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. 2007 Jan 23;104(4):1337-41.
doi: 10.1073/pnas.0606855104. Epub 2007 Jan 16.

Restoration of connexin26 protein level in the cochlea completely rescues hearing in a mouse model of human connexin30-linked deafness

Shoeb Ahmad  1 , Wenxue Tang, Qing Chang, Yan Qu, Jill Hibshman, Yuhua Li, Goran Söhl, Klaus Willecke, Ping Chen, Xi Lin
Affiliations

Restoration of connexin26 protein level in the cochlea completely rescues hearing in a mouse model of human connexin30-linked deafness

Shoeb Ahmad et al. Proc Natl Acad Sci U S A. .

Abstract

Mutations in genes coding for connexin26 (Cx26) and/or Cx30 are linked to approximately half of all cases of human autosomal nonsyndromic prelingual deafness. Cx26 and Cx30 are the two major Cx isoforms found in the cochlea, and they coassemble to form hybrid (heteromeric and heterotypic) gap junctions (GJs). This molecular arrangement implies that homomeric GJs would remain in the cochlea if one of the coassembly partners were mutated resulting in null expression. We generated mice in which extra copies of the Cx26 gene were transgenically expressed from a modified bacterial artificial chromosome in a Cx30-/- background. In the absence of the Cx30 gene, Cx26 expressed from extra alleles completely restored hearing sensitivity and prevented hair cell death in deaf Cx30-/- mice. The results indicated that hybrid GJs consisting of Cx26 and Cx30 were not essential for normal hearing in mice and suggested that up-regulation of Cx26 or slowing down its protein degradation might be a therapeutic strategy to prevent and treat deafness caused by Cx30 mutations.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Structures of Cx30 loci in WT, Cx30−/−, and BACCx26;Cx30−/− mice. (A) Illustration of probe location and expected size of the DNA fragments treated by EcoNI for Cx30−/− (Top), WT (Middle), and BACCx26 (Bottom) mice. (B) Results of Southern blot hybridizations obtained with original and modified BAC, as well as with WT, Cx30+/−, Cx30−/−, and BACCx26;Cx30−/− mice.
Fig. 2.
Fig. 2.
Restoration of Cx26 protein levels in the cochlea by transgenic expressions of BACCx26 in Cx30−/− mice. (A) Western blots showing Cx26 protein expression levels in the cochlea and liver. Results obtained with GADPH are shown as controls for the amount of protein loading in each lane. (B) Quantification of the relative Cx26 protein levels normalized to the WT level in the cochlea and liver in Cx30−/− and BACCx26;Cx30−/− mice. (C) Cx26 protein levels in the cochlea of WT and Cx30−/− mice at different postnatal days. (D) The Cx26 protein expression levels in Cx30−/− mice at different developmental stages were quantified by normalizing to their corresponding WT level.
Fig. 3.
Fig. 3.
Comparisons of immunolabeling patterns of Cx30 (A–C) and Cx26 (D–F) in the cochlea of WT (A and D), Cx30−/− (B and E), and BACCx26;Cx30−/− (C and F) mice. These are not confocal images. The intensities of immunolabeling in different images are not quantitatively comparable because they were obtained from different cochlear samples, and images were acquired with different exposure times. (Scale bar, ≈200 μm.)
Fig. 4.
Fig. 4.
Prevention of apoptosis and hair-cell degeneration by transgenic expression of BACCx26 in Cx30−/− mice. Apoptosis TUNEL staining pattern and morphology of the organ of Corti are given for WT (Left), Cx30−/− (Center), and BACCx26;Cx30−/− (Right) mice. (A) (Upper) Cochlear sections imaged with conventional phase-contrast optics. (Lower) TUNEL staining of corresponding cochlear sections comparing apoptosis in the cochlea of WT, Cx30−/−, and BACCx26;Cx30−/− mice. (Scale bar, ≈200 μm.) (B) Confocal views of whole-mount cochleae obtained from WT, Cx30−/−, and BACCx26;Cx30−/− mice (2 months old). The whole-mount cochlear samples were immunolabeled with a hair-cell-specific antibody against myosin VI. Examples of labeled inner and outer hair cells are indicated by arrowheads and arrows, respectively (Left). Outer hair cells were completely missing in the 2-month-old Cx30−/− mice, and inner hair cells in these mice were degenerated (Center). The cochlear morphology of BACCx26;Cx30−/− mice was indistinguishable from WT mice (comparing Left and Right). (Scale bar, ≈60 μm.)
Fig. 5.
Fig. 5.
Restoration of hearing sensitivities in Cx30−/− mice by transgenic expression of BACCx26. Hearing thresholds of WT, Cx30−/−, BACCx26;Cx30−/−, and BACNeoR−HcRed;Cx30−/− mice were measured by ABR tests across a frequency range of 4–32 kHz. Legends indicate the grouping and age of mice. Upward arrows indicate that the hearing threshold is >90 dB, the loudest sound level that could be generated by the sound transducer used in our experiments.
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