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Hereditary deafness affects approximately 1 in 2,000 children. Mutations in the gene encoding the cochlear gap junction protein connexin 26 (CX26) cause prelingual, nonsyndromic deafness and are responsible for as many as 50% of hereditary deafness cases in certain populations. Connexin-associated deafness is thought to be the result of defective development of auditory sensory epithelium due to connexion dysfunction. Surprisingly, CX26 deficiency is not compensated for by the closely related connexin CX30, which is abundantly expressed in the same cochlear cells. Here, using two mouse models of CX26-associated deafness, we demonstrate that disruption of the CX26-dependent gap junction plaque (GJP) is the earliest observable change during embryonic development of mice with connexin-associated deafness. Loss of CX26 resulted in a drastic reduction in the GJP area and protein level and was associated with excessive endocytosis with increased expression of caveolin 1 and caveolin 2. Furthermore, expression of deafness-associated CX26 and CX30 in cell culture resulted in visible disruption of GJPs and loss of function. Our results demonstrate that deafness-associated mutations in CX26 induce the macromolecular degradation of large gap junction complexes accompanied by an increase in caveolar structures.

Background: Induced pluripotent stem (iPS) cells aremultipotentstem cellsthat can be producedfrom adult cells such as fibroblastsby the induction ofartificial reprogramming. Since such cells can be differentiated to endoderm, mesodermand ectoderm asembryonic stem (ES) cells, theirpotential utility for the regenerative therapy of various organs is expected. The inner ear is composed of various types of functional cells (hair cells, supporting cells, spiral ganglion, cochlear fibrocytes, striavascularis, etc.), and each cell type. Although the degeneration of these cells leads directly to severe hearing loss, there are few reports concerning thedifferentiation of iPS cells to various types of inner ear cells for regenerative therapy. Results: In this study, we co-cultured iPS-derived cells with three different regions (spiral ganglion, the organ of Corti with spiral limbus, lateral wall) of the cochlear tissue and tod attempted to induce their differentiation into various types of inner ear cells. The number of positive colonies (Nanogpositive colony) with reporter GFP controlled by Nanogpromotor was counted to assess the undifferentiated level of the cells. In the co-cultured cells with spiral ganglion, many Nanog-positive colonies were observed, and many cells with neurite-like protrusions were observed among the colonies. In the co-culture with the organ of Cortiand spiral limbus (OC/SL), neuron-like cells were observed on the cell layer with tight junction-like adhesions. In the co-culture with the cochlear lateral wall, many cell layers with tight junction-like adhesions were observed, while undifferentiated Nanogpositive colonies were not observed. Conclusion: In this study, we demonstrated that differentiation of iPS cells to cochlear cells was regulated depending on the part of the co-cultured organs. Co-culture with cochlea tissue and the induced pluripotent stem cells may enable regenerative therapy of various types of inner ear cells.

Background The greater epithelial ridge (GER) is a developmental structure in the maturation of the organ of Corti. Situated near the inner hair cells of neonatal mice, the GER undergoes a wave of apoptosis after postnatal day 8 (P8). We evaluated the GER from P8 to P12 in transgenic mice that carry the R75W + mutation, a dominant-negative mutation of human gap junction protein, beta 2, 26 kDa (GJB2) (also known as connexin 26 or CX26). Cx26 facilitate intercellular communication within the mammalian auditory organ. Results In both non-transgenic (non-Tg) and R75W + mice, some GER cells exhibited apoptotic characteristics at P8. In the GER of non-Tg mice, both the total number of cells and the number of apoptotic cells decreased from P8 to P12. In contrast, apoptotic cells were still clearly evident in the GER of R75W + mice at P12. In R75W + mice, therefore, apoptosis in the GER persisted until a later stage of cochlear development. In addition, the GER of R75W + mice exhibited morphological signs of retention, which may have resulted from diminished levels of apoptosis and/or promotion of cell proliferation during embryogenesis and early postnatal stages of development. Conclusions Here we demonstrate that Cx26 dysfunction is associated with delayed apoptosis of GER cells and GER retention. This is the first demonstration that Cx26 may regulate cell proliferation and apoptosis during development of the cochlea.