Inhibition of connexin 36 hemichannels by glucose contributes to the stimulation of insulin secretion.

The existence of functional connexin36 (Cx36) hemichannels in ß-cells was investigated in pancreatic islets of rat and wild-type (Cx36(+/+)), monoallelic (Cx36(+/-)), and biallelic (Cx36(-/-)) knockout mice. Hemichannel opening by KCl depolarization was studied by measuring ATP release and changes of intracellular ATP (ADP). Cx36(+/+) islets lost ATP after depolarization with 70 mM KCl at 5 mM glucose; ATP loss was prevented by 8 and 20 mM glucose or 50 µM mefloquine (connexin inhibitor). ATP content was higher in Cx36(-/-) than Cx36(+/+) islets and was not decreased by KCl depolarization; Cx36(+/-) islets showed values between that of control and homozygous islets. Five minimolar extracellular ATP increased ATP content and ATP/ADP ratio and induced a biphasic insulin secretion in depolarized Cx36(+/+) and Cx36(+/-) but not Cx36(-/-) islets. Cx36 hemichannels expressed in oocytes opened upon depolarization of membrane potential, and their activation was inhibited by mefloquine and glucose (IC50 ∼8 mM). It is postulated that glucose-induced inhibition of Cx36 hemichannels in islet ß-cells might avoid depolarization-induced ATP loss, allowing an optimum increase of the ATP/ADP ratio by sugar metabolism and a biphasic stimulation of insulin secretion. Gradual suppression of glucose-induced insulin release in Cx36(+/-) and Cx36(-/-) islets confirms that Cx36 gap junction channels are necessary for a full secretory stimulation and might account for the glucose intolerance observed in mice with defective Cx36 expression. Mefloquine targeting of Cx36 on both gap junctions and hemichannels also suppresses glucose-stimulated secretion. By contrast, glucose stimulation of insulin secretion requires Cx36 hemichannels' closure but keeping gap junction channels opened.

Oligodendroglia Generated From Adult Rat Adipose Tissue by Direct Cell Conversion.

Obtaining oligodendroglial cells from dispensable tissues would be of great interest for autologous or immunocompatible cell replacement therapy in demyelinating diseases, as well as for studying myelin-related pathologies or testing therapeutic approaches in culture. We evaluated the feasibility of generating oligodendrocyte precursor cells (OPCs) from adult rat adipose tissue by expressing genes encoding transcription factors involved in oligodendroglial development. Adipose-derived mesenchymal cells were lentivirally transduced with tetracycline-inducible Sox10, Olig2, Zfp536, and/or Nkx6.1 transgenes. Immunostaining with the OPC-specific O4 monoclonal antibody was used to mark oligodendroglial induction. O4- and myelin-associated glycoprotein (MAG)-positive cells emerged after 3 weeks when using the Sox10 + Olig2 + Zfp536 combination, followed in the ensuing weeks by GFAP-, O1 antigen-, p75NTR (low-affinity NGF receptor)-, and myelin proteins-positive cells. The O4+ cell population progressively expanded, eventually constituting more than 70% of cells in culture by 5 months. Sox10 transgene expression was essential for generating O4+ cells but was insufficient for inducing a full oligodendroglial phenotype. Converted cells required continuous transgene expression to maintain their glial phenotype. Some vestigial characteristics of mesenchymal cells were maintained after conversion. Growth factor withdrawal and triiodothyronine (T3) supplementation generated mature oligodendroglial phenotypes, while FBS supplementation produced GFAP+- and p75NTR+-rich cultures. Converted cells also showed functional characteristics of neural-derived OPCs, such as the expression of AMPA, NMDA, kainate, and dopaminergic receptors, as well as similar metabolic responses to differentiation-inducing drugs. When co-cultured with rat dorsal root ganglion neurons, the converted cells differentiated and ensheathed multiple axons. We propose that functional oligodendroglia can be efficiently generated from adult rat mesenchymal cells by direct phenotypic conversion.

Insights into the pathophysiology of DFNA10 hearing loss associated with novel EYA4 variants.

The mutational spectrum of many genes and their contribution to the global prevalence of hereditary hearing loss is still widely unknown. In this study, we have performed the mutational screening of EYA4 gene by DHLPC and NGS in a large cohort of 531 unrelated Spanish probands and one Australian family with autosomal dominant non-syndromic hearing loss (ADNSHL). In total, 9 novel EYA4 variants have been identified, 3 in the EYA4 variable region (c.160G > T; p.Glu54*, c.781del; p.Thr261Argfs*34 and c.1078C > A; p.Pro360Thr) and 6 in the EYA-HR domain (c.1107G > T; p.Glu369Asp, c.1122G > T; p.Trp374Cys, c.1281G > A; p.Glu427Glu, c.1282-1G > A, c.1601C > G; p.S534* and an heterozygous copy number loss encompassing exons 15 to 17). The contribution of EYA4 mutations to ADNSHL in Spain is, therefore, very limited (~1.5%, 8/531). The pathophysiology of some of these novel variants has been explored. Transient expression of the c-myc-tagged EYA4 mutants in mammalian COS7 cells revealed absence of expression of the p.S534* mutant, consistent with a model of haploinsufficiency reported for all previously described EYA4 truncating mutations. However, normal expression pattern and translocation to the nucleus were observed for the p.Glu369Asp mutant in presence of SIX1. Complementary in silico analysis suggested that c.1107G > T (p.Glu369Asp), c.1281G > A (p.Glu427Glu) and c.1282-1G > A variants alter normal splicing. Minigene assays in NIH3T3 cells further confirmed that all 3 variants caused exon skipping resulting in frameshifts that lead to premature stop codons. Our study reports the first likely pathogenic synonymous variant linked to DFNA10 and provide further evidence for haploinsufficiency as the common underlying disease-causing mechanism for DFNA10-related hearing loss.