Construction of translocase of inner mitochondrial membrane 8A gene knockout mice and study of its inner ear function

Abstract

Objective·To explore the hearing phenotype of Timm8a-/- mice and the function of translocase of inner mitochondrial membrane 8A (Timm8a) gene in inner ear by constructing Timm8a gene knockout mice.Methods·Timm8a-/- mice were designed and constructed. PCR and Western blotting were used to verify whether the construction was successful. The body size and weight of Timm8a-/- mice and wild type (WT) mice aged one-month were observed and compared. Immunofluorescence staining was used to observe the expression and distribution of TIMM8A protein in the cochlea of WT mice. Auditory brainstem response (ABR) was used to compare the hearing threshold of Timm8a-/- mice and WT mice. Toluidine blue staining was performed to observe the morphology of organ of Corti (OC), spiral ganglion neuron (SGN) and stria vascularis (SV) in the inner ear of the two kinds of mice. Transmission electron microscope was used to observe the ultrastructure of mitochondria in inner ear hair cells (HCs), SGN cells and SV cells of the two kinds of mice, and the proportion of abnormal mitochondria was counted.Results·The results of PCR showed that Timm8a gene had been successfully knocked out, and the results of Western blotting showed that there was no TIMM8A proteins in the cochlea, brain, heart and skeletal muscle tissues. The both results indicated that Timm8a-/- mice were successfully constructed. The results of immunofluorescence staining showed that TIMM8A was abundantly expressed in the cochlea of WT mice, and was highly expressed in the OC, SGN and SV. Compared with WT mice, Timm8a-/- mice aged one-month developed slower, had lighter body weight and significantly higher hearing threshold (all P<0.05), their amplitude of Ⅰ wave of ABR was decreased and the latency was prolonged. The results of toluidine blue staining showed that compared with WT mice, there was no significant change in the shape and number of SGN cells in the inner ear, but the thickness of SV in the middle turn and basal turn of the cochlea was reduced (both P<0.05). The results of transmission electron microscope showed that compared with WT mice, the structure of mitochondria in inner ear HCs, SGN cells and SV cells of Timm8a-/- mice was abnormal, and the proportion of abnormal mitochondria was significantly increased (all P<0.05).Conclusion·Timm8a-/- mice are successfully constructed in this study, and the elevated hearing threshold of Timm8a-/- mice may be related to the abnormal ultrastructure of mitochondria in inner ear.

Keywords

• translocase of inner mitochondrial membrane 8a (timm8a)
• mohr-tranebjaerg syndrome (mts)
• auditory neuropathy spectrum disorder (ansd)
• mitochondria