Impaired AIF-CHCHD4 interaction and mitochondrial calcium overload contribute to auditory neuropathy spectrum disorder in patient-iPSC-derived neurons with AIFM1 variant

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 (m)Ca(2+) overload. Calpain was activated by (m)Ca(2+) 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 (m)Ca(2+) 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. [Image: see text]