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Bonora M.,University of Ferrara | De Marchi E.,University of Ferrara | Patergnani S.,University of Ferrara | Suski J.M.,University of Ferrara | And 12 more authors.
Cell Death and Differentiation

Mitochondrial defects, affecting parameters such as mitochondrial number and shape, levels of respiratory chain complex components and markers of oxidative stress, have been associated with the appearance and progression of multiple sclerosis. Nevertheless, mitochondrial physiology has never been monitored during oligodendrocyte progenitor cell (OPC) differentiation, especially in OPCs challenged with proinflammatory cytokines. Here, we show that tumor necrosis factor alpha (TNF-α) inhibits OPC differentiation, accompanied by altered mitochondrial calcium uptake, mitochondrial membrane potential, and respiratory complex I activity as well as increased reactive oxygen species production. Treatment with a mitochondrial uncoupler (FCCP) to mimic mitochondrial impairment also causes cells to accumulate at the progenitor stage. Interestingly, AMP-activated protein kinase (AMPK) levels increase during TNF-α exposure and inhibit OPC differentiation. Overall, our data indicate that TNF-α induces metabolic changes, driven by mitochondrial impairment and AMPK activation, leading to the inhibition of OPC differentiation. © 2014 Macmillan Publishers Limited All rights reserved. Source

Decrock E.,Ghent University | Decrock E.,Doctoral Research Fellows | Krysko D.V.,Molecular Signalling and Cell Death Unit | Krysko D.V.,Ghent University | And 21 more authors.
Cell Death and Differentiation

Decades of research have indicated that gap junction channels contribute to the propagation of apoptosis between neighboring cells. Inositol 1,4,5-trisphosphate (IP 3) has been proposed as the responsible molecule conveying the apoptotic message, although conclusive results are still missing. We investigated the role of IP 3 in a model of gap junction-mediated spreading of cytochrome C-induced apoptosis. We used targeted loading of high-molecular-weight agents interfering with the IP 3 signaling cascade in the apoptosis trigger zone and cell death communication zone of C6-glioma cells heterologously expressing connexin (Cx)43 or Cx26. Blocking IP 3 receptors or stimulating IP 3 degradation both diminished the propagation of apoptosis. Apoptosis spread was also reduced in cells expressing mutant Cx26, which forms gap junctions with an impaired IP 3 permeability. However, IP 3 by itself was not able to induce cell death, but only potentiated cell death propagation when the apoptosis trigger was applied. We conclude that IP 3 is a key necessary messenger for communicating apoptotic cell death via gap junctions, but needs to team up with other factors to become a fully pro-apoptotic messenger. © 2012 Macmillan Publishers Limited. All rights reserved. Source

Majumder P.,Istituto Veneto di Medicina Molecolare | Crispino G.,Istituto Veneto di Medicina Molecolare | Rodriguez L.,Istituto Veneto di Medicina Molecolare | Ciubotaru C.D.,Istituto Veneto di Medicina Molecolare | And 9 more authors.
Purinergic Signalling

Connexin 26 (Cx26) and connexin 30 (Cx30) form hemichannels that release ATP from the endolymphatic surface of cochlear supporting and epithelial cells and also form gap junction (GJ) channels that allow the concomitant intercellular diffusion of Ca2+ mobilizing second messengers. Released ATP in turn activates G-protein coupled P2Y2 and P2Y4 receptors, PLC-dependent generation of IP3, release of Ca2+ from intracellular stores, instigating the regenerative propagation of intercellular Ca2+ signals (ICS). The range of ICS propagation is sensitive to the concentration of extracellular divalent cations and activity of ectonucleotidases. Here, the expression patterns of Cx26 and Cx30 were characterized in postnatal cochlear tissues obtained from mice aged between P5 and P6. The expression gradient along the longitudinal axis of the cochlea, decreasing from the basal to the apical cochlear turn (CT), was more pronounced in outer sulcus (OS) cells than in inner sulcus (IS) cells. GJ-mediated dye coupling was maximal in OS cells of the basal CT, inhibited by the nonselective connexin channel blocker carbenoxolone (CBX) and absent in hair cells. Photostimulating OS cells with caged inositol (3,4,5) tri-phosphate (IP3) resulted in transfer of ICS in the lateral direction, from OS cells to IS cells across the hair cell region (HCR) of medial and basal CTs. ICS transfer in the opposite (medial) direction, from IS cells photostimulated with caged IP3 to OS cells, occurred mostly in the basal CT. In addition, OS cells displayed impressive rhythmic activity with oscillations of cytosolic free Ca2+ concentration ([Ca2+]i) coordinated by the propagation of Ca2+ wavefronts sweeping repeatedly through the same tissue area along the coiling axis of the cochlea. Oscillations evoked by uncaging IP3 or by applying ATP differed greatly, by as much as one order of magnitude, in frequency and waveform rise time. ICS evoked by direct application of ATP propagated along convoluted cellular paths in the OS, which often branched and changed dynamically over time. Potential implications of these findings are discussed in the context of developmental regulation and cochlear pathophysiology. © 2010 Springer Science+Business Media B.V. Source

Schutz M.,University of Bonn | Scimemi P.,University of Padua | Majumder P.,Istituto Veneto di Medicina Molecolare | de Siati R.D.,University of Padua | And 14 more authors.
Human Molecular Genetics

Mutations in the GJB2 and GJB6 genes, respectively, coding for connexin26 (Cx26) and connexin30 (Cx30) proteins, are the most common cause for prelingual non-syndromic deafness in humans. In the inner ear, Cx26 and Cx30 are expressed in different non-sensory cell types, where they largely co-localize and may form heteromeric gap junction channels. Here, we describe the generation and characterization of a mouse model for human bilateral middle/high-frequency hearing loss based on the substitution of an evolutionarily conserved threonine by a methionine residue at position 5 near the N-terminus of Cx30 (Cx30T5M). The mutation was inserted in the mouse genome by homologous recombination in mouse embryonic stem cells. Expression of the mutated Cx30T5M protein in these transgenic mice is under the control of the endogenous Cx30 promoter and was analysed via activation of the lacZ reporter gene. When probed by auditory brainstem recordings, Cx30T5M/T5M mice exhibited a mild, but significant increase in their hearing thresholds of about 15 dB at all frequencies. Immunolabelling with antibodies to Cx26 or Cx30 suggested normal location of these proteins in the adult inner ear, but western blot analysis showed significantly down-regulated the expression levels of Cx26 and Cx30. In the developing cochlea, electrical coupling, probed by dual patch-clamp recordings, was normal. However, transfer of the fluorescent tracer calcein between cochlear non-sensory cells was reduced, as was intercellular Ca2+ signalling due to spontaneous ATP release from connexin hemichannels. Our findings link hearing loss to decreased biochemical coupling due to the point-mutated Cx30 in mice. © The Author 2010. Published by Oxford University Press. Source

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