Connexin32 gap junction channels deliver miR155-3p to mediate pyroptosis in renal ischemia-reperfusion injury.

OBJECTIVES: To explore whether the gap junction (GJ) composed by connexin32(Cx32) mediated pyroptosis in renal ischemia-reperfusion(I/R) injury via transmitting miR155-3p, with aim to provide new strategies for the prevention and treatment of acute kidney injury (AKI) after renal I/R. METHODS: 8-10 weeks of male C57BL/ 6 wild-type mice and Cx32 knockdown mice were divided into two groups respectively: control group and renal I/R group. MCC950 (50 mg/kg. ip.) was used to inhibit NLRP3 in vivo. Human kidney tubular epithelial cells (HK - 2) and rat kidney tubular epithelial cells (NRK-52E) were divided into high-density group and low-density group, and treated with hypoxia reoxygenation (H/R) to mimic I/R. The siRNA and plasmid of Cx32, mimic and inhibitor of miR155-3p were transfected into HK - 2 cells respectively. Kidney pathological and functional injuries were measured. Western Blot and immunofluorescent staining were used to observe the expression of NLRP3, GSDMD, GSDMD-N, IL - 18, and mature IL-18. The secretion of IL-18 and IL-1ß in serum, kidney tissue and cells supernatant were detected by enzyme-linked immuno sorbent assay (ELISA) kit, and the expression of NLPR3 and miR155-3p were detected by RT-qPCR and fluorescence in situ hybridization (FISH). RESULTS: Tubular pyroptosis were found to promote AKI after I/R in vivo and Cx32-GJ regulated pyroptosis by affecting the expression of miR155-3p after renal I/R injury. In vitro, H/R could lead to pyroptosis in HK-2 and NRK-52E cells. When the GJ channels were not formed, and Cx32 was inhibited or knockdown, the expression of miR155-3p was significantly reduced and the pyroptosis was obviously inhibited, leading to the reduction of injury and the increase of survival rate. Moreover, regulating the level of miR155-3p could affect survival rate and pyroptosis in vitro after H/R. CONCLUSIONS: The GJ channels composed of Cx32 regulated tubular pyroptosis in renal I/R injury by transmitting miR155-3p. Inhibition of Cx32 could reduce the level of miR155-3p further to inhibit pyroptosis, leading to alleviation of renal I/R injury which provided a new strategy for preventing the occurrence of AKI. Video Abstract.

Genetic analysis and natural history of Charcot-Marie-Tooth disease CMTX1 due to GJB1 variants.

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.

Role of connexin 32 in the directional differentiation of induced pluripotent stem cells into hepatocytes.

This study aimed to explore the role of connexin 32 (Cx32) in the directional differentiation of induced pluripotent stem cells (iPSCs) into hepatocytes. Urine-derived epithelial cells were collected from the fresh urine of a healthy donor and transducted with reprogramming plasmid mixture to generate iPSCs. The iPSCs were then directionally differentiated into hepatocytes. During the differentiation, the upregulated and downregulated groups were treated with vitamin K2 (VK2) and 2-aminoethoxyboronate diphenylester (2-APB) to increase and inhibit Cx32 expression, respectively. The control group was not treated with the regulatory factor. Expression of Cx32 and hepatocyte-specific markers, including AFP, hepatocyte nuclear factor 4α (HNF-4α), albumin (ALB) and cytokeratin 18 (CK18) were detected. It indicated that Cx32 expression was not observed in iPSCs, but gradually increased during the process of hepatic differentiation from iPSCs. Upregulation of Cx32 expression by VK2 treatment promoted hepatocyte maturation and enhanced the expression of the aforementioned hepatic specific markers, whereas downregulation of Cx32 expression by 2-APB treatment had the opposite effects. In conclusion, urine-derived iPSCs could be directionally differentiated into hepatocytes. Up-regulation of Cx32 improves the efficiency and maturity of differentiation of iPSCs into hepatocytes, and Cx32 may be a promoting factor during the process of hepatic differentiation from iPSCs.

Connexin Expression in Human Minor Salivary Glands: An Immunohistochemical Microscopy Study.

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.

Investigating oligodendrocyte connexins: Heteromeric interactions between Cx32 and mutant or wild-type forms of Cx47 do not contribute to or modulate gap junction function.

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.

Connexin32 activates necroptosis through Src-mediated inhibition of caspase 8 in hepatocellular carcinoma.

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.

GJB1 mutations c.212T>G and c.311A>C induce apoptosis and inwardly rectifying potassium current changes in X-linked Charcot-Marie-Tooth type 1.

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.

Nitric oxide affects cisplatin cytotoxicity oppositely in A2780 and A2780-CDDP cells via the connexin32/gap junction.

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.

Evidence for Cognitive Deficits in X-Linked Charcot-Marie-Tooth Disease.

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.

Characterization of three connexin32 genes and their role in inflammation-induced ATP release in the Japanese flounder Paralichthys olivaceus.

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.

X linked Charcot-Marie-Tooth disease and multiple sclerosis: emerging evidence for an association.

OBJECTIVE: X linked Charcot-Marie-Tooth disease (CMTX) is a hereditary neuropathy caused by mutations in GJB1 coding for connexin-32, a gap junction protein expressed in Schwann cells, but also found in oligodendrocytes. Four patients with CMTX developing central nervous system (CNS) demyelination compatible with multiple sclerosis (MS) have been individually published. We presently sought to systematically investigate the relationship between CMTX and MS. METHODS: Over 20 years, 70 consecutive patients (36 men) with GJB1 mutations were identified at our Neurogenetics Unit, Athens, Greece, and assessed for clinical features suggestive of MS. Additionally, 18 patients with CMTX without CNS symptoms and 18 matched controls underwent brain MRI to investigate incidental findings. Serum from patients with CMTX and MS was tested for CNS immunoreactivity. RESULTS: We identified three patients with CMTX who developed clinical features suggestive of inflammatory CNS demyelination fulfilling MS diagnostic criteria. The resulting 20-year MS incidence (4.3%) differed significantly from the highest background 20-year MS incidence ever reported from Greece (p=0.00039). The search for incidental brain MRI findings identified two CMTX cases (11%) with lesions suggestive of focal demyelination compared with 0 control. Moreover, 10 cases in the CMTX cohort had hyperintensity in the splenium of the corpus callosum compared with 0 control (p=0.0002). No specific CNS-reactive humoral factors were identified in patients with CMTX and MS. CONCLUSIONS: We have demonstrated a higher than expected frequency of MS in patients with CMTX and identified incidental focal demyelinating lesions on brain MRI in patients with CMTX without CNS symptoms. This provides circumstantial evidence for GJB1 mutations acting as a possible MS risk factor.

New novel mutations in Brazilian families with X-linked Charcot-Marie-Tooth disease.

Mutations in the GJB1 gene are the second most frequent cause of Charcot-Marie-Tooth disease (CMT), accounting for approximately 10% of CMT cases worldwide. We retrospectively analyzed detailed clinical and neurophysiological data on four Brazilian families carrying novel mutations of the GJB1 gene. Mutations were identified by bidirectional Sanger sequence analysis on the GJB1 coding region. We identified a total of 12 subjects from four different kindred. There was no male-to-male transmission, and their clinical pictures were within the expected spectrum for GJB1-related neuropathy. Males were more severely affected than females. Five out of the eight females only had subclinical neuropathy. Nerve conduction velocities were in the intermediate range in the male patients and higher in the females affected. These mutations increase the genotypic variability associated with GJB1.

Intrathecal gene therapy rescues a model of demyelinating peripheral neuropathy.

Inherited demyelinating peripheral neuropathies are progressive incurable diseases without effective treatment. To develop a gene therapy approach targeting myelinating Schwann cells that can be translatable, we delivered a lentiviral vector using a single lumbar intrathecal injection and a myelin-specific promoter. The human gene of interest, GJB1, which is mutated in X-linked Charcot-Marie-Tooth Disease (CMT1X), was delivered intrathecally into adult Gjb1-null mice, a genetically authentic model of CMT1X that develops a demyelinating peripheral neuropathy. We obtained widespread, stable, and cell-specific expression of connexin32 in up to 50% of Schwann cells in multiple lumbar spinal roots and peripheral nerves. Behavioral and electrophysiological analysis revealed significantly improved motor performance, quadriceps muscle contractility, and sciatic nerve conduction velocities. Furthermore, treated mice exhibited reduced numbers of demyelinated and remyelinated fibers and fewer inflammatory cells in lumbar motor roots, as well as in the femoral motor and sciatic nerves. This study demonstrates that a single intrathecal lentiviral gene delivery can lead to Schwann cell-specific expression in spinal roots extending to multiple peripheral nerves. This clinically relevant approach improves the phenotype of an inherited neuropathy mouse model and provides proof of principle for treating inherited demyelinating neuropathies.

Connexin32 plays a crucial role in ROS-mediated endoplasmic reticulum stress apoptosis signaling pathway in ischemia reperfusion-induced acute kidney injury.

BACKGROUND: Ischemia-reperfusion (I/R)-induced acute kidney injury (AKI) not only prolongs the length of hospital stay, but also seriously affects the patient's survival rate. Although our previous investigation has verified that reactive oxygen species (ROS) transferred through gap junction composed of connexin32 (Cx32) contributed to AKI, its underlying mechanisms were not fully understood and viable preventive or therapeutic regimens were still lacking. Among various mechanisms involved in organs I/R-induced injuries, endoplasmic reticulum stress (ERS)-related apoptosis is currently considered to be an important participant. Thus, in present study, we focused on the underlying mechanisms of I/R-induced AKI, and postulated that Cx32 mediated ROS/ERS/apoptosis signal pathway activation played an important part in I/R-induced AKI. METHODS: We established renal I/R models with Cx32+/+ and Cx32-/- mice, which underwent double kidneys clamping and recanalization. ROS scavenger (N-acetylcysteine, NAC) and ERS inhibitors (4-phenyl butyric acid, 4-PBA, and tauroursodeoxycholic acid, TUDCA) were used to decrease the content of ROS and attenuate ERS activation, respectively. RESULTS: Renal damage was progressively exacerbated in a time-dependent manner at the reperfusion stage, that was consistent with the alternation of ERS activation, including glucose regulated protein 78 (BiP/GRP78), X box-binding protein1, and C/EBP homologous protein expression. TUDCA or 4-PBA application attenuated I/R-induced ERS activation and protected against renal tubular epithelial cells apoptosis and renal damage. Cx32 deficiency decreased ROS generation and distribution between the neighboring cells, which attenuated I/R-induced ERS activation, and improved cell apoptosis and renal damage. CONCLUSION: Cx32 mediated ROS/ERS/apoptosis signal pathway activation played an important part in I/R-induced AKI. Cx32 deficiency, ROS elimination, and ERS inhibition all could protect against I/R-induced AKI.

An 8-generation family with X-linked Charcot-Marie-Tooth: Confirmation Of the pathogenicity Of a 3' untranslated region mutation in GJB1 and its clinical features.

INTRODUCTION: Mutations in gap junction protein beta 1 (GJB1) on the X chromosome represent one of the most common causes of hereditary neuropathy. We assessed manifestations associated with a rare 3' untranslated region mutation (UTR) of GJB1 in a large family with X-linked Charcot-Marie-Tooth disease (CMTX). METHODS: Clinical, electrophysiological, and molecular genetic analyses were performed on an 8-generation family with CMTX. RESULTS: There were 22 affected males and 19 symptomatic females, including an 83-year-old woman followed for 40 years. Electrophysiological studies showed a primarily axonal neuropathy. The c.*15C>T mutation in the GJB1 3' UTR was identified in 4 branches of the family with a log of odds (LOD) of 4.91. This created a BstE II enzyme recognition site that enabled detection by restriction digestion. DISCUSSION: The c.*15C>T mutation in the GJB1 3' UTR segregates with CMTX1 in 8 generations. Penetrance in males and females is essentially complete. A straightforward genetic method to detect this mutation is described. Muscle Nerve 57: 859-862, 2018.

Connexin32 deficiency is associated with liver injury, inflammation and oxidative stress in experimental non-alcoholic steatohepatitis.

Non-alcoholic steatohepatitis is a highly prevalent liver pathology featured by hepatocellular fat deposition and inflammation. Connexin32, which is the major building block of hepatocellular gap junctions, has a protective role in hepatocarcinogenesis and is downregulated in chronic liver diseases. However, the role of connexin32 in non-alcoholic steatohepatitis remains unclear. Connexin32-/- mice and their wild-type littermates were fed a choline-deficient high-fat diet. The manifestation of non-alcoholic steatohepatitis was evaluated based on a battery of clinically relevant read-outs, including histopathological examination, diverse indicators of inflammation and liver damage, in-depth lipid analysis, assessment of oxidative stress, insulin and glucose tolerance, liver regeneration and lipid-related biomarkers. Overall, more pronounced liver damage, inflammation and oxidative stress were observed in connexin32-/- mice compared to wild-type animals. No differences were found in insulin and glucose tolerance measurements and liver regeneration. However, two lipid-related genes, srebf1 and fabp3, were upregulated in Cx32-/- mice in comparison with wild-type animals. These findings suggest that connexin32-based signalling is not directly involved in steatosis as such, but rather in the sequelae of this process, which underlie progression of non-alcoholic steatohepatitis.

Connexin32: a mediator of acetaminophen-induced liver injury?

Connexin32 is the building block of hepatocellular gap junctions, which control direct intercellular communication and thereby act as goalkeepers of liver homeostasis. This study was set up to investigate whether connexin32 is involved in hepatotoxicity induced by the analgesic and antipyretic drug acetaminophen. To this end, whole body connexin32 knock-out mice were overdosed with acetaminophen followed by sampling at different time points within a 24-h time frame. Evaluation was done based upon a series of clinically and mechanistically relevant read-outs, including protein adduct formation, histopathological examination, measurement of alanine aminotransferase activity, cytokine production, levels of reduced and oxidized glutathione and hepatic protein amounts of proliferating cell nuclear antigen. In essence, it was found that genetic ablation of connexin32 has no influence on several key events in acetaminophen-induced hepatotoxicity, including cell death, inflammation or oxidative stress, yet it does affect production of protein adducts as well as proliferating cell nuclear antigen steady-state protein levels. This outcome is not in line with previous studies, which are contradicting on their own, as both amplification and alleviation of this toxicological process by connexin32 have been described. This could question the suitability of the currently available models and tools to investigate the role of connexin32 in acetaminophen-triggered hepatotoxicity.

Connexin32 deficiency exacerbates carbon tetrachloride-induced hepatocellular injury and liver fibrosis in mice.

OBJECTIVE: Liver fibrosis results from the perpetuation of the normal wound healing response to several types of injury. Despite the wealth of knowledge regarding the involvement of intracellular and extracellular signaling pathways in liver fibrogenesis, information about the role of intercellular communication mediated by gap junctions is scarce. METHODS: In this study, liver fibrosis was chemically induced by carbon tetrachloride in mice lacking connexin32, the major liver gap junction constituent. The manifestation of liver fibrosis was evaluated based on a series of read-outs, including collagen morphometric and mRNA analysis, oxidative stress, apoptotic, proliferative and inflammatory markers. RESULTS: More pronounced liver damage and enhanced collagen deposition were observed in connexin32 knockout mice compared to wild-type animals in experimentally triggered induced liver fibrosis. No differences between both groups were noticed in apoptotic signaling nor in inflammation markers. However, connexin32 deficient mice displayed decreased catalase activity and increased malondialdehyde levels. CONCLUSION: These findings could suggest that connexin32-based signaling mediates tissue resistance against liver damage by the modulation of the antioxidant capacity. In turn, this could point to a role for connexin32 signaling as a therapeutic target in the treatment of liver fibrosis.

Downregulation of connexin 32 attenuates hypoxia/reoxygenation injury in liver cells.

Gap junction intercellular communication is involved in ischemia-reperfusion (IR) injury of organs. Connexins are proteins that are critical to the function of gap junctions. To clarify the role of gap junctions in IR injury in liver cells, the function of gap junctions was modulated in an in vitro hypoxia/reoxygenation (H/R) model. BRL-3A rat liver cells, endogenously expressing connexins Cx32 and Cx43, were used to model the process of hepatic IR injury. Suppression of gap junction activity was achieved genetically, using Cx32-specific small interfering RNA (siRNA), or chemically, with pharmacological inhibitors, oleamide, and 18-α-GA. BRL-3A cells subjected to H/R exhibited reduced cell survival and pathologies indicative of IR injury. Cx32-specific siRNA, oleamide, and 18-α-GA, respectively, decreased gap junction permeability, as assessed by the parachute assay. Pretreatment with Cx32-specific siRNA increased cell survival. Pretreatment with oleamide or 18-α-GA did not improve cell survival. Modulating gap junction by Cx32 gene silencing protected BRL-3A liver cells from H/R.

Recurrent central nervous system white matter changes in charcot-Marie-tooth type X disease.

INTRODUCTION: X-linked Charcot-Marie-Tooth (CMT1X) disease is caused by mutations in the GJB1 gene. We describe a young man who presented with recurrent central nervous symptoms and transient white matter changes in the setting of a novel mutation in the GJB1 gene. METHODS: Evaluation included clinical examination, neuroimaging, electrophysiological, and molecular genetic studies. RESULTS: Clinical examination on 2 admissions 5 years apart demonstrated hemiparesis with findings of underlying peripheral neuropathy. Electrophysiologic studies revealed a sensorimotor polyneuropathy. MRI studies from both admissions revealed white matter changes, with improvement on an intervening study. Mutation analysis showed a novel mutation (c.98T>A; p.Ile33Asn) in the GJB1 gene. CONCLUSIONS: Mutations in GJB1 can result in recurrent central nervous system symptoms with transient white matter signal changes on MRI. In patients presenting with hemiparesis, the presence of signs of a peripheral neuropathy may facilitate identification of CMT1X, and is likely to affect clinical management.