GJB2 Mutations Linked to Hearing Loss Exhibit Differential Trafficking and Functional Defects as Revealed in Cochlear-Relevant Cells

Abstract

GJB2 gene (that encodes Cx26) mutations are causal of hearing loss highlighting the importance of Cx26-based channel signaling amongst the supporting cells in the organ of Corti. While the majority of these GJB2 mutations are inherited in an autosomal recessive manner, others are inherited in an autosomal dominant manner and lead to syndromic hearing loss as well as skin diseases. To assess if common or divergent mechanisms are at the root of GJB2-linked hearing loss, we expressed several mutants in cochlear-relevant HEI-OC1 cells derived from the developing organ of Corti. Since supporting cells of the mature mammalian organ of Corti have negligible Cx43, but HEI-OC1 cells are rich in Cx43, we first used CRISPR-Cas9 to ablate endogenous Cx43, thus establishing a connexin-deficient platform for controlled reintroduction of hearing-relevant connexins and Cx26 mutants. We found three distinct outcomes and cellular phenotypes when hearing loss-linked Cx26 mutants were expressed in cochlear-relevant cells. The dominant syndromic Cx26 mutant N54K had trafficking defects and did not fully prevent wild-type Cx26 gap junction plaque formation but surprisingly formed gap junctions when co-expressed with Cx30. In contrast, the dominant syndromic S183F mutant formed gap junctions incapable of transferring dye and, as expected, co-localized in the same gap junctions as wild-type Cx26 and Cx30, but also gained the capacity to intermix with Cx43 within gap junctions. Both recessive non-syndromic Cx26 mutants (R32H and R184P) were retained in intracellular vesicles including early endosomes and did not co-localize with Cx30. As might be predicted, none of the Cx26 mutants prevented Cx43 gap junction plaque formation in Cx43-rich HEI-OC1 cells while Cx43-ablation had little effect on the expression of reference genes linked to auditory cell differentiation. We conclude from our studies in cochlear-relevant cells that the selected Cx26 mutants likely evoke hearing loss via three unique connexin defects that are independent of Cx43 status.

Functional Evaluation of a Rare Variant c.516G>C (p.Trp172Cys) in the GJB2 (Connexin 26) Gene Associated with Nonsyndromic Hearing Loss

“…Press et al (2017) revealed that S183F mutant has defects in connexin trafficking as well as impaired gap junction and hemichannel function and exhibited transdominant effects on co-expressed wild type Cx26, Cx30, and Cx43 [ 48 ]. Consistent with previous studies of this mutation, Beach et al [ 19 ] reported that the dominant syndromic S183F mutant was able to traffic to the plasma membrane (although some intracellular reservoirs of S183F were found) and to form gap junctions incapable of transferring a dye [ 48 , 70 ]. In addition, the S183F mutant co-localized in the same gap junctions as wild type Cx26 and Cx30, and gained a capacity to intermix with Cx43 within gap junctions [ 70 ].…”

“…Mani et al (2009) described defective localization of the R184P mutant protein found largely in the cytoplasm [ 40 ]. The results of Bruzzone et al (2003) in the paired Xenopus oocytes expression system, as well as of Beach et al (2020) in the cochlear-relevant HEI-OC1 cells, supported the R184P inability to form intercellular channels [ 19 , 28 ]. The recessive mutation p.C169Y previously classified as a polymorphism, has been identified as causative of severe hearing loss in two Qatari families.…”

“…Although about 30 different ”pathogenic” and ”likely pathogenic” missense variants have been reported in E2, the functional consequences have been examined only for a fraction of them [ 19 , 25 , 27 , 28 , 38 , 40 , 41 , 43 , 46 , 48 , 69 , 70 ] ( Table S1 , Figure 5 A). Ten mutations were analyzed in these functional studies: dominant mutations (nonsyndromic p.M163L, p.D179N, p.R184Q, and syndromic p.S183F associated with deafness and focal palmoplantar keratoderma), mutations linked to nonsyndromic recessive deafness (p.F161S, p.C169Y, p.W172R, p.P173R, and p.R184P), and an ambiguously defined (recessive or dominant) mutation p.M163V ( Figure 5 A).…”

“…Different pathogenic mutations in the GJB2 gene can affect all stages of the Cx26 life cycle: biosynthesis, post-translational modifications, and degradation of Cx26 molecules, their oligomerization into connexons, intracellular trafficking of connexons to the plasma membrane, normal docking of the connexons from neighboring cells to form gap junction channels, and interactions between other co-expressed connexins [ 4 , 16 , 17 , 18 , 19 ]. The majority of GJB2 mutations are recessively inherited and, being in homozygous or compound heterozygous states, result in nonsyndromic recessive deafness (DFNB1) while some of GJB2 mutations are dominantly inherited and lead to nonsyndromic autosomal dominant deafness (DFNA3) or several different syndromes associating hearing loss with skin disorders [ 2 , 4 , 20 , 21 , 22 , 23 , 24 , 25 ].…”

Hearing Phenotypes of Patients with Hearing Loss Homozygous for the GJB2 c.235delc Mutation

“…Sensorineural HL (SNHL) is the most common form of HL and typically is caused by a loss of functional sensory hair cells (HCs) and supporting cells (SCs) within the cochlea [3,4]. HCs and SCs develop from common progenitor cells within the prosensory domain of the developing cochlea [5]. HCs transfer mechanical vibration into an acoustic electrical signal, which is then transmitted to the auditory cortex via spiral ganglion neurons (SGNs).…”