BAAV mediated GJB2 gene transfer restores gap junction coupling in cochlear organotypic cultures from deaf Cx26Sox10Cre mice.

Giulia Crispino,Laura Rodriguez,Edoardo Arslan,Mario Bortolozzi,Rosa Maria Santarelli,Pietro Scimemi,John A Chiorini,Giovanni Di Pasquale,Fabio Mammano,Romolo Daniele De Siati,Fabian Galindo Ramirez,Bruce Riley

doi:10.1371/journal.pone.0023279

Abstract

The deafness locus DFNB1 contains GJB2, the gene encoding connexin26 and GJB6, encoding connexin30, which appear to be coordinately regulated in the inner ear. In this work, we investigated the expression and function of connexin26 and connexin30 from postnatal day 5 to adult age in double transgenic Cx26Sox10Cre mice, which we obtained by crossing connexin26 floxed mice with a deleter Sox10–Cre line. Cx26Sox10Cre mice presented with complete connexin26 ablation in the epithelial gap junction network of the cochlea, whereas connexin30 expression was developmentally delayed; immunolabeling patterns for both connexins were normal in the cochlear lateral wall. In vivo electrophysiological measurements in Cx26Sox10Cre mice revealed profound hearing loss accompanied by reduction of endocochlear potential, and functional experiments performed in postnatal cochlear organotypic cultures showed impaired gap junction coupling. Transduction of these cultures with a bovine adeno associated virus vector restored connexin26 protein expression and rescued gap junction coupling. These results suggest that restoration of normal connexin levels by gene delivery via recombinant adeno associated virus could be a way to rescue hearing function in DFNB1 mouse models and, in future, lead to the development of therapeutic interventions in humans.

Highlights

Connexins are tetraspan transmembrane proteins that form hexameric assemblies in the plasma membrane called connexons; head–to–head docking of two connexons in adjacent cells establishes intercellular channels that cluster into a plaque, and the two adjoining plasma membranes in the plaque remain separated by a narrow extracellular gap of 2–3 nm [1].GJB2, the gene encoding connexin26 (Cx26) was the first gene to be linked to an autosomal recessive form of deafness, DFNB1 [2], as well as to a rare dominant form of deafness, DFNA3 [3]
Auditory function in Cx26Sox10Cre mice and in Cx26loxP/loxP mice, taken as controls, was quantified by recording auditory brainstem responses (ABR) which are electrical signals evoked from the brainstem following the presentation of sound stimuli (Figure 1a)
Compared to Cx26loxP/loxP mice, thresholds were significantly elevated in Cx26Sox10Cre mice (P,0.001, ANOVA) and in excess of 90 dB sound pressure level (SPL) for tone bursts as well as for click stimuli, whereas endocochlear potential (Figure 1c) was significantly reduced (3862 mV in Cx26Sox10Cre mice, n = 7, vs. 10963 mV in Cx26loxP/loxP mice, n = 11; P,0.001, ANOVA)

Summary

Introduction

Connexins are tetraspan transmembrane proteins that form hexameric assemblies in the plasma membrane called connexons; head–to–head docking of two connexons in adjacent cells establishes intercellular channels that cluster into a plaque, and the two adjoining plasma membranes in the plaque remain separated by a narrow extracellular gap of 2–3 nm [1].GJB2, the gene encoding connexin (Cx26) was the first gene to be linked to an autosomal recessive form of deafness, DFNB1 [2], as well as to a rare dominant form of deafness, DFNA3 [3]. More than 90 distinct recessive mutations of GJB2 have been described, including nonsense, missense, splicing, frame–shift mutations and inframe deletions [4] (see http://davinci.crg.es/deafness/ index.php). These mutations account for approximately 50% of congenital, recessively inherited, sensorineural nonsyndromic hearing loss in several populations, with approximate carrier frequency of 1 in 33 and up to 1 in 28 amongst Mediterraneans [5] (see http://hereditaryhearingloss.org/). DFNB1–linked familial cases with no mutation in GJB2 have been reported and shown to be associated with two large deletions occurring upstream of GJB2 in GJB6, the gene encoding connexin (Cx30) which lies 30 kb telomeric to GJB2 on human chromosome (chromosome in the mouse) [4]. A threonine–to–methionine substitution at position 5 is the only Cx30 mutation (Cx30T5M) associated to DFNA3 [6]

Methods

Results

Conclusion