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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.)

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.