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Journal of Zhejiang University SCIENCE B
ISSN 1673-1581(Print), 1862-1783(Online), Monthly
2023 Vol.24 No.2 P.172-184
AIFM1 variants associated with auditory neuropathy spectrum disorder cause apoptosis due to impaired apoptosis-inducing factor dimerization
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.
Key words: Auditory neuropathy spectrum disorder; Apoptosis-inducing factor (AIF) mitochondria-associated 1 (AIFM1) variants; Dimerization; Caspase-independent apoptosis; Nicotinamide adenine dinucleotide (NADH) treatment
1浙江大学生命科学学院，中国杭州市，310058
2中国人民解放军总医院耳鼻咽喉研究所，中国北京市，100853
3浙江大学医学院第一附属医院儿科，中国杭州市，310003
4浙江大学医学院儿童医院，中国杭州市，310052
5浙江省细胞与基因工程重点实验室，中国杭州市，310058
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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DOI:
10.1631/jzus.B2200081
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On-line Access:
2023-02-05
Received:
2022-02-22
Revision Accepted:
2022-08-30
Crosschecked:
2023-02-09