Gap-Junction Channels Dysfunction in Deafness and Hearing Loss

Gap-junction channels connect the cytoplasm of adjacent cells, allowing the diffusion of ions and small metabolites. They are formed at the appositional plasma membranes by a family of related proteins named connexins. Mutations in connexins 26, 31, 30, 32, and 43 have been associated with nonsyndromic or syndromic deafness. The majority of these mutations are inherited in an autosomal recessive manner, but a few of them have been associated with dominantly inherited hearing loss. Mutations in the connexin26 gene (GJB2) are the most common cause of genetic deafness. This review summarizes the most relevant and recent information about different mutations in connexin genes found in human patients, with emphasis on GJB2. The possible effects of the mutations on channel expression and function are discussed, in addition to their possible physiologic consequences for inner ear physiology. Finally, we propose that connexin channels (gap junctions and hemichannels) may be targets for age-related hearing loss induced by oxidative damage. Antioxid. Redox Signal. 11, 309–322

Gap-junctional channel and hemichannel activity of two recently identified connexin 26 mutants associated with deafness

Gap-junction channels (GJCs) are formed by headto-head association of two hemichannels (HCs, connexin hexamers). HCs and GJCs are permeable to ions and hydrophilic molecules of up to Mr ~1 kDa. Hearing impairment of genetic origin is common, and mutations of connexin 26 (Cx26) are its major cause. We recently identified two novel Cx26 mutations in hearing-impaired subjects, L10P and G109V. L10P forms functional GJCs with slightly altered voltage dependence and HCs with decrease ATP/cationic dye selectivity. G109V does not form functional GJCs, but forms functional HCs with enhanced extracellular Ca2+ sensitivity and subtle alterations in voltage dependence and ATP/ cationic dye selectivity. Deafness associated with G109V could result from decreased GJCs activity, whereas deafness associated to L10P may have a more complex mechanism that involves changes in HC permeability.Fil: Dalamon, Viviana Karina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Fiori, Mariana C.. Texas Tech University; Estados UnidosFil: Figueroa, Vania A.. Universidad del Desarrollo; ChileFil: Oliva, Carolina A.. Universidad del Desarrollo; ChileFil: del Rio, Rodrigo. Universidad Autónoma de Chile; ChileFil: Gonzalez, Wendy. Universidad de Talca; ChileFil: Canan, Jonathan. Universidad de Talca; ChileFil: Elgoyhen, Ana Belen. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Farmacología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Altenberg, Guillermo A.. Texas Tech University; Estados UnidosFil: Retamal, Mauricio A.. Universidad del Desarrollo; Chile. Texas Tech University; Estados Unido

Intravenous Administration of Bone Marrow-Derived Mesenchymal Stem Cells Induces a Switch from Classical to Atypical Symptoms in Experimental Autoimmune Encephalomyelitis

Potent immunosuppressive and regenerative properties of mesenchymal stem cells (MSCs) position them as a novel therapy for autoimmune diseases. This research examines the therapeutic effect of MSCs administration at different disease stages in experimental autoimmune encephalomyelitis (EAE). Classical and atypical scores of EAE, associated with Th1 and Th17 response, respectively, and also Treg lymphocytes, were evaluated. MSCs administration at the onset (EAE+MSC onset ) induced an important amelioration of the clinical signs and less lasting effect at the peak of EAE (EAE+MSC peak ). No effect was observed when MSCs were applied after EAE stabilization (EAE+MSC late ). Surprisingly, EAE atypical signs were detected in EAE+MSC peak and EAE+MSC late mice. However, no correlation was found in Th17/Th1 ratio. Interestingly, regardless of time administration, MSCs significantly reduced IL-6 and also T-bet, ROR T, and Foxp3 mRNA levels in brain samples of EAE mice. The downregulation of IL-6 could restore the well-functioning of the blood-brain barrier of EAE mice, correlated with a decreased number of brain infiltrating leukocytes. These results suggest that the inflammatory status is important to be considered for administering MSCs in autoimmune pathologies, leading to a further research to clarify the effect of MSCs for multiple sclerosis

Disruption in Connexin-Based Communication Is Associated with Intracellular Ca²⁺ Signal Alterations in Astrocytes from Niemann-Pick Type C Mice

<div><p>Reduced astrocytic gap junctional communication and enhanced hemichannel activity were recently shown to increase astroglial and neuronal vulnerability to neuroinflammation. Moreover, increasing evidence suggests that neuroinflammation plays a pivotal role in the development of Niemann-Pick type C (NPC) disease, an autosomal lethal neurodegenerative disorder that is mainly caused by mutations in the <i>NPC1</i> gene. Therefore, we investigated whether the lack of NPC1 expression in murine astrocytes affects the functional state of gap junction channels and hemichannels. Cultured cortical astrocytes of NPC1 knock-out mice (Npc1^−/−) showed reduced intercellular communication via gap junctions and increased hemichannel activity. Similarly, astrocytes of newborn Npc1^−/− hippocampal slices presented high hemichannel activity, which was completely abrogated by connexin 43 hemichannel blockers and was resistant to inhibitors of pannexin 1 hemichannels. Npc1^−/− astrocytes also showed more intracellular Ca²⁺ signal oscillations mediated by functional connexin 43 hemichannels and P2Y₁ receptors. Therefore, Npc1^−/− astrocytes present features of connexin based channels compatible with those of reactive astrocytes and hemichannels might be a novel therapeutic target to reduce neuroinflammation in NPC disease.</p></div

Npc1^−/− astrocytes exhibit larger Cx43 aggregates.

<p>(<b>A–B</b>) Representative confocal images depicting Cx43 (green) and GFAP (red) immunolabeling in Npc1^+/+ (<b>A</b>) and Npc1^−/− (<b>B</b>) astrocytes. Bar = 15 µm. (<b>C</b>) Quantification of the frequency of Cx43 spots of different diameters in Npc1^+/+ and Npc1^−/− (white and black bars, respectively) astrocytes. *p<0.05. Averaged data were obtained from at least three independent experiments.</p

Lack of NPC1 protein reduces surface but not total levels of Cx43 in astrocytes.

<p>(<b>A</b>) Representative western blots of the relative levels of total Cx43 (left panel) and total Cx43 (right panel) present in cultures of Npc1^+/+, Npc1^+/− and Npc1^−/− astrocytes. Ponceau red (PR) was used as a loading control. The phosphorylated (P1–P2) and non-phosphorylated (NP) forms of Cx43 are indicated on the left. (<b>B</b>) Quantification normalized to Npc1^+/+ levels (wild type, dashed line) of total and surface (white and black bars, respectively) Cx43 levels in Npc1^+/− and Npc1^−/− astrocytes. Total and surface levels of Cx43 were normalized according to the PR levels detected in each lane. *p<0.05 compared to Npc1^+/+. The averaged data were obtained from at least three independent experiments.</p

Npc1^−/− astrocytes exhibit increased Etd uptake mediated by Cx43 hemichannels.

<p>(<b>A</b>) Time-lapse measurements of ethidium (Etd) uptake in Npc1^+/+ and Npc1^−/− (white and black circles, respectively) astrocytes under control conditions or pre-incubated for 15 min with the Cx43 HC blocking antibody Cx43^(E2) (white and black triangles, respectively). (<b>B–C</b>) Fluorescence micrographs of Etd uptake (5 min exposure to dye) in Npc1^+/+ (<b>B</b>) and Npc1^−/− (<b>C</b>) astrocytes.(<b>D</b>) Etd uptake rate of Npc1^+/+ (white bar); Npc1^+/− (gray bar) and NPC1^−/− (black bar) astrocytes under control conditions or treated with 200 µM La³⁺ (acutely added during experiment) or Cx43^(E2) antibody (pre-incubated 15 min before experiments). *<i>p</i><0.05; **<i>p</i><0.005 and ***<i>p</i><0.001 compared to Npc1^+/+ astrocytes. Each value corresponds to the mean ± S.E. of 20 cells in a representative of five experiments. Calibration bar = 60 µm.</p

Npc1^−/− astrocytes exhibit reduced gap junctional communication.

<p>(A-B) Fluorescence micrographs of Lucifer yellow (LY) coupling in Npc1^+/+ (A) and Npc1^−/− (B) astrocytes. The (*) in panels A-B denote the cell microinjected with LY. Calibration bar = 25 µm. (C) Average of incidence of coupling to LY in Npc1^+/+ (white bar); Npc1^+/− (gray bar) and NPC1^−/− (black bar) astrocytes. *p<0.05 and ***p<0.005; compared to Npc1^+/+ condition. The values are the means ± S.E. of 10 cells in a representative experiment of four separate cultures were used for each time point.</p