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.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Al-Wardy NM, Al-Kindi MN, Al-Khabouri MJ, et al., 2016. A novel missense mutation in the C2C domain of otoferlin causes profound hearing impairment in an Omani family with auditory neuropathy. Saudi Med J, 37(10):1068–1075. https://doi.org/10.15537/smj.2016.10.14967
Ardissone A, Piscosquito G, Legati A, et al., 2015. A slowly progressive mitochondrial encephalomyopathy widens the spectrum of AIFM1 disorders. Neurology, 84(21):2193–2195. https://doi.org/10.1212/wnl.0000000000001613
Bano D, Prehn JHM, 2018. Apoptosis-inducing factor (AIF) in physiology and disease: the tale of a repented natural born killer. eBioMedicine, 30:29–37. https://doi.org/10.1016/j.ebiom.2018.03.016
Berger I, Ben-Neriah Z, Dor-Wolman T, et al., 2011. Early prenatal ventriculomegaly due to an AIFM1 mutation identified by linkage analysis and whole exome sequencing. Mol Genet Metab, 104(4):517–520. https://doi.org/10.1016/j.ymgme.2011.09.020
Birkmayer JGD, Vrecko C, Volc D, et al., 1993. Nicotinamide adenine dinucleotide (NADH)—a new therapeutic approach to Parkinson’s disease. Comparison of oral and parenteral application. Acta Neurol Scand, 87(S146):32–35. https://doi.org/10.1111/j.1600-0404.1993.tb00018.x
Brosey CA, Ho C, Long WZ, et al., 2016. Defining NADH-driven allostery regulating apoptosis-inducing factor. Structure, 24(12):2067–2079. https://doi.org/10.1016/j.str.2016.09.012
Castro-Marrero J, Sáez-Francàs N, Segundo MJ, et al., 2016. Effect of coenzyme Q10 plus nicotinamide adenine dinucleotide supplementation on maximum heart rate after exercise testing in chronic fatigue syndrome—a randomized, controlled, double-blind trial. Clin Nutr, 35(4):826–834. https://doi.org/10.1016/j.clnu.2015.07.010
Cheng X, Li L, Brashears S, et al., 2005. Connexin 26 variants and auditory neuropathy/dys-synchrony among children in schools for the deaf. Am J Med Genet, 139A(1): 13–18. https://doi.org/10.1002/ajmg.a.30929
Cheung ECC, Joza N, Steenaart NAE, et al., 2006. Dissociating the dual roles of apoptosis-inducing factor in maintaining mitochondrial structure and apoptosis. EMBO J, 25(17):4061–4073. https://doi.org/10.1038/sj.emboj.7601276
Chiu YH, Wu CC, Lu YC, et al., 2010. Mutations in the OTOF gene in Taiwanese patients with auditory neuropathy. Audiol Neurootol, 15(6):364–374. https://doi.org/10.1159/000293992
Delmaghani S, del Castillo FJ, Michel V, et al., 2006. Mutations in the gene encoding pejvakin, a newly identified protein of the afferent auditory pathway, cause DFNB59 auditory neuropathy. Nat Genet, 38(7):770–778. https://doi.org/10.1038/ng1829
Demarin V, Podobnik SS, Storga-Tomic D, et al., 2004. Treatment of Alzheimer’s disease with stabilized oral nicotin-amide adenine dinucleotide: a randomized, double-blind study. Drugs Exp Clin Res, 30(1):27–33.
Diodato D, Tasca G, Verrigni D, et al., 2016. A novel AIFM1 mutation expands the phenotype to an infantile motor neuron disease. Eur J Hum Genet, 24(3):463–466. https://doi.org/10.1038/ejhg.2015.141
Ferreira P, Villanueva R, Martínez-Júlvez M, et al., 2014. Structural insights into the coenzyme mediated monomerdimer transition of the pro-apoptotic apoptosis inducing factor. Biochemistry, 53(25):4204–4215. https://doi.org/10.1021/bi500343r
Ghezzi D, Sevrioukova I, Invernizzi F, et al., 2010. Severe X-linked mitochondrial encephalomyopathy associated with a mutation in apoptosis-inducing factor. Am J Hum Genet, 86(4):639–649. https://doi.org/10.1016/j.ajhg.2010.03.002
Hangen E, Blomgren K, Bénit P, et al., 2010. Life with or without AIF. Trends Biochem Sci, 35(5):278–287. https://doi.org/10.1016/j.tibs.2009.12.008
Hangen E, Féraud O, Lachkar S, et al., 2015. Interaction between AIF and CHCHD4 regulates respiratory chain biogenesis. Mol Cell, 58(6):1001–1014. https://doi.org/10.1016/j.molcel.2015.04.020
Heimer G, Eyal E, Zhu X, et al., 2018. Mutations in AIFM1 cause an X-linked childhood cerebellar ataxia partially responsive to riboflavin. Eur J Paediatr Neurol, 22(1):93–101. https://doi.org/10.1016/j.ejpn.2017.09.004
Herrmann JM, Riemer J, 2020. Apoptosis inducing factor and mitochondrial NADH dehydrogenases: redox-controlled gear boxes to switch between mitochondrial biogenesis and cell death. Biol Chem, 402(3):289–297. https://doi.org/10.1515/hsz-2020-0254
Hu B, Wang M, Castoro R, et al., 2017. A novel missense mutation in AIFM1 results in axonal polyneuropathy and mis-assembly of OXPHOS complexes. Eur J Neurol, 24(12): 1499–1506. https://doi.org/10.1111/ene.13452
Lang-Roth R, Fischer-Krall E, Kornblum C, et al., 2017. AUNA2: a novel type of non-syndromic slowly progressive auditory synaptopathy/auditory neuropathy with autosomal-dominant inheritance. Audiol Neurootol, 22(1): 30–40. https://doi.org/10.1159/000474929
Li C, Brant E, Budak H, et al., 2021. CRISPR/Cas: a Nobel Prize award-winning precise genome editing technology for gene therapy and crop improvement. J Zhejiang Univ-Sci B (Biomed & Biotechnol), 22(4):253–284. https://doi.org/10.1631/jzus.B2100009
Matsuoka AJ, Morrissey ZD, Zhang CY, et al., 2017. Directed differentiation of human embryonic stem cells toward placode-derived spiral ganglion-like sensory neurons. Stem Cells Transl Med, 6(3):923–936. https://doi.org/10.1002/sctm.16-0032
Mierzewska H, Rydzanicz M, Biegański T, et al., 2017. Spondyloepimetaphyseal dysplasia with neurodegeneration associated with AIFM1 mutation—a novel phenotype of the mitochondrial disease. Clin Genet, 91(1):30–37. https://doi.org/10.1111/cge.12792
Penido RC, Isaac ML, 2013. Prevalence of auditory neuropathy spectrum disorder in an auditory health care service. Braz J Otorhinolaryngol, 79(4):429–433. https://doi.org/10.5935/1808-8694.20130077
Pittelli M, Felici R, Pitozzi V, et al., 2011. Pharmacological effects of exogenous NAD on mitochondrial bioenergetics, DNA repair, and apoptosis. Mol Pharmacol, 80(6): 1136–1146. https://doi.org/10.1124/mol.111.073916
Reinhardt C, Arena G, Nedara K, et al., 2020. AIF meets the CHCHD4/Mia40-dependent mitochondrial import pathway. Biochim Biophys Acta Mol Basis Dis, 1866(6): 165746. https://doi.org/10.1016/j.bbadis.2020.165746
Rinaldi C, Grunseich C, Sevrioukova IF, et al., 2012. Cow-chock syndrome is associated with a mutation in apoptosis-inducing factor. Am J Hum Genet, 91(6): 1095–1102. https://doi.org/10.1016/j.ajhg.2012.10.008
Romanos J, Kimura L, Fávero ML, et al., 2009. Novel OTOF mutations in Brazilian patients with auditory neuropathy. J Hum Genet, 54(7):382–385. https://doi.org/10.1038/jhg.2009.45
Romero-Tamayo S, Laplaza R, Velazquez-Campoy A, et al., 2021. W196 and the β-hairpin motif modulate the redox switch of conformation and the biomolecular interaction network of the apoptosis-inducing factor. Oxid Med Cell Longev, 2021:6673661. https://doi.org/10.1155/2021/6673661
Sánchez-Martínez A, Benito-Orejas JI, Tellería-Orriols JJ, et al., 2017. Autosomal dominant auditory neuropathy and variant DIAPH3 (c.-173C>T). Acta Otorrinolaringol Esp, 68(3): 183–185. https://doi.org/10.1016/j.otorri.2016.06.004
Sancho P, Sánchez-Monteagudo A, Collado A, et al., 2017. A newly distal hereditary motor neuropathy caused by a rare AIFM1 mutation. Neurogenetics, 18(4):245–250. https://doi.org/10.1007/s10048-017-0524-6
Santarelli R, Cama E, Scimemi P, et al., 2008. Audiological and electrocochleography findings in hearing-impaired children with connexin 26 mutations and otoacoustic emissions. Eur Arch Otorhinolaryngol, 265(1):43–51. https://doi.org/10.1007/s00405-007-0412-z
Schoen CJ, Emery SB, Thorne MC, et al., 2010. Increased activity of Diaphanous homolog 3 (DIAPH3)/diaphanous causes hearing defects in humans with auditory neuropathy and in Drosophila. Proc Natl Acad Sci USA, 107(30): 13396–13401. https://doi.org/10.1073/pnas.1003027107
Sevrioukova IF, 2011. Apoptosis-inducing factor: structure, function, and redox regulation. Antioxid Redox Signal, 14(12):2545–2579. https://doi.org/10.1089/ars.2010.3445
Sevrioukova IF, 2016. Structure/function relations in AIFM1 variants associated with neurodegenerative disorders. J Mol Biol, 428(18):3650–3665. https://doi.org/10.1016/j.jmb.2016.05.004
Starr A, Sininger YS, Pratt H, 2000. The varieties of auditory neuropathy. J Basic Clin Physiol Pharmacol, 11(3):215–230. https://doi.org/10.1515/jbcpp.2000.11.3.215
Starr A, Isaacson B, Michalewski HJ, et al., 2004. A dominantly inherited progressive deafness affecting distal auditory nerve and hair cells. J Assoc Res Otolaryngol, 5(4): 411–426. https://doi.org/10.1007/s10162-004-5014-5
Susin SA, Lorenzo HK, Zamzami N, et al., 1999. Molecular characterization of mitochondrial apoptosis-inducing factor. Nature, 397(6718):441–446. https://doi.org/10.1038/17135
Tang DL, Kang R, Berghe TV, et al., 2019. The molecular machinery of regulated cell death. Cell Res, 29(5):347–364. https://doi.org/10.1038/s41422-019-0164-5
Tekin M, Akcayoz D, Incesulu A, 2005. A novel missense mutation in a C2 domain of OTOF results in autosomal recessive auditory neuropathy. Am J Med Genet, 138A(1):6–10. https://doi.org/10.1002/ajmg.a.30907
Tranebjærg L, Strenzke N, Lindholm S, et al., 2018. The CAPOS mutation in ATP1A3 alters Na/K-ATPase function and results in auditory neuropathy which has implications for management. Hum Genet, 137(2):111–127. https://doi.org/10.1007/s00439-017-1862-z
Varga R, Kelley PM, Keats BJ, et al., 2003. Non-syndromic recessive auditory neuropathy is the result of mutations in the otoferlin (OTOF) gene. J Med Genet, 40(1):45–50. https://doi.org/10.1136/jmg.40.1.45
Wang QJ, Li RH, Zhao H, et al., 2005. Clinical and molecular characterization of a Chinese patient with auditory neuropathy associated with mitochondrial 12S rRNA T1095C mutation. Am J Med Genet, 133A(1):27–30. https://doi.org/10.1002/ajmg.a.30424
Wang QJ, Li QZ, Rao SQ, et al., 2006. AUNX1, a novel locus responsible for X linked recessive auditory and peripheral neuropathy, maps to Xq23-27.3. J Med Genet, 43(7):e33. https://doi.org/10.1136/jmg.2005.037929
Yang W, Li SS, Zhang X, et al., 2015. Gene editing and cell therapy based on induced pluripotent stem cells. Chin J Cell Biol, 37(1):90–99.
Ying WH, 2008. NAD+/NADH and NADP+/NADPH in cellular functions and cell death: regulation and biological consequences. Antioxid Redox Signal, 10(2):179–206. https://doi.org/10.1089/ars.2007.1672
Yu SW, Wang HM, Poitras MF, et al., 2002. Mediation of poly (ADP-ribose) polymerase-1-dependent cell death by apoptosis-inducing factor. Science, 297(5579):259–263. https://doi.org/10.1126/science.1072221
Yu SW, Andrabi SA, Wang HM, et al., 2006. Apoptosis-inducing factor mediates poly(ADP-ribose) (PAR) polymer-induced cell death. Proc Natl Acad Sci USA, 103(48): 18314–18319. https://doi.org/10.1073/pnas.0606528103
Zhu KQ, Swanson RA, Ying WH, 2005. NADH can enter into astrocytes and block poly(ADP-ribose) polymerase-1-mediated astrocyte death. NeuroReport, 16(11):1209–1212. https://doi.org/10.1097/00001756-200508010-00015
Zong L, Guan J, Ealy M, et al., 2015. Mutations in apoptosis-inducing factor cause X-linked recessive auditory neuropathy spectrum disorder. J Med Genet, 52(8):523–531. https://doi.org/10.1136/jmedgenet-2014-102961
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.
Author information
Authors and Affiliations
Contributions
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.
Corresponding authors
Ethics declarations
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.
This article does not contain any studies with human or animal subjects performed by any of the authors.
Additional information
Materials and methods
The detailed methods are provided in the electronic supplementary materials of this paper.
Supplementary information
Materials and methods; Figs. S1–S3
Supplementary information
11585_2023_676_MOESM1_ESM.pdf
AIFM1 variants associated with auditory neuropathy spectrum disorder cause apoptosis due to impaired apoptosis-inducing factor dimerization
Rights and permissions
About this article
Cite this article
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
Received:
Accepted:
Published:
Issue Date:
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