Diaphanous homolog 3 (Diap3) Overexpression Causes Progressive Hearing Loss and Inner Hair Cell Defects in a Transgenic Mouse Model of Human Deafness

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.我们此前已证实，Diaphanous同源物3（DIAPH3）的5′非翻译区（5′ untranslated region）发生突变可导致该基因过表达2至3倍，进而引发一种迟发性、进行性人类耳聋，命名为AUNA1（非综合征性常染色体显性遗传性听神经病1型，auditory neuropathy, nonsyndromic, autosomal dominant, 1）。为探究该耳聋的发病机制，我们在FVB/NJ小鼠背景上构建了两株过表达Diap3的转基因小鼠——Diap3为DIAPH3的小鼠同源物。771品系小鼠在4周和8周龄时，于12 kHz频率下表现出轻度听力损失（20 dB）；随年龄增长，在16周龄和24周龄时，该损失分别进展至40 dB和60 dB（检测频率为12 kHz和24 kHz）。924品系小鼠在4周和8周龄时无听力损失，但在16周龄和24周龄时，于12 kHz和24 kHz频率下分别出现35 dB和50 dB的听阈偏移。值得注意的是，两株转基因小鼠均保留畸变产物耳声发射（distortion product otoacoustic emissions），提示尽管听阈升高，其耳蜗外毛细胞（outer hair cell, OHC）功能仍正常。对柯蒂氏器（organ of Corti）的扫描电子显微镜观察显示，其内毛细胞（inner hair cell, IHC）静纤毛出现显著异常，而外毛细胞基本完好。随着时间推移，两品系小鼠的内毛细胞静纤毛均出现伸长，并与相邻静纤毛发生融合，这一变化与各品系小鼠听力损失的时间进程相一致。此外，我们观察到，两品系小鼠在24周内的内毛细胞带状突触（ribbon synapses）数量均显著减少，但该减少的时间进程与听力损失的发生、进展以及静纤毛异常均无相关性。综上，在两株转基因小鼠中过表达野生型Diap3可引发听力损失，该表型重现了人类AUNA1耳聋的特征。本研究结果表明，Diap3在调控内毛细胞静纤毛内肌动蛋白丝（actin filaments）的组装和/或维持过程中发挥关键作用，同时可能在内毛细胞带状突触中也具有潜在功能。