Structural and molecular characterization of astrocyte and vasculature connectivity in the mouse hippocampus and cortex.

The relation of astrocytic endfeet to the vasculature plays a key functional role in the neuro-glia-vasculature unit. We characterize the spatial organization of astrocytes and the structural aspects that facilitate their involvement in molecular exchanges. Using double transgenic mice, we performed co-immunostaining, confocal microscopy, and three-dimensional digital segmentation to investigate the biophysical and molecular organization of astrocytes and their intricate endfoot network at the micrometer level in the isocortex and hippocampus. The results showed that hippocampal astrocytes had smaller territories, reduced endfoot dimensions, and fewer contacts with blood vessels compared with those in the isocortex. Additionally, we found that both connexins 43 and 30 have a higher density in the endfoot and the former is overexpressed relative to the latter. However, due to the limitations of the method, further studies are needed to determine the exact localization on the endfoot. The quantitative information obtained in this study will be useful for modeling the interactions of astrocytes with the vasculature.

Fluid shear stress stimulates ATP release without regulating purinergic gene expression in the renal inner medullary collecting duct.

In the kidney, the flow rate of the pro-urine through the renal tubules is highly variable. The tubular epithelial cells sense these variations in pro-urinary flow rate in order to regulate various physiological processes, including electrolyte reabsorption. One of the mechanosensitive pathways activated by flow is the release of ATP, which can then act as a autocrine or paracrine factor. Increased ATP release is observed in various kidney diseases, among others autosomal dominant polycystic kidney disease (ADPKD). However, the mechanisms underlying flow-induced ATP release in the collecting duct, especially in the inner medullary collecting duct, remain understudied. Using inner medullary collecting duct 3 (IMCD3) cells in a microfluidic setup, we show here that administration of a high flow rate for 1 min results in an increased ATP release compared to a lower flow rate. Although the ATP release channel pannexin-1 contributed to flow-induced ATP release in Pkd1-/- IMCD3 cells, it did not in wildtype IMCD3 cells. In addition, flow application increased the expression of the putative ATP release channel connexin-30.3 (CX30.3) in wildtype and Pkd1-/- IMCD3 cells. However, CX30.3 knockout IMCD3 cells exhibited a similar flow-induced ATP release as wildtype IMCD3 cells, suggesting that CX30.3 does not drive flow-induced ATP release in wildtype IMDC3 cells. Collectively, our results show differential mechanisms underlying flow-induced ATP release in wildtype and Pkd1-/- IMCD3 cells and further strengthen the link between ADPKD and pannexin-1-dependent ATP release.

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.

Cytomembrane Trafficking Pathways of Connexin 26, 30, and 43.

The connexin gene family is the most prevalent gene that contributes to hearing loss. Connexins 26 and 30, encoded by GJB2 and GJB6, respectively, are the most abundantly expressed connexins in the inner ear. Connexin 43, which is encoded by GJA1, appears to be widely expressed in various organs, including the heart, skin, the brain, and the inner ear. The mutations that arise in GJB2, GJB6, and GJA1 can all result in comprehensive or non-comprehensive genetic deafness in newborns. As it is predicted that connexins include at least 20 isoforms in humans, the biosynthesis, structural composition, and degradation of connexins must be precisely regulated so that the gap junctions can properly operate. Certain mutations result in connexins possessing a faulty subcellular localization, failing to transport to the cell membrane and preventing gap junction formation, ultimately leading to connexin dysfunction and hearing loss. In this review, we provide a discussion of the transport models for connexin 43, connexins 30 and 26, mutations affecting trafficking pathways of these connexins, the existing controversies in the trafficking pathways of connexins, and the molecules involved in connexin trafficking and their functions. This review can contribute to a new way of understanding the etiological principles of connexin mutations and finding therapeutic strategies for hereditary deafness.

Erythrokeratodermia variabilis et progressiva due to a novel mutation in GJB4.

Erythrokeratodermia variabilis et progressiva (EKVP) is a rare genodermatosis of clinical and genetic heterogeneity, characterized by the manifestations of localized or disseminated persistent hyperkeratotic plagues and stationary to migratory transient erythematous patches. The majority of EKVP cases display an autosomal dominant mode of inheritance with incomplete penetrance, although recessive transmission has also been described. Mutations associated with EKVP have been primarily detected in connexin (Cx) genes. We herein reported a Chinese sporadic case of late-onset EKVP with a novel heterozygous missense mutation c.109G>A (p.V37M) in GJB4 (Cx30.3) gene, which resulted in a significant reduction of GJB4 expression in the epidermis of the patient. In accordance, while wild-type GJB4 localized at the cell membrane of HeLa cells forming intercellular junctions and intracellular puncta, V37M mutant variant was diffusely expressed within HeLa cells at a considerably lower level. Our findings reveal an essential role of GJB4 in the pathogenesis of EKVP and provides insights into the therapeutic potential of the disease.

Does astrocyte gap junction protein expression differ during development in absence epileptic rats?

Intercellular communication via gap junctions (GJs) has a wide variety of complex and essential functions in the CNS. In the present developmental study, we aimed to quantify the number of astrocytic GJs protein connexin 30 (Cx30) of genetic model of absence epilepsy rats from Strasbourg (GAERS) at postnatal P10, P30, and P60 days in the epileptic focal areas involved in the cortico-thalamic circuit. We compared the results with Wistar rats using immunohistochemistry and western blotting. The number of Cx30 immunopositive astrocytes per unit area were quantified for the somatosensory cortex (SSCx), ventrobasal (VB), and lateral geniculate (LGN) thalamic nuclei of the two strains and Cx30 western blot was applied to the tissue samples from the same regions. Both immunohistochemical and western blot results revealed the presence of Cx30 in all regions studied at P10 in both Wistar and GAERS animals. The SSCx, VB, and LGN of Wistar animals showed progressive increase in the number of Cx30 immunopositive labeled astrocytes from P10 to P30 and reached a peak at P30; then a significant decline was observed from P30 to P60 for the SSCx and VB. However, in GAERS Cx30 immunopositive labeled astrocytes showed a progressive increase from P10 to P60 for all brain regions studied. The immunohistochemical data highly corresponded with western blotting results. We conclude that the developmental disproportional expression of Cx30 in the epileptic focal areas in GAERS may be related to the onset of absence seizures or may be related to the neurogenesis of absence epilepsy.

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.

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.

Structural determinants underlying permeant discrimination of the Cx43 hemichannel.

Connexin (Cx) gap junction channels comprise two hemichannels in neighboring cells, and their permeability is well-described, but permeabilities of the single Cx hemichannel remain largely unresolved. Moreover, determination of isoform-specific Cx hemichannel permeability is challenging because of concurrent expression of other channels with similar permeability profiles and inhibitor sensitivities. The mammalian Cx hemichannels Cx30 and Cx43 are gated by extracellular divalent cations, removal of which promotes fluorescent dye uptake in both channels but atomic ion conductance only through Cx30. To determine the molecular determinants of this difference, here we employed chimeras and mutagenesis of predicted pore-lining residues in Cx43. We expressed the mutated channels in Xenopus laevis oocytes to avoid background activity of alternative channels. Oocytes expressing a Cx43 hemichannel chimera containing the N terminus or the first extracellular loop from Cx30 displayed ethidium uptake and, unlike WT Cx43, ion conduction, an observation further supported by molecular dynamics simulations. Additional C-terminal truncation of the chimeric Cx43 hemichannel elicited an even greater ion conductance with a magnitude closer to that of Cx30. The inhibitory profile for the connexin hemichannels depended on the permeant, with conventional connexin hemichannel inhibitors having a higher potency toward the ion conductance pathway than toward fluorescent dye uptake. Our results demonstrate a permeant-dependent, isoform-specific inhibition of connexin hemichannels. They further reveal that the outer segments of the pore-lining region, including the N terminus and the first extracellular loop, together with the C terminus preclude ion conductance of the open Cx43 hemichannel.

Connexin 30 mediated rewiring of glucose metabolism in rat C6 xenograft and grades of glioma.

Connexin 30 (Cx30), a tumour-suppressive gap junctional protein, impacts on insulin-like growth factor receptor 1-mediated progression and stemness of glioma. Of late, metabolic reprogramming, a recently adjudged hall mark of malignancy, could reasonably associated with the changes in gap junctional communication in glioma. This newly recognized hallmark of reprogramming of metabolism to maintain the rapid proliferation necessitates further probing to establish the stronger hall marks. Hence, the current study attempted to link the association between the expression of Cx30 with glucose uptake and glucose metabolism in glioma. We have transfected Cx30 in C6 glioma cells, characterized by a low level of intercellular communication and developed xenografts to study the status of glucose transporters (GLUTs), hexokinase 2 and Pyruvate dehydrogenase kinase 1 (PDK 1) along with human glioma tissues by RT-PCR and immunoblotting. The results showed a significant increase in the levels of GLUTs, hexokinase 2 and PDK 1 in C6-implanted rat xenografts and high grades compared to their respective controls, whereas Cx30-transfected C6-implanted rat xenograft and low grades show no significant change compared to that of controls supporting the association between Gap junctional communications and glucose metabolism. We strongly speculate the impact of Cx30 over the glucose metabolism that might provide therapeutic prospects and challenges for anti-glycolytic cancer therapy.

Astroglial Cx30 sustains neuronal population bursts independently of gap-junction mediated biochemical coupling.

Astroglial networks mediated by gap junction channels contribute to neurotransmission and promote neuronal coordination. Connexin 30, one of the two main astroglial gap junction forming protein, alters at the behavioral level the reactivity of mice to novel environment and at the synaptic level excitatory transmission. However, the role and function of Cx30 at the neuronal network level remain unclear. We thus investigated whether Cx30 regulates neuronal population bursts and associated convulsive behavior. We found in vivo that Cx30 is upregulated by kainate-induced seizures and that it regulates in turn the severity of associated behavioral seizures. Using electrophysiology ex vivo, we report that Cx30 regulates aberrant network activity via control of astroglial glutamate clearance independently of gap-junction mediated biochemical coupling. Altogether, our results indicate that astroglial Cx30 is an important player in orchestrating neuronal network activity.

Connexin 30 deficiency attenuates A2 astrocyte responses and induces severe neurodegeneration in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride Parkinson's disease animal model.

BACKGROUND: The first pathology observed in Parkinson's disease (PD) is 'dying back' of striatal dopaminergic (DA) terminals. Connexin (Cx)30, an astrocytic gap junction protein, is upregulated in the striatum in PD, but its roles in neurodegeneration remain elusive. We investigated Cx30 function in an acute PD model by administering 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to wild-type (WT) and Cx30 knockout (KO) mice. METHODS: On days 1 and 7 after MPTP administration, we evaluated changes in astrocytic Cx30, Cx43, glial fibrillary acidic protein, and ionised calcium-binding adapter molecule 1 expression by immunostaining and biochemical analysis. Loss of DA neurons was evaluated by tyrosine hydroxylase immunostaining. Gene expression was analysed using A1, A2, pan-reactive astrocyte microarray gene sets, and M1, M2, and M1/M2 mixed microglial microarray gene sets. Real-time PCR and in situ hybridisation were performed to evaluate glial cell-derived neurotrophic factor (Gdnf) and S100a10 expression. Striatal GDNF protein levels were determined by enzyme-linked immunosorbent assay. RESULTS: MPTP treatment induced upregulation of Cx30 and Cx43 levels in the striatum of WT and KO mice. DA neuron loss was accelerated in Cx30 KO compared with WT mice after MPTP administration, despite no change in the striatal concentration of methyl-4-phenylpyridinium+. Astrogliosis in the striatum of Cx30 KO mice was attenuated by MPTP, whereas microglial activation was unaffected. Microarrays of the striatum showed reduced expression of pan-reactive and A2 astrocyte genes after MPTP treatment in Cx30 KO compared with WT mice, while M1, M2, and M1/M2 mixed microglial gene expression did not change. MPTP reduced the number of striatal astrocytes co-expressing Gdnf mRNA and S100ß protein or S100a10 mRNA and S100ß protein and also reduced the level of GDNF in the striatum of Cx30 KO compared with WT mice. CONCLUSIONS: These findings indicate that Cx30 plays critical roles in astrocyte neuroprotection in an MPTP PD model.

Cx30 exhibits unique characteristics including a long half-life when assembled into gap junctions.

In the present study we investigated the life cycle, trafficking, assembly and cell surface dynamics of a poorly characterized connexin family member, connexin 30 (Cx30; also known as GJB6), which plays a critical role in skin health and hearing. Unexpectedly, Cx30 localization at the cell surface and gap junctional intercellular communication was not affected by prolonged treatments with the endoplasmic reticulum (ER)-Golgi transport inhibitor brefeldin A or the protein synthesis inhibitor cycloheximide, whereas Cx43 (also known as GJA1) was rapidly cleared. Fluorescent recovery after photobleaching revealed that Cx30 plaques were rebuilt from the outer edges in keeping with older channels residing in the inner core of the plaque. Expression of a dominant-negative form of Sar1 GTPase led to the accumulation of Cx30 within the ER, in contrast to a report that Cx30 traffics via a Golgi-independent pathway. Co-expression of Cx30 with Cx43 revealed that these connexins segregate into distinct domains within common gap junction plaques, suggesting that their assembly is governed by different mechanisms. In summary, Cx30 was found to be an unusually stable, long-lived connexin (half-life >12 h), which may underlie its specific role in the epidermis and cochlea.

Implication of connexin30 on the stemness of glioma: connexin30 reverses the malignant phenotype of glioma by modulating IGF-1R, CD133 and cMyc.

Gap-junctional intercellular communication (GJIC) plays a major role in the malignant growth of glioma. Although the mechanistic aspects of GJIC have been extensively studied, the role of connexins in the regulation of the malignant behavior of glioma stem cells (GSCs) remains unclear. In our previous studies, we have shown that connexin30 can interfere with the insulin-like growth factor 1 receptor (IGF-1R), which is known for self-renewal and pluripotency. Following our earlier in vitro observation, in this work, we aimed to study the consequence of this influence of Cx30 on IGF-1R by evaluating the marker of GSCs, CD133 and oncoprotein, cMyc. We strengthened our basis by examining human glioma samples of different grades as well as rat C6 xenografts (Cx30-transfected and -non-transfected C6 cells) along with the sphere formation assays in vitro. Investigation of stemness-related CD133 and cMyc in human samples and rat xenografts exhibited a reciprocal relationship between Cx30 and IGF-1R in the low and high grades (HG) of glioma. Cx30 was completely abolished in HG; levels of IGF-1R, CD133 and cMyc expression were positively correlated with HG. Cx30 transfection could attenuate the malignant burden of glioma in rat xenografts. Cx30 transfection also altered the tumor sphere formation of C6 glioma cells in vitro, an important property of GSCs, and there was a significant reduction of CD133 and cMyc expression by Cx30 both in vitro and in vivo. These factors indicate that dysfunction of Cx30 plays a crucial role in the prevention of the stemness of glioma, and the exploitation of this feature will help in the management of glioma.

Diverse pattern of gap junction beta-2 and gap junction beta-4 genes mutations and lack of contribution of DFNB21, DFNB24, DFNB29, and DFNB42 loci in autosomal recessive nonsyndromic hearing loss patients in Hormozgan, Iran.

BACKGROUND: We aimed to determine the contribution of four DFNB loci and mutation analysis of gap junction beta-2 (GJB2) and GJB4 genes in autosomal recessive nonsyndromic hearing loss (ARNSHL) in South of Iran. MATERIALS AND METHODS: A total of 36 large ARNSHL pedigrees with at least two affected subjects were enrolled in the current study. The GJB2 and GJB4 genes mutations were screened using direct sequencing method. The GJB2 and GJB4 negative families were analyzed for the linkage to DFNB21, DFNB24, DFNB29, and DFNB42 loci by genotyping the corresponding STR markers using polymerase chain reaction-PAGE method. RESULTS: We found a homozygous nonsense mutation W77X and a homozygous missense mutation C169W in 5.55% of studied families in GJB2 and GJB4 genes, respectively. Five heterozygous mutations including V63G, A78T, and R127H in GJB2 gene, and R103C and R227W in GJB4 gene were detected. We identified two novel variations V63G in GJB2 and R227W in GJB4. In silico analysis predicted that both novel variations are deleterious mutations. We did not unveil any linkage between DFNB21, DFNB24, DFNB29, and DFNB42 loci and ARNSHL among studied families. CONCLUSION: This is the first report of GJB2 and GJB4 mutations from Hormozgan population. According to the previous publications regarding GJB2 and GJB4 mutations, the distribution of the mutations is different from other parts of Iran that should be considered in primary health-care programs. Further investigations are needed to evaluate the contribution of other loci in ARNSHL subjects in South of Iran.

Functional Analysis of a Novel Connexin30 Mutation in a Large Family with Hearing Loss, Pesplanus, Ichthyosis, Cutaneous Nodules, and Keratoderma.

Mutations in the gap-junction gene Cx30 (Connexin30, GJB6) are a known cause of hearing loss. Here, we report our findings on a large multigeneration family in which severe to profound sensorineural hearing impairment is associated with a variety of skin-related anomalies. Genome-wide analysis of the family showed that the locus maps to chromosome region 13ptel-q12.1 and that a novel mutation, p.N54K, in Cx30, cosegregates with the phenotype. Unlike wild-type Cx30, p.N54K Cx30 is predominantly localized in the cytoplasm and does not permit transfer of neurobiotin, suggesting improper cellular localization and abolishment of gap-junction activity.

Prevalence of Deafness-Associated Connexin-26 (GJB2) and Connexin-30 (GJB6) Pathogenic Alleles in a Large Patient Cohort from Eastern Sicily.

Mutations in the gene encoding the gap junction protein connexin 26 (GJB2) and connexin 30 (GJB6) have been shown to be a major contributor to prelingual, sensorineural, nonsyndromic deafness. The aim of this study was to characterize and establish the prevalence of GJB2 and GJB6 gene alterations in 196 patients affected by sensorineural, nonsyndromic hearing loss, from Eastern Sicily. We performed sequence analysis of GJB2 and identified sequence variants in 68 out of 196 patients (34.7%); (28 homozygous for c.35delG, 22 compound heterozygous and 11 with only one variant allele). We found 12 different allelic variants, the most prevalent being c.35delG, which was found on 89 chromosomes (65.5%), followed by other alleles with different frequencies (p.E47X, c.-23+1G>A, p.L90P, p.R184W, p.M34T, c.167delT, p.R127H, p.M163V, p.V153I, p.W24X, and p.T8M). Importantly, for the first time we present the frequency and spectrum of GJB2 mutations in NSHL patients from Eastern Sicily. No alterations were found in the GJB6 gene, confirming that alterations in this gene are uncommon in our geographic area. Note that 65.3% and 23.5% of our patients, respectively were found to be negative or carriers by GJB2 molecular screening. This emphasizes the need to broaden the genetic analysis to other genes involved in hearing loss.

Protector Role of Cx30.2 in Pancreatic ß-Cell against Glucotoxicity-Induced Apoptosis.

Glucotoxicity may exert its deleterious effects on pancreatic ß-cell function via a myriad of mechanisms, leading to impaired insulin secretion and, eventually, type 2 diabetes. ß-cell communication requires gap junction channels to be present among these cells. Gap junctions are constituted by transmembrane proteins of the connexins (Cxs) family. Two Cx genes have been identified in ß cells, Cx36 and Cx30.2. We have found evidence that the glucose concentration on its own is sufficient to regulate Cx30.2 gene expression in mouse islets. In this work, we examine the involvement of the Cx30.2 protein in the survival of ß cells (RIN-m5F). METHODS: RIN-m5F cells were cultured in 5 mM D-glucose (normal) or 30 mM D-glucose (high glucose) for 24 h. Cx30.2 siRNAs was used to downregulate Cx30.2 expression. Apoptosis was measured by means of TUNEL, an annexin V staining method, and the cleaved form of the caspase-3 protein was determined using Western blot. RESULTS: High glucose did not induce apoptosis in RIN-m5F ß cells after 24 h; interestingly, high glucose increased the Cx30.2 total protein levels. Moreover, this work found that the downregulation of Cx30.2 expression in high glucose promoted apoptosis in RIN-m5F cells. CONCLUSION: The data suggest that the upregulation of Cx30.2 protects ß cells from hyperglycemia-induced apoptosis. Furthermore, Cx30.2 may be a promising avenue of therapeutic investigation for the treatment of glucose metabolic disorders.

Role of Impaired Astrocyte Gap Junction Coupling in Epileptogenesis.

The gap-junction-coupled astroglial network plays a central role in the regulation of neuronal activity and synchronisation, but its involvement in the pathogenesis of neuronal diseases is not yet understood. Here, we present the current state of knowledge about the impact of impaired glial coupling in the development and progression of epilepsy and discuss whether astrocytes represent alternative therapeutic targets. We focus mainly on temporal lobe epilepsy (TLE), which is the most common form of epilepsy in adults and is characterised by high therapy resistance. Functional data from TLE patients and corresponding experimental models point to a complete loss of astrocytic coupling, but preservation of the gap junction forming proteins connexin43 and connexin30 in hippocampal sclerosis. Several studies further indicate that astrocyte uncoupling is a causal event in the initiation of TLE, as it occurs very early in epileptogenesis, clearly preceding dysfunctional changes in neurons. However, more research is needed to fully understand the role of gap junction channels in epilepsy and to develop safe and effective therapeutic strategies targeting astrocytes.