Abstract
We previously demonstrated that a mutation in the 5′ untranslated region of Diaphanous homolog 3 (DIAPH3) results in 2 to 3-fold overexpression of the gene, leading to a form of delayed onset, progressive human deafness known as AUNA1 (auditory neuropathy, nonsyndromic, autosomal dominant, 1). To investigate the mechanism of deafness, we generated two lines of transgenic mice overexpressing Diap3, the murine ortholog of DIAPH3, on an FVB/NJ background. Line 771 exhibits a relatively mild 20 dB hearing loss at 12 kHz at 4 and 8 weeks of age, progressing to 40 dB and 60 dB losses at 16 and 24 weeks, respectively, at 12 and 24 kHz. Line 924 shows no hearing loss at 4 or 8 weeks, but manifests 35 and 50 dB threshold shifts at 16 and 24 weeks, respectively, at both 12 and 24 kHz. Notably, mice from the two transgenic lines retain distortion product otoacoustic emissions, indicative of normal cochlear outer hair cell (OHC) function despite elevation of auditory thresholds. Scanning electron microscopy of the organ of Corti demonstrates striking anomalies of the inner hair cell (IHC) stereocilia, while OHCs are essentially intact. Over time, IHCs of both lines develop elongated stereocilia that appear fused with neighboring stereocilia, in parallel to the time course of hearing loss in each line. Furthermore, we observe significant reduction in the number of IHC ribbon synapses over 24 weeks in both lines, although this reduction does not correlate temporally with onset and progression of hearing loss or stereociliary anomalies. In summary, overexpression of wild-type Diap3 in two lines of transgenic mice results in hearing loss that recapitulates human AUNA1 deafness. These findings suggest an essential role of Diap3 in regulating assembly and/or maintenance of actin filaments in IHC stereocilia, as well as a potential role at the IHC ribbon synapse.
Highlights
Genetic studies of families with hereditary hearing impairment have allowed the identification of dozens of genes and their encoded proteins that are essential for the development and maintenance of normal hearing
Diaphanous homolog 3 (Diap3) is widely expressed in tissues of wild-type mice We examined Diap3 expression in mRNA from a variety of tissues using quantitative reverse transcription-PCR
Generation of Diap3-overexpressing transgenic mice To investigate the effects of Diap3 overexpression in the cochlea, we generated transgenic mice in which Diap3 was under the control of the human cytomegalovirus (HCMV) immediate early promoter enhancer with chicken beta-actin/rabbit beta-globin hybrid (CAG) promoter
Summary
Genetic studies of families with hereditary hearing impairment have allowed the identification of dozens of genes and their encoded proteins that are essential for the development and maintenance of normal hearing. We previously identified Diaphanous homolog 3 (DIAPH3) as the gene responsible for a form of progressive human hearing loss called AUNA1 (auditory neuropathy, nonsyndromic, autosomal dominant, 1) [1] notable for preservation of cochlear outer hair cell (OHC) function decades after onset of profound deafness [2]. Affected individuals segregate a nucleotide substitution (c.-172G.A) occurring in a highly conserved region of the DIAPH3 59 untranslated region (59UTR), which is sufficient to drive overexpression of a luciferase reporter. This substitution results in 2–3 fold overexpression of DIAPH3 mRNA. DIAPH3 belongs to a family of genes encoding diaphanous-related formins (DRFs). We demonstrate that overexpression of Diap leads to profound abnormalities of the IHC stereocilia and loss of IHC ribbons, associated with delayedonset, progressive hearing loss, with relative preservation of OHC function
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