Abstract
Auditory neuropathy spectrum disorder (ANSD) represents a variety of sensorineural deafness conditions characterized by abnormal inner hair cells and/or auditory nerve function, but with the preservation of outer hair cell function. ANSD represents up to 15% of individuals with hearing impairments. Through mutation screening, bioinformatic analysis and expression studies, we have previously identified several apoptosis-inducing factor (AIF) mitochondria-associated 1 (AIFM1) variants in ANSD families and in some other sporadic cases. Here, to elucidate the pathogenic mechanisms underlying each AIFM1 variant, we generated AIF-null cells using the clustered regularly interspersed short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system and constructed AIF-wild type (WT) and AIF-mutant (mut) (p.T260A, p.R422W, and p.R451Q) stable transfection cell lines. We then analyzed AIF structure, coenzyme-binding affinity, apoptosis, and other aspects. Results revealed that these variants resulted in impaired dimerization, compromising AIF function. The reduction reaction of AIF variants had proceeded slower than that of AIF-WT. The average levels of AIF dimerization in AIF variant cells were only 34.5%–49.7% of that of AIF-WT cells, resulting in caspase-independent apoptosis. The average percentage of apoptotic cells in the variants was 12.3%–17.9%, which was significantly higher than that (6.9%–7.4%) in controls. However, nicotinamide adenine dinucleotide (NADH) treatment promoted the reduction of apoptosis by rescuing AIF dimerization in AIF variant cells. Our findings show that the impairment of AIF dimerization by AIFM1 variants causes apoptosis contributing to ANSD, and introduce NADH as a potential drug for ANSD treatment. Our results help elucidate the mechanisms of ANSD and may lead to the provision of novel therapies.
概要
听神经病谱系障碍 (ANSD) 属于感音神经性耳聋, 其特征为内毛细胞和/或听觉神经元的功能异常, 但外毛细胞的功能正常. 在听力障碍患者中, 听神经病谱系障碍的发病率高达 15%. 我们前期通过突变筛查、 生物信息学分析和蛋白表达等检测, 在 ANSD 家系和某些散发病例中发现了凋亡诱导因子 1 (AIFM1)基因的几种点突变. 为阐明 AIFM1突变体的致病机制, 本文使用 CRISPR/Cas9 系统构建了凋亡诱导因子 (AIF) 蛋白敲除的细胞系, 及其稳定转染野生型和突变型 AIF 蛋白 (p.T260A、 p.R422W 和 p.R451Q) 的细胞系, 并且分析了 AIF 蛋白结构、 AIF 与辅酶的亲和力及细胞凋亡等情况. 结果显示, 上述 AIF 突变体可导致 AIF 蛋白二聚体形成障碍, 损害 AIF 蛋白的生理功能. 突变型 AIF 蛋白的还原速率显著低于野生型 AIF 蛋白. 且在 AIF 突变型细胞系中, AIF 蛋白的二聚体含量仅为 AIF 野生型细胞系的 34.5%~49.7%, 导致非 caspase 依赖性细胞凋亡. AIF 突变型细胞系中凋亡细胞的平均百分比为 12.3%~17.9%, 显著高于对照组的 6.9%~7.4%. 特别是, 烟酰胺腺嘌呤二核苷酸 (NADH) 处理显著提高 AIF 突变型细胞中的 AIF 蛋白二聚体含量, 从而降低细胞凋亡. 结果表明: AIFM1 突变引起 AIF 蛋白二聚体形成障碍, 使得细胞凋亡增加, 导致 ANSD 发生; NADH 是 ANSD 的潜在治疗药物. 我们的研究结果有助于阐明 ANSD 的发病机制, 并提供新的治疗方案.
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Acknowledgments
This work was supported by the National Natural Science Foundation of China (Nos. 32070584, 81830028, 31771398, 82222016, and 8207040100), the Zhejiang Provincial Natural Science Foundation of China (No. LZ19C060001), and the Fundamental Research Funds for the Central Universities (No. 2019QNA6001). We thank Chris WOOD from the College of Life Sciences, Zhejiang University for the proof reading of this manuscript. We thank Fangliang HUANG and Shelong ZHANG from the Instrument and Technical Service Platform, College of Life Sciences, Zhejiang University for their technical support.
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Qingfeng YAN and Qiuju WANG conceived and designed the research. Yue QIU, Hongyang WANG, and Huaye PAN performed the experiments and analyzed the data. Lei YAN and Dong ZHANG performed molecular dynamics analysis. Hongyang WANG, Jing GUAN, Mingjie FAN, Hui ZHOU, Kaiwen WU, and Zexiao JIA provided technical support. Xuanhao ZHOU, Qianqian ZHUANG, Zhaoying LEI, Mengyao LI, and Xue DING verified the reproducibility of results. Aifu LIN, Yong FU, and Dong ZHANG contributed to discussion and data interpretation. Qingfeng YAN, Qiuju WANG, Yue QIU, and Huaye PAN wrote the manuscript. All authors have read and approved the final manuscript, and therefore, have full access to all the data in the study and take responsibility for the integrity and security of the data.
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Yue QIU, Hongyang WANG, Huaye PAN, Jing GUAN, Lei YAN, Mingjie FAN, Hui ZHOU, Xuanhao ZHOU, Kaiwen WU, Zexiao JIA, Qianqian ZHUANG, Zhaoying LEI, Mengyao LI, Xue DING, Aifu LIN, Yong FU, Dong ZHANG, Qiuju WANG, and Qingfeng YAN declare that they have no conflict of interest.
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AIFM1 variants associated with auditory neuropathy spectrum disorder cause apoptosis due to impaired apoptosis-inducing factor dimerization
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Qiu, Y., Wang, H., Pan, H. et al. AIFM1 variants associated with auditory neuropathy spectrum disorder cause apoptosis due to impaired apoptosis-inducing factor dimerization. J. Zhejiang Univ. Sci. B 24, 172–184 (2023). https://doi.org/10.1631/jzus.B2200081
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DOI: https://doi.org/10.1631/jzus.B2200081
Key words
- Auditory neuropathy spectrum disorder
- Apoptosis-inducing factor (AIF) mitochondria-associated 1 (AIFM1) variants
- Dimerization
- Caspase-independent apoptosis
- Nicotinamide adenine dinucleotide (NADH) treatment