The opening of connexin hemichannels (HCs) expressed at the plasma membrane of mammalian cells is regulated by a number of physiological parameters, including extracellular and intracellular Ca2+ ions. Submicromolar variations of the cytosolic Ca2+ concentration ([Ca2+]c) are per se sufficient to trigger extracellular bursts of messenger molecules through connexin HCs, thus mediating paracrine signaling. In this chapter, we present a quantitative method to measure the opening dynamics of connexin HCs expressed in a single HeLa cell upon stimulation by a canonical InsP3-mediated [Ca2+]c transient. The protocol relies on a combination of Ca2+ imaging and patch-clamp techniques. The insights gained from our method are expected to make a significant contribution to understanding the structure-function relationship of connexin HCs. The protocol is also suitable to screen candidate therapeutic compounds to treat connexin-related diseases linked to HC dysfunction.

X-linked Charcot-Marie-Tooth disease type 1 (CMT1X) is a demyelinating neuropathy resulting from loss-of-function mutations affecting the GJB1/connexin 32 (Cx32) gene. We previously showed functional and morphological improvement in Gjb1-null mice following AAV9-mediated delivery of human Cx32 driven by the myelin protein zero (Mpz) promoter in Schwann cells. However, CMT1X mutants may interfere with virally delivered wild-type (WT) Cx32. To confirm the efficacy of this vector also in the presence of CMT1X mutants, we delivered AAV9-Mpz-GJB1 by lumbar intrathecal injection in R75W/Gjb1-null and N175D/Gjb1-null transgenic lines expressing Golgi-retained mutations, before and after the onset of the neuropathy. Widespread expression of virally delivered Cx32 was demonstrated in both genotypes. Re-establishment of WT Cx32 function resulted in improved muscle strength and increased sciatic nerve motor conduction velocities in all treated groups from both mutant lines when treated before as well as after the onset of the neuropathy. Furthermore, morphological analysis showed improvement of myelination and reduction of inflammation in lumbar motor roots and peripheral nerves. In conclusion, this study provides proof of principle for a clinically translatable gene therapy approach to treat CMT1X before and after the onset of the neuropathy, even in the presence of endogenously expressed Golgi-retained Cx32 mutants.

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.

BACKGROUND: Nonalcoholic steatohepatitis (NASH) is characterized by fat deposition, inflammation, and hepatocellular damage. The diagnosis of NASH is confirmed pathologically, and hepatocyte ballooning is an important finding for definite diagnosis. Recently, α-synuclein deposition in multiple organs was reported in Parkinson\'s disease. Since it was reported that α-synuclein is taken up by hepatocytes via connexin 32, the expression of α-synuclein in the liver in NASH is of interest. The accumulation of α-synuclein in the liver in NASH was investigated. Immunostaining for p62, ubiquitin, and α-synuclein was performed, and the usefulness of immunostaining in pathological diagnosis was examined.
METHODS: Liver biopsy tissue specimens from 20 patients were evaluated. Several antibodies against α-synuclein, as well as antibodies against connexin 32, p62, and ubiquitin were used for immunohistochemical analyses. Staining results were evaluated by several pathologists with varying experience, and the diagnostic accuracy of ballooning was compared.
RESULTS: Polyclonal α-synuclein antibody, not the monoclonal antibody, reacted with eosinophilic aggregates in ballooning cells. Expression of connexin 32 in degenerating cells was also demonstrated. Antibodies against p62 and ubiquitin also reacted with some of the ballooning cells. In the pathologists\' evaluations, the highest interobserver agreement was obtained with hematoxylin and eosin (H&E)-stained slides, followed by slides immunostained for p62 and α-synuclein, and there were cases with different results between H&E staining and immunostaining CONCLUSION: These results indicate the incorporation of degenerated α-synuclein into ballooning cells, suggesting the involvement of α-synuclein in the pathogenesis of NASH. The combination of immunostaining including polyclonal α-synuclein may contribute to improving the diagnosis of NASH.

Pathogenic variants in Gap Junction Protein Beta 1 (GJB1) cause X-linked Charcot-Marie-Tooth (CMT) disease type 1 (CMTX1), which is a common hereditary motor and sensory neuropathy. A 45-year-old man presented with progressive muscle weakness, atrophy, sensory disturbance of all limbs from childhood, and visual field defects in both eyes at 40 years old. A segregation analysis revealed a novel variant, c.173C>A (p.P58H), in the GJB1 gene. Patients with variants at codon 58 in GJB1 showed clinically varied phenotypes, ranging from demyelinating neuropathy to cerebellar ataxia. This patient may represent one of the various clinical phenotypes of GJB1 variants.

Connexins (Cxs) are transmembrane proteins involved in the formation of hemichannels and gap junctions (GJs). GJs are involved in various physiological functions, including secretion in glandular tissue. It has been demonstrated that Cx26, Cx32, and Cx43 are mainly expressed in glands, but no data are available in human salivary glands to date. The aim of our study was to investigate the presence and the localization of Cxs in human minor labial salivary glands. Immunofluorescence and immunoelectron microscopy were employed to evaluate the Cx26, Cx32, and Cx43 protein in human labial salivary gland biopsies (hLSGBs). RT-PCR was also used to detect their mRNA expression. Cx expression was found at both the mRNA and protein levels in all hLSGBs analysed. Cxs were observed at the level of the duct and acinar cells, as well as in myoepithelial cells. The localization of the three Cx types was very similar, suggesting colocalization of these Cxs in the same connexons. These results demonstrated the presence of Cxs in human salivary glands for the first time. Moreover, the few samples with primary Sjögren\'s Syndrome analysed only by immunofluorescence showed an alteration of the Cx expression, indicating that these proteins could be involved in salivary gland dysfunctions.

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.

Connexins (Cx) are primary components of gap junctions that selectively allow molecules to be exchanged between adjacent cells, regulating multiple cellular functions. Along with their channel forming functions, connexins play a variety of roles in different stages of tumorigenesis and their roles in tumor initiation and progression is isoform- and tissue-specific. While Cx26 and Cx43 were downregulated during breast tumorigenesis, Cx32 was accumulated in the cytoplasm of the cells in lymph node metastasis of breast cancers and Cx32 was further upregulated in metastasis. Cx32\'s effect on cell proliferation, gap junctional communication, hemichannel activity, cellular motility and epithelial-to-mesenchymal transition (EMT) were investigated by overexpressing Cx32 in Hs578T and MCF7 breast cancer cells. Additionally, the expression and localization of Cx26 and Cx43 upon Cx32 overexpression were examined by Western blot and immunostaining experiments, respectively. We observed that MCF7 cells had endogenous Cx32 while Hs578T cells did not and when Cx32 was overexpressed in these cells, it caused a significant increase in the percentages of Hs578T cells at the S phase in addition to increasing their proliferation. Further, while Cx32 overexpression did not induce hemichannel activity in either cell, it decreased gap junctional communication between Hs578T cells. Additionally, Cx32 was mainly observed in the cytoplasm in both cells, where it did not form gap junction plaques but Cx32 overexpression reduced Cx43 levels without affecting Cx26. Moreover, migration and invasion potentials of Hs578T and migration in MCF7 were reduced upon Cx32 overexpression. Finally, the protein level of mesenchymal marker N-cadherin decreased while epithelial marker ZO-1 and E-cadherin increased in Hs578T cells. We observed that Cx32 overexpression altered cell proliferation, communication, migration and EMT in Hs578T, suggesting a tumor suppressor role in these cells while it had minor effects on MCF7 cells.

With ever-increasing production and use of nanoparticles (NPs), there is a necessity to evaluate the probability of consequential adverse effects in individuals exposed to these particles. It is now understood that a proportion of NPs can translocate from primary sites of exposure to a range of secondary organs, with the liver, kidneys and spleen being some of the most important. In this study, we carried out a comprehensive toxicological profiling (inflammation, changes in serum biochemistry, oxidative stress, acute phase response and histopathology) of Ag NP induced adverse effects in the three organs of interest following acute exposure of the materials at identical doses via intravenous (IV), intratracheal (IT) instillation and oral administration. The data clearly demonstrated that bioaccumulation and toxicity of the particles were most significant following the IV route of exposure, followed by IT. However, oral exposure to the NPs did not result in any changes that could be interpreted as toxicity in any of the organs of interest within the confines of this investigation. The finding of this study clearly indicates the importance of the route of exposure in secondary organ hazard assessment for NPs. Finally, we identify Connexin 32 (Cx32) as a novel biomarker of NP-mediated hepatic damage which is quantifiable both (in vitro) and in vivo following exposure of physiologically relevant doses.

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 Cx32I71S (GJB1 c.212T > G) and Cx32K104T (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 Cx32K104T mutant, leading to an increase in endoplasmic reticulum stress, resulting in apoptosis. Furthermore, whole-cell patch clamp experiments in oligodendrocytes revealed that the Cx32K104T 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.