made through the use of a myelin-specific promoter to restrict expression in Schwann cells, and by lumbar intrathecal delivery of lentiviral viral vectors to achieve more widespread biodistribution in the peripheral nervous system. This review summarizes the molecular-genetic mechanisms of selected demyelinating CMT neuropathies and the progress made so far, as well as the remaining challenges in the path towards a gene therapy to treat these disorders through the use of optimal gene therapy tools including clinically translatable delivery methods and adeno-associated viral (AAV) vectors., (Copyright © 2019 Elsevier B.V. All rights reserved.)