br Materials and methods br

Materials and methods
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Introduction Hearing loss is the most common congenital sensory deficit. About 1–3 in 1000 children are affected at birth or during early childhood by severe hearing loss, which is defined as prelingual deafness, with at least half of all cases attributable to genetic causes (Korver et al., 2017). Despite extraordinary genetic heterogeneity, mutations in one gene, GJB2, which encodes the connexin 26 protein (Cx26) and is involved in inner ear homeostasis, are found in up to 50% of patients with autosomal recessive non-syndromic hearing loss (Kelsell et al., 1997). Beside this non-syndromic form of deafness, GJB2 mutations cause several types of syndromic hearing loss associated with skin diseases with variable prognosis (Avshalumova et al., 2014; Lilly et al., 2016; Xu and Nicholson, 2013). In mammals, sounds are perceived through mechanosensory hair cells located within the sensory epithelium of the cochlea (i.e. the organ of Corti). Within the organ of Corti, sensory inner and outer hair cells and non-sensory supporting cells are organized in a regular mosaic pattern that extends along the basal-to-apical axis of the cochlear duct. Cx26 gap junction protein, which assembles to form channels between cochlear supporting cells, allows the rapid removal of K+ away from the Arecaidine but-2-ynyl ester tosylate of sensory hair cells, resulting in the recycling of this ion back to the endolymph to maintain cochlear homeostasis (Kikuchi et al., 2000). However, gap junctions may serve additional roles in the cochlea, such as providing networks for nutrient transfer (Chang et al., 2008; Jagger and Forge, 2015). Cx26 and Cx30 (encoded by the deafness gene GJB6 (Grifa et al., 1999)) are the two most abundantly expressed gap junction proteins in the cochlea and form heteromeric and heterotypic channels between adjacent supporting cells, from the spiral limbus to the cochlear spiral ligament (Ahmad et al., 2003; Sun et al., 2005) (Fig. 1A). Although the two channel components Cx26 and Cx30 are well characterized, the gap junction plaque (GJP) assembly mechanisms occurring in situ remain largely unknown. Of note, however, is the fact that these two proteins are not functionally equivalent, since Cx26 has been shown as the key organizer of the gap junction macromolecular complex (Kamiya et al., 2014). Beside mutations that affect the Cx26 channel function itself, many of the disease-causing mutations in GJB2 impair the trafficking and delivery of Cx26 to the cell surface, what prevents the formation of gap junctions (Ambrosi et al., 2013; Hoang Dinh et al., 2009; Xu and Nicholson, 2013). Thus, deciphering the trafficking pathway of cochlear Cx26 should represent an advance in understanding the pathogenic significance of these mutations. Gap junction assembly usually occurs in a “two-step mechanism” which requires successively microtubules and actin cytoskeletal components. First, hexameric connexons assembled in the trans-Golgi network are trafficked along microtubules to the non-junctional plasma membrane (Koval et al., 1997; Lauf et al., 2002; Musil and Goodenough, 1993). Secondly, hemichannels associate with cortical actin through actin-binding proteins zonula occludens (ZOs) which regulate delivery of connexins from the periphery to the GJP (Hervé et al., 2014; Thévenin et al., 2013). The membrane lipid environment is critical for gap junction assembly and function (Cascio, 2005; Defamie and Mesnil, 2012). Junctional connexins assemble into lipid raft microdomains whereas non-junctional connexins are present in non-lipid raft fractions (Defamie and Mesnil, 2012; Hunter et al., 2005; Musil and Goodenough, 1991). Because of the relatively short half-life of connexins (usually 1 – 5 h), the junctional plaque is in a dynamic state, constantly remodeled through both recruitment of newly synthesized connexons to the periphery and endocytosis of older components from the center of the plaque (Gaietta et al., 2002).