PDF(Pubmed)
Abstract：
Auditory neuropathy spectrum disorder (ANSD) is a hearing impairment caused by dysfunction of inner hair cells, ribbon synapses, spiral ganglion neurons and/or the auditory nerve itself. Approximately 1/7000 newborns have abnormal auditory nerve function, accounting for 10%-14% of cases of permanent hearing loss in children. Although we previously identified the AIFM1 c.1265 G > A variant to be associated with ANSD, the mechanism by which ANSD is associated with AIFM1 is poorly understood. We generated induced pluripotent stem cells (iPSCs) from peripheral blood mononuclear cells (PBMCs) via nucleofection with episomal plasmids. The patient-specific iPSCs were edited via CRISPR/Cas9 technology to generate gene-corrected isogenic iPSCs. These iPSCs were further differentiated into neurons via neural stem cells (NSCs). The pathogenic mechanism was explored in these neurons. In patient cells (PBMCs, iPSCs, and neurons), the AIFM1 c.1265 G > A variant caused a novel splicing variant (c.1267-1305del), resulting in AIF p.R422Q and p.423-435del proteins, which impaired AIF dimerization. Such impaired AIF dimerization then weakened the interaction between AIF and coiled-coil-helix-coiled-coil-helix domain-containing protein 4 (CHCHD4). On the one hand, the mitochondrial import of ETC complex subunits was inhibited, subsequently leading to an increased ADP/ATP ratio and elevated ROS levels. On the other hand, MICU1-MICU2 heterodimerization was impaired, leading to mCa2+ overload. Calpain was activated by mCa2+ and subsequently cleaved AIF for its translocation into the nucleus, ultimately resulting in caspase-independent apoptosis. Interestingly, correction of the AIFM1 variant significantly restored the structure and function of AIF, further improving the physiological state of patient-specific iPSC-derived neurons. This study demonstrates that the AIFM1 variant is one of the molecular bases of ANSD. Mitochondrial dysfunction, especially mCa2+ overload, plays a prominent role in ANSD associated with AIFM1. Our findings help elucidate the mechanism of ANSD and may lead to the provision of novel therapies.
摘要：
听觉神经病变谱系障碍(ANSD)是由内毛细胞功能障碍引起的听力障碍,带状突触，螺旋神经节神经元和/或听觉神经本身。约有1/7000的新生儿有听觉神经功能异常，占儿童永久性听力损失病例的10%-14%。尽管我们之前确定了AIFM1c.1265G>A变体与ANSD相关，ANSD与AIFM1相关的机制知之甚少。我们通过游离质粒的核转染从外周血单核细胞（PBMC）产生了诱导多能干细胞（iPSC）。通过CRISPR/Cas9技术编辑患者特异性iPSC以产生基因校正的等基因iPSC。这些iPSC通过神经干细胞(NSC)进一步分化成神经元。在这些神经元中探索了致病机制。在患者细胞中（PBMC，iPSC,和神经元)，AIFM1c.1265G>A变体引起了一个新的剪接变体（c.1267-1305del），产生AIFp.R422Q和p.423-435del蛋白，这损害了AIF二聚化。这种受损的AIF二聚化然后削弱了AIF与含卷曲螺旋-螺旋-卷曲螺旋-螺旋结构域的蛋白4(CHCHD4)之间的相互作用。一方面,ETC复杂亚基的线粒体输入受到抑制，随后导致ADP/ATP比率增加和ROS水平升高。另一方面,MICU1-MICU2异源二聚化受损，导致mCa2+过载。钙蛋白酶被mCa2激活，随后被切割的AIF易位到细胞核中，最终导致不依赖caspase的细胞凋亡。有趣的是,校正AIFM1变体显着恢复了AIF的结构和功能，进一步改善患者特异性iPSC衍生神经元的生理状态。该研究表明AIFM1变体是ANSD的分子碱基之一。线粒体功能障碍,尤其是mCa2+过载,在与AIFM1相关的ANSD中起着重要作用。我们的发现有助于阐明ANSD的机制，并可能导致提供新疗法。
