A murine model for the del(GJB6-D13S1830) deletion recapitulating the phenotype of human DFNB1 hearing impairment: generation and functional and histopathological study.

Inherited hearing impairment is a remarkably heterogeneous monogenic condition, involving hundreds of genes, most of them with very small (< 1%) epidemiological contributions. The exception is GJB2, the gene encoding connexin-26 and underlying DFNB1, which is the most frequent type of autosomal recessive non-syndromic hearing impairment (ARNSHI) in most populations (up to 40% of ARNSHI cases). DFNB1 is caused by different types of pathogenic variants in GJB2, but also by large deletions that keep the gene intact but remove an upstream regulatory element that is essential for its expression. Such large deletions, found in most populations, behave as complete loss-of-function variants, usually associated with a profound hearing impairment. By using CRISPR-Cas9 genetic edition, we have generated a murine model (Dfnb1em274) that reproduces the most frequent of those deletions, del(GJB6-D13S1830). Dfnb1em274 homozygous mice are viable, bypassing the embryonic lethality of the Gjb2 knockout, and present a phenotype of profound hearing loss (> 90 dB SPL) that correlates with specific structural abnormalities in the cochlea. We show that Gjb2 expression is nearly abolished and its protein product, Cx26, is nearly absent all throughout the cochlea, unlike previous conditional knockouts in which Gjb2 ablation was not obtained in all cell types. The Dfnb1em274 model recapitulates the clinical presentation of patients harbouring the del(GJB6-D13S1830) variant and thus it is a valuable tool to study the pathological mechanisms of DFNB1 and to assay therapies for this most frequent type of human ARNSHI.

Astrocyte-derived neurons provide excitatory input to the adult striatal circuitry.

Astrocytes have emerged as a potential source for new neurons in the adult mammalian brain. In mice, adult striatal neurogenesis can be stimulated by local damage, which recruits striatal astrocytes into a neurogenic program by suppression of active Notch signaling (J. P. Magnusson et al., Science 346, 237-241 [2014]). Here, we induced adult striatal neurogenesis in the intact mouse brain by the inhibition of Notch signaling in astrocytes. We show that most striatal astrocyte-derived neurons are confined to the anterior medial striatum, do not express established striatal neuronal markers, and exhibit dendritic spines, which are atypical for striatal interneurons. In contrast to striatal neurons generated during development, which are GABAergic or cholinergic, most adult astrocyte-derived striatal neurons possess distinct electrophysiological properties, constituting the only glutamatergic striatal population. Astrocyte-derived neurons integrate into the adult striatal microcircuitry, both receiving and providing synaptic input. The glutamatergic nature of these neurons has the potential to provide excitatory input to the striatal circuitry and may represent an efficient strategy to compensate for reduced neuronal activity caused by aging or lesion-induced neuronal loss.

Recent insights into gap junction biogenesis in the cochlea.

In the cochlea, connexin 26 (Cx26) and connexin 30 (Cx30) co-assemble into two types of homomeric and heteromeric gap junctions between adjacent non-sensory epithelial cells. These channels provide a mechanical coupling between connected cells, and their activity is critical to maintain cochlear homeostasis. Many of the mutations in GJB2 or GJB6, which encode Cx26 and Cx30 in humans, impair the formation of membrane channels and cause autosomal syndromic and non-syndromic hearing loss. Thus, deciphering the connexin trafficking pathways in situ should represent a major step forward in understanding the pathogenic significance of many of these mutations. A growing body of evidence now suggests that Cx26/Cx30 heteromeric and Cx30 homomeric channels display distinct assembly mechanisms. Here, we review the most recent advances that have been made toward unraveling the biogenesis and stability of these gap junctions in the cochlea.

A Gap-Junction Mutation Reveals That Outer Hair Cell Extracellular Receptor Potentials Drive High-Frequency Cochlear Amplification.

Cochlear amplification enables the enormous dynamic range of hearing through amplifying cochlear responses to low- to moderate-level sounds and compressing them to loud sounds. Amplification is attributed to voltage-dependent electromotility of mechanosensory outer hair cells (OHCs) driven by changing voltages developed across their cell membranes. At low frequencies, these voltage changes are dominated by intracellular receptor potentials (RPs). However, OHC membranes have electrical low-pass filter properties that attenuate high-frequency RPs, which should potentially attenuate amplification of high-frequency cochlear responses and impede high-frequency hearing. We made in vivo intracellular and extracellular electrophysiological measurements from the organ of Corti of male and female mice of the CBA/J strain, with excellent high-frequency hearing, and from the CD-1 mouse strain, which has sensitive hearing below 12 kHz but loses high-frequency hearing within a few weeks postpartum. The CD-1 mouse strain was transfected with an A88V mutation of the connexin 30 gap-junction protein. By blocking the action of the GJ protein to reduce input resistance, the mutation increased the OHC extracellular RP (ERP) magnitude and rescued high-frequency hearing. However, by increasing the organ of Corti resistance, the mutation rescued high-frequency hearing through preserving the OHC extracellular RP (ERP) magnitude. We measured the voltage developed across the basolateral membranes of OHCs, which controls their electromotility, for low- to high-frequency sounds in male and female mice of the CD-1 strain that expressed the A88V mutation. We demonstrate that ERPs, not RPs, drive OHC motility and cochlear amplification at high frequencies because at high frequencies, ERPs are not frequency attenuated, exceed RPs in magnitude, and are appropriately timed to provide cochlear amplification.SIGNIFICANCE STATEMENT Cochlear amplification, which enables the enormous dynamic range of hearing, is attributed to voltage-dependent electromotility of the mechanosensory outer hair cells (OHCs) driven by sound-induced voltage changes across their membranes. OHC intracellular receptor potentials are electrically low-pass filtered, which should hinder high-frequency hearing. We measured the intracellular and extracellular voltages that control OHC electromotility in vivo in a mouse strain with impaired high-frequency hearing. A gap-junction mutation of the strain rescued high-frequency hearing, increased organ of Corti resistance, and preserved large OHC extracellular receptor potentials but reduced OHC intracellular receptor potentials and impaired low-frequency hearing. We concluded intracellular potentials drive OHC motility at low frequencies and extracellular receptor potentials drive OHC motility and cochlear amplification at high frequencies.

Connexin 30 Deficiency Ameliorates Disease Progression at the Early Phase in a Mouse Model of Amyotrophic Lateral Sclerosis by Suppressing Glial Inflammation.

Connexin 30 (Cx30), which forms gap junctions between astrocytes, regulates cell adhesion and migration, and modulates glutamate transport. Cx30 is upregulated on activated astroglia in central nervous system inflammatory lesions, including spinal cord lesions in mutant superoxide dismutase 1 (mSOD1) transgenic amyotrophic lateral sclerosis (ALS) model mice. Here, we investigated the role of Cx30 in mSOD1 mice. Cx30 was highly expressed in the pre-onset stage in mSOD1 mice. mSOD1 mice with knockout (KO) of the Cx30 gene (Cx30KO-mSOD1 mice) showed delayed disease onset and tended to have an extended survival period (log-rank, p = 0.09). At the progressive and end stages of the disease, anterior horn cells were significantly preserved in Cx30KO-mSOD1 mice. In lesions of these mice, glial fibrillary acidic protein/C3-positive inflammatory astroglia were decreased. Additionally, the activation of astrocytes in Cx30KO-mSOD1 mice was reduced compared with mSOD1 mice by gene expression microarray. Furthermore, expression of connexin 43 at the pre-onset stage was downregulated in Cx30KO-mSOD1 mice. These findings suggest that reduced expression of astroglial Cx30 at the early disease stage in ALS model mice protects neurons by attenuating astroglial inflammation.

Polydisperse molecular architecture of connexin 26/30 heteromeric hemichannels revealed by atomic force microscopy imaging.

Connexin (Cx) protein forms hemichannels and gap junctional channels, which play diverse and profound roles in human physiology and diseases. Gap junctions are arrays of intercellular channels formed by the docking of two hemichannels from adjacent cells. Each hexameric hemichannel contains the same or different Cx isoform. Although homomeric Cxs forms have been largely described functionally and structurally, the stoichiometry and arrangement of heteromeric Cx channels remain unknown. The latter, however, are widely expressed in human tissues and variation might have important implications on channel function. Investigating properties of heteromeric Cx channels is challenging considering the high number of potential subunit arrangements and stoichiometries, even when only combining two Cx isoforms. To tackle this problem, we engineered an HA tag onto Cx26 or Cx30 subunits and imaged hemichannels that were liganded by Fab-epitope antibody fragments via atomic force microscopy. For Cx26-HA/Cx30 or Cx30-HA/Cx26 heteromeric channels, the Fab-HA binding distribution was binomial with a maximum of three Fab-HA bound. Furthermore, imaged Cx26/Cx30-HA triple liganded by Fab-HA showed multiple arrangements that can be derived from the law of total probabilities. Atomic force microscopy imaging of ringlike structures of Cx26/Cx30-HA hemichannels confirmed these findings and also detected a polydisperse distribution of stoichiometries. Our results indicate a dominant subunit stoichiometry of 3Cx26:3Cx30 with the most abundant subunit arrangement of Cx26-Cx26-Cx30-Cx26-Cx30-Cx30. To our knowledge, this is the first time that the molecular architecture of heteromeric Cx channels has been revealed, thus providing the basis to explore the functional effect of these channels in biology.

Astroglial Cx30 differentially impacts synaptic activity from hippocampal principal cells and interneurons.

Astrocytes play important roles in brain function via dynamic structural and functional interactions with neurons. Yet the underlying mechanisms remain poorly defined. A typical feature of astrocytes is the high expression of connexins, which mediate their extensive intercellular communication and regulate their structural properties. In particular, connexin 30 (Cx30), one of the two connexins abundantly expressed by astrocytes, was recently shown to be a critical regulator of excitatory synaptic transmission by controlling the astroglial coverage of synapses. However, the role of Cx30 in the regulation of inhibitory synaptic transmission and excitatory/inhibitory balance remains elusive. Here, we investigated the role of astroglial Cx30 on the electrophysiological and morphological properties of five classes of hippocampal CA1 stratum oriens and pyramidale neurons, defined by the unsupervised Ward's clustering. Using Cx30 knockout mice, we found that Cx30 alters specific properties of some subsets of CA1 interneurons, such as resting membrane potential and sag ratio, while other parameters, such as action potential threshold and saturation frequency, were more frequently altered among the different classes of neurons. The excitation-inhibition balance was also differentially and selectively modulated among the different neuron subtypes. Only slight morphological differences were observed on reconstructed neurons. Altogether, these data indicate that Cx30 differentially alters the electrophysiological and morphological properties of hippocampal cell populations, and modulates both their excitatory and inhibitory inputs. Astrocytes, via Cx30, are thus active modulators of both excitatory and inhibitory synapses in the hippocampus.

The connexin 30 A88V mutant reduces cochlear gap junction expression and confers long-term protection against hearing loss.

Mutations in the genes that encode the gap junction proteins connexin 26 (Cx26, encoded by GJB2) and Cx30 (GJB6) are the leading cause of hereditary hearing loss. That said, the Cx30 p.Ala88Val (A88V) mutant causes Clouston syndrome, but not hearing loss. Here, we report that the Cx30-A88V mutant, despite being toxic to inner ear-derived HEI-OC1 cells, conferred remarkable long-term protection against age-related high frequency hearing loss in Cx30A88V/A88V mice. During early development, there were no overt structural differences in the cochlea between genotypes, including a normal complement of hair cells; however, the supporting cell Cx30 gap junction plaques in mutant mice were reduced in size. In adulthood, Cx30A88V/A88V mutant mice had a reduction of cochlear Cx30 mRNA and protein, yet a full complement of hair cells. Conversely, the age-related high frequency hearing loss in Cx30+/+ and Cx30+/A88V mice was due to extensive loss of outer hair cells. Our data suggest that the Cx30-A88V mutant confers long-term hearing protection and prevention of hair cell death, possibly via a feedback mechanism that leads to the reduction of total Cx30 gap junction expression in the cochlea.

Frequency of Usher gene mutations in non-syndromic hearing loss: higher variability of the Usher phenotype.

Non-syndromic hearing loss (NSHL) is characterized by a vast genetic heterogeneity; some syndromic forms as Usher syndrome (USH) have onset as isolated deafness and then evolve later in life. We developed an NGS targeted gene-panel containing 59 genes and a customized bioinformatic pipeline for the analysis of DNA samples from clinically highly selected subjects with sensorineural hearing loss, previously resulted negative for GJB2 mutations/GJB6 deletions. Among the 217 tested subjects, 24 (11.1%) were found to carry mutations in genes involved both in NSHL and USH. For 6 out of 24 patients a diagnosis of USH was performed. Eleven subjects out of 24 had hearing loss without vestibular or ocular dysfunction and, due to their young age, it was not possible to establish whether their phenotype could be NSHL or USH. Seven subjects were diagnosed with NSHL, due to their age and phenotype. A total of 41 likely pathogenic/pathogenic mutations were identified, among which 17 novel ones. We report a high frequency of mutations in genes involved both in NSHL and in USH in a cohort of individuals tested for seemingly isolated deafness. Our data also highlight a wider than expected phenotypic variability in the USH phenotype.

A recurrent mutation of GJB6 in a big Chinese family with Hidrotic ectodermal dysplasia.

Hidrotic ectodermal dysplasia (HED) is a rare inherited syndrome characterised by nail dystrophy, palmoplantar hyperkeratosis and alopecia. Four mutations (p.G11R, p.A88V, p.V37E and p.D50N) in gap junction beta 6 (GJB6) gene, which codes connexin30 protein, have been found to cause HED in different populations. Here, we reported a big Chinese family in which 24 patients over five generations were suffered with HED. Sequence analysis identified all 24 patients carry a recurrent missense mutation c.263C > T (p.A88V) in GJB6. Our results reveal gene testing of GJB6 is important for diagnosis, prenatal diagnosis and future gene treatment of HED.

Altered Gap Junction Network Topography in Mouse Models for Human Hereditary Deafness.

Anisotropic gap junctional coupling is a distinct feature of astrocytes in many brain regions. In the lateral superior olive (LSO), astrocytic networks are anisotropic and oriented orthogonally to the tonotopic axis. In CaV1.3 knock-out (KO) and otoferlin KO mice, where auditory brainstem nuclei are deprived from spontaneous cochlea-driven neuronal activity, neuronal circuitry is disturbed. So far it was unknown if this disturbance is also accompanied by an impaired topography of LSO astrocyte networks. To answer this question, we immunohistochemically analyzed the expression of astrocytic connexin (Cx) 43 and Cx30 in auditory brainstem nuclei. Furthermore, we loaded LSO astrocytes with the gap junction-permeable tracer neurobiotin and assessed the network shape and orientation. We found a strong elevation of Cx30 immunoreactivity in the LSO of CaV1.3 KO mice, while Cx43 levels were only slightly increased. In otoferlin KO mice, LSO showed a slight increase in Cx43 as well, whereas Cx30 levels were unchanged. The total number of tracer-coupled cells was unaltered and most networks were anisotropic in both KO strains. In contrast to the WTs, however, LSO networks were predominantly oriented parallel to the tonotopic axis and not orthogonal to it. Taken together, our data demonstrate that spontaneous cochlea-driven neuronal activity is not required per se for the formation of anisotropic LSO astrocyte networks. However, neuronal activity is required to establish the proper orientation of networks. Proper formation of LSO astrocyte networks thus necessitates neuronal input from the periphery, indicating a critical role of neuron-glia interaction during early postnatal development in the auditory brainstem.

Role of GJB2 and GJB6 in Iranian Nonsyndromic Hearing Impairment: From Molecular Analysis to Literature Reviews.

Background: Hearing impairment (HI) is a heterogeneous disorder. GJB2 and GJB6 genes are typically the first line of genetic screening before proceeding to any massive parallel sequencing. We evaluated the clinical utility of GJB2 and GJB6 testing in the Iranian population. Methods: GJB2 and GJB6 were sequenced. PubMed and Google Scholar were searched for Iranian publications on deletions in the DFNB1 locus. Results: We detected mutations of GJB2 in 16.5%, and no mutations of GJB6. Literature review revealed no reports of mutations of GJB6 in the Iranian population. Conclusion: This data and literature reviews indicate that GJB6 is not commonly responsible for Iranian nonsyndromic HI. Hence, the clinical utility of GJB6 genetic analysis as a first line for HI evaluation does not have the same utility as GJB2. The study is consistent with recent studies emphasizing the role of ethnicity in the selection of HI genetic testing strategy.

A novel homozygous frame-shift mutation in the SLC29A3 gene: a new case report and review of literature.

BACKGROUND: The SLC29A3 gene, encoding a nucleoside transporter protein, is found in intracellular membranes. Based on the literatures, mutations in this gene cause a wide range of clinical manifestations including H syndrome, pigmented hypertrichosis with insulin dependent diabetes, Faisalabad histiocytosis, and dysosteosclerosis. However, all these disorders with their different names and terminologies are actually the same entity termed H syndrome. CASE PRESENTATION: We report four GJB2 and GJB6 negative deaf patients from two Iranian related families who present the associated symptoms of SLC29A3-disorder. Whole Exome Sequencing (WES) using Next Generation Illumina Sequencing was used to enrich all exons of protein-coding genes as well as some other important genomic regions in one of studied patients. A novel homozygous frame-shift mutation c.307-308delTT (p.Phe103fs) in exon 3 of SLC29A3 gene was identified in a 35 years old man with profound hearing loss, camptodactyly, rheumatoid arthritis and delayed puberty without any skin changes, short stature and insulin dependent diabetes mellitus. The mutation found was also confirmed by Sanger sequencing in other studied patients and their healthy parents. In compared to proband, however the clinical manifestations of these patients were different, indicating variable expressivity of mutant SLC29A3 gene as well as possible involvement of other modifier genes. CONCLUSION: The present study uncovered a rare novel homozygous frame-shift mutation c.307-308delTT in SLC29A3 gene of four related patients with various manifestation of SLC29A3-disorder. Such studies can help to conduct genetic counseling and subsequently, prenatal diagnosis more accurately for individuals at the high risk of these types of genetic disorders.

Connexin30 and Connexin43 show a time-of-day dependent expression in the mouse suprachiasmatic nucleus and modulate rhythmic locomotor activity in the context of chronodisruption.

BACKGROUND: The astroglial connexins Cx30 and Cx43 contribute to many important CNS functions including cognitive behaviour, motoric capacity and regulation of the sleep-wake cycle. The sleep wake cycle, is controlled by the circadian system. The central circadian rhythm generator resides in the suprachiasmatic nucleus (SCN). SCN neurons are tightly coupled in order to generate a coherent circadian rhythm. The SCN receives excitatory glutamatergic input from the retina which mediates entrainment of the circadian system to the environmental light-dark cycle. Connexins play an important role in electric coupling of SCN neurons and astrocytic-neuronal signalling that regulates rhythmic SCN neuronal activity. However, little is known about the regulation of Cx30 and Cx43 expression in the SCN, and the role of these connexins in light entrainment of the circadian system and in circadian rhythm generation. METHODS: We analysed time-of-day dependent as well as circadian expression of Cx30 and Cx43 mRNA and protein in the mouse SCN by means of qPCR and immunohistochemistry. Moreover, we analysed rhythmic spontaneous locomotor activity in mice with a targeted deletion of Cx30 and astrocyte specific deletion of Cx43 (DKO) in different light regimes by means of on-cage infrared detectors. RESULTS: Fluctuation of Cx30 protein expression is strongly dependent on the light-dark cycle whereas fluctuation of Cx43 protein expression persisted in constant darkness. DKO mice entrained to the light-dark cycle. However, re-entrainment after a phase delay was slightly impaired in DKO mice. Surprisingly, DKO mice were more resilient to chronodisruption. CONCLUSION: Circadian fluctuation of Cx30 and Cx43 protein expression in the SCN is differently regulated. Cx30 and astroglial Cx43 play a role in rhythm stability and re-entrainment under challenging conditions.

Site specific hypermethylation of CpGs in Connexin genes 30, 26 and 43 in different grades of glioma and attenuated levels of their mRNAs.

AIM: Gliomas, the intracranial tumours are considered the deadliest malignancies. The gap junctional Connexins (Cxs) that maintain cellular homeostasis perform a unique function in glial tumour suppression. However, the differential methylation patterns of Cxs were not revealed in glioma so far. The current study attempts to categorise promoter methylation of Cx30 and Cx26 and intron methylation of Cx43 in different grades of human glioma. MATERIALS AND METHODS: About 85 glioma patients with pathologically confirmed grades and 15 control brain tissues were recruited in the study. Bisulphite-PCR-Single Stranded Conformation analysis(SSCA), Bisulphite sequencing and MeDIP-qPCR were carried out to assess methylation status and Cx mRNA levels were also analysed to evaluate the effect of methylation. RESULTS: We found that promoter CpG islands(CpGs) reside in Sp1 and Ap2 sites of Cx30 and 26 were hypermethylated in high grades (HG) of glioma rather than low grades. The input % of both was significantly increased (p < 0.03) in progressive grades. Interestingly, Cx43 could exhibit a significant increase (p < 0.05) in input % only in grade IV. While, Cx30 and 26 mRNAs were downregulated according to their methylation status in progressive fashion with grades, Cx43 was downregulated irrespective of intron methylation. CONCLUSION: Thus, we suggest that the sites and extent of methylation of Cxs (30 and 26 but not in 43) are found to be altered. In different grades of glioma can provide better appreciation of the grade of the patient and might help in strategies based on epigenetic approaches.

Comparison of Predictive In Silico Tools on Missense Variants in GJB2, GJB6, and GJB3 Genes Associated with Autosomal Recessive Deafness 1A (DFNB1A).

In silico predictive software allows assessing the effect of amino acid substitutions on the structure or function of a protein without conducting functional studies. The accuracy of in silico pathogenicity prediction tools has not been previously assessed for variants associated with autosomal recessive deafness 1A (DFNB1A). Here, we identify in silico tools with the most accurate clinical significance predictions for missense variants of the GJB2 (Cx26), GJB6 (Cx30), and GJB3 (Cx31) connexin genes associated with DFNB1A. To evaluate accuracy of selected in silico tools (SIFT, FATHMM, MutationAssessor, PolyPhen-2, CONDEL, MutationTaster, MutPred, Align GVGD, and PROVEAN), we tested nine missense variants with previously confirmed clinical significance in a large cohort of deaf patients and control groups from the Sakha Republic (Eastern Siberia, Russia): Сх26: p.Val27Ile, p.Met34Thr, p.Val37Ile, p.Leu90Pro, p.Glu114Gly, p.Thr123Asn, and p.Val153Ile; Cx30: p.Glu101Lys; Cx31: p.Ala194Thr. We compared the performance of the in silico tools (accuracy, sensitivity, and specificity) by using the missense variants in GJB2 (Cx26), GJB6 (Cx30), and GJB3 (Cx31) genes associated with DFNB1A. The correlation coefficient (r) and coefficient of the area under the Receiver Operating Characteristic (ROC) curve as alternative quality indicators of the tested programs were used. The resulting ROC curves demonstrated that the largest coefficient of the area under the curve was provided by three programs: SIFT (AUC = 0.833, p = 0.046), PROVEAN (AUC = 0.833, p = 0.046), and MutationAssessor (AUC = 0.833, p = 0.002). The most accurate predictions were given by two tested programs: SIFT and PROVEAN (Ac = 89%, Se = 67%, Sp = 100%, r = 0.75, AUC = 0.833). The results of this study may be applicable for analysis of novel missense variants of the GJB2 (Cx26), GJB6 (Cx30), and GJB3 (Cx31) connexin genes.

Permeant-specific gating of connexin 30 hemichannels.

Gap junctions confer interconnectivity of the cytoplasm in neighboring cells via docking of two connexons expressed in each of the adjacent membranes. Undocked connexons, referred to as hemichannels, may open and connect the cytoplasm with the extracellular fluid. The hemichannel configuration of connexins (Cxs) displays isoform-specific permeability profiles that are not directly determined by the size and charge of the permeant. To further explore Ca2+-mediated gating and permeability features of connexin hemichannels, we heterologously expressed Cx30 hemichannels in Xenopus laevis oocytes. The sensitivity toward divalent cation-mediated gating differed between small atomic ions (current) and fluorescent dye permeants, indicating that these permeants are distinctly gated. Three aspartate residues in Cx30 (Asp-50, Asp-172, and Asp-179) have been implicated previously in the Ca2+ sensitivity of other hemichannel isoforms. Although the aspartate at position Asp-50 was indispensable for divalent cation-dependent gating of Cx30 hemichannels, substitutions of the two other residues had no significant effect on gating, illustrating differences in the gating mechanisms between connexin isoforms. Using the substituted cysteine accessibility method (SCAM), we evaluated the role of possible pore-lining residues in the permeation of ions and ethidium through Cx30 hemichannels. Of the cysteine-substituted residues, interaction of a proposed pore-lining cysteine at position 37 with the positively charged compound [2-(trimethylammonium)ethyl] methane thiosulfonate bromide (MTS-ET) increased Cx30-mediated currents with unperturbed ethidium permeability. In summary, our results demonstrate that the permeability of hemichannels is regulated in a permeant-specific manner and underscores that hemichannels are selective rather than non-discriminating and freely diffusable pores.

Blocking TNFα-driven astrocyte purinergic signaling restores normal synaptic activity during epileptogenesis.

Epilepsy is characterized by unpredictable recurrent seizures resulting from abnormal neuronal excitability. Increasing evidence indicates that aberrant astrocyte signaling to neurons plays an important role in driving the network hyperexcitability, but the underlying mechanism that alters glial signaling in epilepsy remains unknown. Increase in glutamate release by astrocytes participates in the onset and progression of seizures. Epileptic seizures are also accompanied by increase of tumor necrosis factor alpha (TNFα), a cytokine involved in the regulation of astrocyte glutamate release. Here we tested whether TNFα controls abnormal astrocyte glutamate signaling in epilepsy and through which mechanism. Combining Ca2+ imaging, optogenetics, and electrophysiology, we report that TNFα triggers a Ca2+ -dependent glutamate release from astrocytes that boosts excitatory synaptic activity in the hippocampus through a mechanism involving autocrine activation of P2Y1 receptors by astrocyte-derived ATP/ADP. In a mouse model of temporal lobe epilepsy, such TNFα-driven astrocytic purinergic signaling is permanently active, promotes glial glutamate release, and drives abnormal synaptic activity in the hippocampus. Blocking this pathway by inhibiting P2Y1 receptors restores normal excitatory synaptic activity in the inflamed hippocampus. Our findings indicate that targeting the coupling of TNFα with astrocyte purinergic signaling may be a therapeutic strategy for reducing glial glutamate release and normalizing synaptic activity in epilepsy.

mCCDcl1 cells show plasticity consistent with the ability to transition between principal and intercalated cells.

The cortical collecting duct of the mammalian kidney plays a critical role in the regulation of body volume, sodium pH, and osmolarity and is composed of two distinct cells types, principal cells and intercalated cells. Each cell type is detectable in the kidney by the localization of specific transport proteins such as aquaporin 2 (Aqp2) and epithelial sodium channel (ENaC) in principal cells and V-ATPase B1 and connexin 30 (Cx30) in intercalated cells. mCCDcl1 cells have been widely used as a mouse principal cell line on the basis of their physiological characteristics. In this study, the mCCDcl1 parental cell line and three sublines cloned from isolated single cells (Ed1, Ed2, and Ed3) were grown on filters to assess their transepithelial resistance, transepithelial voltage, equivalent short circuit current and expression of the cell-specific markers Aqp2, ENaC, V-ATPaseB1, and Cx30. The parental mCCDcl1 cell line presented amiloride-sensitive electrogenic sodium transport indicative of principal cell function; however, immunocytochemistry and RT-PCR showed that some cells expressed the intercalated cell-specific markers V-ATPase B1 and Cx30, including a subset of cells also positive for Aqp2 and ENaC. The three subclonal lines contained cells that were positive for both intercalated and principal cell-specific markers. The vertical transmission of both principal and intercalated cell characteristics via single cell cloning reveals the plasticity of mCCDcl1 cells and a direct lineage relationship between these two physiologically important cell types and is consistent with mCCDcl1 cells being precursor cells.

Exome Sequencing Identifies a Novel Nonsense Mutation of MYO6 as the Cause of Deafness in a Brazilian Family.

We investigated 313 unrelated subjects who presented with hearing loss to identify the novel genetic causes of this condition in Brazil. Causative GJB2/GJB6 mutations were found in 12.7% of the patients. Among the familial cases (100/313), four were selected for exome sequencing. In one case, two novel heterozygous variants were found and were predicted to be pathogenic based on bioinformatics tools, that is, p.Ser906* (MYO6) and p.Arg42Cys (GJB3). We confirmed that this nonsense MYO6 mutation segregated with deafness in this family. Only the proband and her unaffected mother exhibited the GJB3 mutation, which is in the same amino acid of a known Erythrokeratodermia variabilis mutation. None of the patients exhibited this skin disease, but the proband exhibited a more severe hearing loss. Hence, the GJB3 mutation was considered to be a variant of uncertain significance. In conclusion, we described a novel nonsense MYO6 mutation that was responsible for the hearing loss in a Brazilian family. This mutation resides in the neck domain of myosin-VI after the motor domain. Thus, our data give further support for genotype-phenotype correlations, which state that when the motor domain of the protein is functioning, the hearing loss is milder and has a later onset. The three remaining families without mutations in the known genes suggest that there are still deafness genes to be revealed.