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Fabio Mammano"Calcium waves in the inner ear"

Abstract :

Propagating Ca2+ signals in the cochlea (Gale et al., Current Biology, 14: 526-529, 2004), depend on ATP release at the endolymphatic surface of the neurosensory epithelium and have been recently linked by others to the development of afferent synapses in the cochlea (Tritsch NX et al., Nature, 450: 50, 2007).
Using four types of genetically targeted mice, we show unequivocally that Connexin 26 (Cx26) and connexin (Cx30), the two most abundant connexins in the inner ear, contribute in two strictly interdependent ways to the generation and propagation of cochlear Ca2+ waves, acting as conduits for both ATP and second messengers. Cx26 and Cx30 are encoded by two genes (GJB2 and GJB6, respectively) that are found within 50 kb in the same complex deafness locus, named DFNB1. By showing that a specific defect of Cx26 affects metabolic coupling mediated by IP3 we have recently offered a mechanistic explanation for the pathogenesis of deafness due to connexin mutations (Beltramello et al, Nature Cell Biology, 7:63-69, 2005; Hernandez et al. Nature Methods 4(4):353-8). Abnormal or impaired connexin function has been linked to several other diseases, including skin disease, peripheral neuropathies, and cataracts, thus our results may have a more general impact. Here we show that Cx26 is down–regulated in Cx30 KO cultures and consequently Ca2+ signals evoked by UV photolysis of caged IP3 fail to propagate from cell to cell in this DFNB1 model system, generated by Teubner B. et al. (Hum Mol Genet 12:13-21, 2003). The reciprocal is also true, as ablation of Cx26 causes Cx30 downregulation. We showed this by targeting Cx26 in cochlear cultures of the Cx26loxP/loxP model (Cohen-Salmon M, et al. Current Biology 12:1106-1111, 2002) using a novel viral vector (BAAV) that transduces cells of the inner ear with high efficacy (Di Pasquale et al. Mol Therapy, Mol Ther 11, 849-55, 2005). Thus our data offer critical insight into the hitherto unexplained observation that some deafness associated DFNB1 alleles are characterized by hereditable significant reduction of both GJB2 and GJB6 expression. Reintroduction of Cx30 in Cx30 KO cultures by the BAAV gene delivery system upregulates also Cx26 and restores Ca2+ signaling. Finally we demonstrate that Ca2+ oscillations evoked by threshold levels of ATP (20 nM) in cochlear cultures cause upregulation of Cx26 mRNA. This effect is suppressed by transducing cultures with a BAAV vector that expresses IkB(sr), a stable form of the IkB repressor protein resistant to degradation which blocks the activation/translocation of NFkB to the nucleus.
In summary, we demonstrate that Cx26 and Cx30 of the inner ear play a dual role in Ca2+ signaling: as hemichannels, they promote ATP release to the endolymphatic compartment; as gap junction channels, they allow diffusion second messenger across coupled cells. In turn, Ca2+ signals evoked by ATP regulate connexin expression through NFkB, a Ca2+-dependent transcription factor. Thus our data reveal a Ca2+-dependent co-regulation in the expression of inner ear connexins implicated in hereditary deafness, as well as the potential of a gene therapy approach to treat connexin dysfunction.

Selected publications

Mammano F, Bortolozzi M, Ortolano S, Anselmi F.
Ca2+ signaling in the inner ear.
Physiology (Bethesda). 2007 Apr;22:131-44. Review.
Hernandez VH, Bortolozzi M, Pertegato V, Beltramello M, Giarin M, Zaccolo M, Pantano S, Mammano F.
Unitary permeability of gap junction channels to second messengers measured by FRET microscopy.
Nat Methods. 2007 Apr;4(4):353-8. Epub 2007 Mar 11.
Ficarella R, Di Leva F, Bortolozzi M, Ortolano S, Donaudy F, Petrillo M, Melchionda S, Lelli A, Domi T, Fedrizzi L, Lim D, Shull GE, Gasparini P, Brini M, Mammano F, Carafoli E.
A functional study of plasma-membrane calcium-pump isoform 2 mutants causing digenic deafness.
Proc Natl Acad Sci U S A. 2007 Jan 30;104(5):1516-21. Epub 2007 Jan 18.