Auditory neuropathy spectrum disorder (ANSD) is a type of hearing disorder that disrupts the function of the auditory micro circuitry. Its associated lesion sites can involve inner hair cells (IHCs), ribbon synapses, spiral ganglion neurons (SGNs), and/or the auditory nerve itself (Chinese clinical practice, 2022). ANSD can be caused by either genetic or environmental factors (Starr et al., 2000) and can be classified as presynaptic, synaptic, or postsynaptic auditory neuropathy, dependent upon the specific sites of the lesions (De Siati et al., 2020). Along with the discovery of genes associated with ANSD, some of the underlying pathogenesis has been initially summarized (Wang et al., 2005, Roux et al., 2006, Surel et al., 2016, Han et al., 2017, Defourny et al., 2019, Zong et al., 2020, Qiu et al., 2023, Qiu et al., 2023). Nevertheless, hearing rehabilitation has proven extremely challenging due to the varied locations of the lesion sites.

At present, clinical interventions for ANSD mainly focus upon hearing aids (HA) and cochlear implants (CI). Despite this, conventional hearing aids cannot provide sufficient rehabilitation in most cases (Berlin et al., 2010, Roush et al., 2011, Ching et al., 2013, Ramanathan et al., 2023, Morlet et al., 2023). As a result, cochlear implantation is currently the most commonly used intervention option for ANSD patients (Morlet et al., 2023). However, the effect of CI in ANSD patients remains comparably worse, and with a high degree of variability, than in patients with sensorineural hearing loss from other etiologies (Hu et al., 2022, Kurt and Akgül, 2023). Again, this seems attributable to the varied nature of the differing lesion sites (Rance and Starr, 2015, Shearer and Hansen, 2019, Lin et al., 2022). For patients with postsynaptic auditory neuropathy, such as those with SGNs and auditory nerve/brainstem lesions, the effect of a CI remains insufficient, and poor hearing performance persists (Lin et al., 2022, Buchman et al., 2006, Brookes et al., 2008, Cif et al., 2013, Young et al., 2012, Goswamy et al., 2012). Some studies reported that mitochondrial drugs, glycine, glucocorticoids, or taurine could be potential agents for ANSD (Feng et al., 2020, Muñoz-Carlin Mde et al., 2010, Zhang et al., 2014, Zhou et al., 2012, Doostkam et al., 2021, Gedik Soyuyuce et al., 2022, Huang et al., 2019, Nevoux et al., 2021, Piecuch et al., 2023). However, pharmacological intervention for ANSD remains in its early stages, requiring more effective candidate drugs and more clinical trials with a large number of cases.

We previously reported several novel AIFM1 variants associated with ANSD (Zong et al., 2015). Given that cochlear nerve deficiency was detected in these patients, CI would be expected to have poor outcomes (Shearer and Hansen, 2019, Zong et al., 2015). In this way, it became evident to us that there is a particularly urgent requirement to explore the mechanism of ANSD and find effective drugs for patients with postsynaptic auditory neuropathy.

Patient-specific induced pluripotent stem cells (iPSCs) and iPSC-derived differentiation cells have been widely recognized as novel cell models of disease (Hu et al., 2019). We previously constructed control-iPSCs (Con-iPSCs), patient-specific iPSCs (AN-iPSCs), and gene-corrected iPSCs (CORR-iPSCs) to explore the pathogenesis of ANSD, as associated with the AIF p.R422Q variant. The study indicated that the AIF variant protein showed impaired dimerization and caused mitochondrial dysfunctions, such as upregulated ADP/ATP ratio, elevated mitochondrial ROS level, and mCa2+ overload. Calpain, activated by mCa2+, cleaved AIF for its translocation into the nucleus, ultimately increased caspase-independent apoptosis, and resulted in the occurrence of ANSD (Qiu et al., 2023). NADH is a main electron donor in the ETC (Heikal, 2010) and a key coenzyme for the redox reaction (Bouamama et al., 2017). Some studies reported that NADH could be a potential agent for neurodegenerative diseases (Bouamama et al., 2017, Birkmayer et al., 1993, Braidy et al., 2019, Demarin et al., 2004, Rex et al., 2004). Furthermore, NADH has been noted to be able to significantly improve AIF dimerization in 293T stable transfection cell lines carrying some AIF mutations (p.T260A, p.R422W, and p.R451Q) (Qiu et al., 2023). We therefore contrived to test the therapeutic effect of NADH in patient-specific iPSCs-derived neurons.

Here, 293T stable transfection cell lines were used to detect the effects of NADH on ANSD associated with the AIF p.R422Q variant. AN-iPSCs-derived auditory neurons were then utilized as more reliable models for further confirmation. We finally compared the therapeutic effects of gene correction and NADH treatment on hereditary ANSD.