线粒体内膜转位酶8A基因敲除小鼠的构建及其内耳功能研究

doi:10.3969/j.issn.1674-8115.2023.03.001

上海交通大学学报（医学版） ›› 2023, Vol. 43 ›› Issue (3): 261-268.doi: 10.3969/j.issn.1674-8115.2023.03.001

• 创新团队成果专栏 •

线粒体内膜转位酶8A基因敲除小鼠的构建及其内耳功能研究

洪晗馨¹^,²^,³(), 王龙昊¹^,²^,³, 刘辉辉¹^,²^,³, 彭浒⁴, 吴皓¹^,²^,³(), 杨涛¹^,²^,³()

^1.上海交通大学医学院附属第九人民医院耳鼻咽喉头颈外科，上海 200011
^2.上海交通大学医学院耳科学研究所，上海 200125
^3.上海市耳鼻疾病转化医学重点实验室，上海 200125
^4.海军军医大学长征医院耳鼻喉科，上海 200003

收稿日期:2022-12-15 接受日期:2023-02-22 出版日期:2023-03-28 发布日期:2023-03-28
通讯作者: 吴皓,杨涛 E-mail:h764274629@sjtu.edu.cn;wuhao@shsmu.edu.cn;yangtfxl@sina.com
作者简介:洪晗馨（1997—），女，硕士生；电子信箱：h764274629@sjtu.edu.cn。
基金资助:
国家自然科学基金(82101938);上海市科学技术委员会科技创新行动计划(21JC1404000);上海市耳鼻疾病转化医学重点实验室(14DZ2260300)

Construction of translocase of inner mitochondrial membrane 8A gene knockout mice and study of its inner ear function

HONG Hanxin¹^,²^,³(), WANG Longhao¹^,²^,³, LIU Huihui¹^,²^,³, PENG Hu⁴, WU Hao¹^,²^,³(), YANG Tao¹^,²^,³()

^1.Department of Otolaryngology-Head and Neck Surgery, Shanghai Ninth People′s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China
^2.Ear Institute, Shanghai Jiao Tong University School of Medicine, Shanghai 200125, China
^3.Shanghai Key Laboratory of Translation Medicine on Ear and Nose Diseases, Shanghai 200125, China
^4.Department of Otolaryngology, Changzheng Hospital, Naval Medical University, Shanghai 200003, China

Received:2022-12-15 Accepted:2023-02-22 Online:2023-03-28 Published:2023-03-28
Contact: WU Hao,YANG Tao E-mail:h764274629@sjtu.edu.cn;wuhao@shsmu.edu.cn;yangtfxl@sina.com
Supported by:
National Natural Science Foundation of China(82101938);Science and Technology Innovation Action Plan of Shanghai Municipal Science and Technology Commission(21JC1404000);Shanghai Key Laboratory of Translational Medicine on Ear and Nose Diseases(14DZ2260300)

摘要/Abstract

摘要：

目的·通过构建线粒体内膜转位酶8A（translocase of inner mitochondrial membrane 8A，Timm8a）基因敲除小鼠（即Timm8a^-^/- 小鼠），探究其听力表型及该基因在内耳的功能。方法·设计并构建Timm8a^-^/- 小鼠，采用PCR及蛋白质印迹法（Western blotting）验证是否构建成功。观察并比较1月龄时Timm8a^-^/- 小鼠与野生型（wild type，WT）小鼠的体型及体质量。采用免疫荧光染色观察WT小鼠耳蜗组织中TIMM8A的表达分布。通过听性脑干反应（auditory brainstem response，ABR）比较Timm8a^-^/- 小鼠和WT小鼠的听力阈值。采用甲苯胺蓝染色法观察该2种小鼠内耳螺旋器、螺旋神经节与血管纹形态。通过透射电镜观察该2种小鼠的内耳毛细胞、螺旋神经节细胞与血管纹细胞中的线粒体超微结构，并统计异常线粒体所占比例。结果·PCR结果显示Timm8a基因已成功敲除，Western blotting结果显示Timm8a^-^/- 小鼠的耳蜗、大脑、心肌、骨骼肌组织中已无TIMM8A表达，表明Timm8a^-^/- 小鼠构建成功。免疫荧光染色结果提示TIMM8A在WT小鼠耳蜗组织中呈泛表达，且高表达于螺旋器、螺旋神经节与血管纹区域。与WT小鼠相比，1月龄时的Timm8a^-^/- 小鼠发育较慢，体质量明显偏轻，且ABR阈值有所升高（均P<0.05）；该小鼠的ABR的Ⅰ波波幅出现下降、Ⅰ波潜伏期有所延长。甲苯胺蓝染色结果显示，与WT小鼠相比，Timm8a^-^/- 小鼠的螺旋神经节细胞的形态与数量均无明显改变，耳蜗中圈及底圈的血管纹厚度减薄（均P<0.05）。透射电镜的观察结果显示，与WT小鼠相比，Timm8a^-^/- 小鼠内耳毛细胞、螺旋神经节细胞与血管纹细胞中线粒体结构出现异常，且异常线粒体比例明显增多（均P<0.05）。结论·该研究成功构建了Timm8a^-^/- 小鼠，其听力阈值的升高可能与其内耳线粒体超微形态结构异常有关。

关键词: 线粒体内膜转位酶8A, Mohr-Tranebjaerg综合征, 听神经病谱系障碍, 线粒体

Abstract:

Objective ·To explore the hearing phenotype of Timm8a^-^/- mice and the function of translocase of inner mitochondrial membrane 8A(Timm8a)gene in inner ear by constructing Timm8a gene knockout mice. Methods ·Timm8a^-^/- mice were designed and constructed. PCR and Western blotting were used to verify whether the construction was successful. The body size and weight of Timm8a^-/- mice and wild type (WT) mice aged one-month were observed and compared. Immunofluorescence staining was used to observe the expression and distribution of TIMM8A protein in the cochlea of WT mice. Auditory brainstem response (ABR) was used to compare the hearing threshold of Timm8a^-^/- mice and WT mice. Toluidine blue staining was performed to observe the morphology of organ of Corti (OC), spiral ganglion neuron (SGN) and stria vascularis (SV) in the inner ear of the two kinds of mice. Transmission electron microscope was used to observe the ultrastructure of mitochondria in inner ear hair cells (HCs), SGN cells and SV cells of the two kinds of mice, and the proportion of abnormal mitochondria was counted. Results ·The results of PCR showed that Timm8a gene had been successfully knocked out, and the results of Western blotting showed that there was no TIMM8A proteins in the cochlea, brain, heart and skeletal muscle tissues. The both results indicated that Timm8a^-^/- mice were successfully constructed. The results of immunofluorescence staining showed that TIMM8A was abundantly expressed in the cochlea of WT mice, and was highly expressed in the OC, SGN and SV. Compared with WT mice, Timm8a^-^/- miceaged one-month developed slower, had lighter body weight and significantly higher hearing threshold (all P<0.05), their amplitude of Ⅰ wave of ABR was decreased and the latency was prolonged. The results of toluidine blue staining showed that compared with WT mice, there was no significant change in the shape and number of SGN cells in the inner ear, but the thickness of SV in the middle turn and basal turn of the cochlea was reduced (both P<0.05). The results of transmission electron microscope showed that compared with WT mice, the structure of mitochondria in inner ear HCs, SGN cells and SV cells of Timm8a^-^/- mice was abnormal, and the proportion of abnormal mitochondria was significantly increased (all P<0.05). Conclusion ·Timm8a^-^/- mice are successfully constructed in this study, and the elevated hearing threshold of Timm8a^-^/- mice may be related to the abnormal ultrastructure of mitochondria in inner ear.

Key words: translocase of inner mitochondrial membrane 8A (TIMM8A), Mohr-Tranebjaerg syndrome (MTS), auditory neuropathy spectrum disorder (ANSD), mitochondria

中图分类号:

R764.43

洪晗馨, 王龙昊, 刘辉辉, 彭浒, 吴皓, 杨涛. 线粒体内膜转位酶8A基因敲除小鼠的构建及其内耳功能研究[J]. 上海交通大学学报（医学版）, 2023, 43(3): 261-268.

HONG Hanxin, WANG Longhao, LIU Huihui, PENG Hu, WU Hao, YANG Tao. Construction of translocase of inner mitochondrial membrane 8A gene knockout mice and study of its inner ear function[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2023, 43(3): 261-268.

图/表 6

图1 Timm8a-/-小鼠构建的示意图Note： Cas9/gRNA—CRISPR-associated protein 9/guide RNA.

Fig 1 Schematic diagram of the construction of the Timm8a-/- mice

表1 Timm8a-/- 小鼠基因型鉴定的引物序列

Tab 1 Primer sequences for Timm8a-/- mouse genotyping

Primer	Forward sequence (5'→3')	Reverse sequence (5'→3')
Timm8a	TTGGGGCACCTAACTGAT	CAAAGCAAGGCAATGGAATA
ACTB-Cre 1	GCGGTCTGGCAGTAAAAACTATC	GTAGGTGGAAATTCTAGCATCATCC
ACTB-Cre 2	CTAGGCCACAGAATTGAAAGATCT	GTGAAACAGCATTGCTGTCACTT

图2 Timm8a-/- 小鼠构建的验证及其与WT小鼠的体型观察Note： A/B. Identification of Timm8a loxP (A) and ACTB-Cregenotype (B) of mice by PCR amplification. 421 bp is band of Timm8a loxP band, 361 bp is WT band, 100 bp is Cre positive band, 324 bp is positive control band. C. Detection of TIMM8A in different tissues of Timm8a-/- mice and WT mice by Western blotting. D. Comparison of body size between Timm8a-/- mice and WT mice. E. Comparison of weight between Timm8a-/- mice and WT mice.

Fig 2 Verification of Timm8a-/- mouse construction and observation of its body size with WT mice

图3 TIMM8A在耳蜗组织中的表达定位及 Timm8a-/-小鼠与WT小鼠听力表型对比Note： A. Expression of TIMM8A in cochlea tissue of WT mice. DAPI—4', 6-diamidino-2-phenylindole. B. Comparison of the hearing thresholds between Timm8a-/- mice and WT mice by ABR. ①P=0.002, ②P=0.005, ③P=0.003, ④P=0.008, ⑤P=0.000, ⑥P=0.007, ⑦P=0.001. C. Comparison of the ABR waveforms between Timm8a-/- miceand WT mice.

Fig 3 Expression and localization of TIMM8A in cochlear tissue and comparison of the hearing phenotype between Timm8a-/- mice and WT mice

图4 Timm8a-/- 小鼠与WT小鼠的内耳病理形态学分析Note： A. Observation of the structural morphology of inner ear in Timm8a-/- mice and WT mice. B. Comparison of the number of SGN cells between Timm8a-/- mice and WT mice. SGN cells per 1 000 μm2 were counted C. Comparison of SV thickness between Timm8a-/- mice and WT mice.

Fig 4 Pathological analysis of inner ear between Timm8a-/- mice and WT mice

图5 Timm8a-/- 小鼠与野生型小鼠内耳HCs、SGN细胞及SV细胞中线粒体超微结构观察及异常结构占比分析Note： A. Observation of mitochondrial ultrastructure in different types of cells in Timm8a-/- mice and WT mice. B. Proportion of abnormal mitochondria in different types of cells in Timm8a-/- mice and WT mice.

Fig 5 Observation of mitochondrial ultrastructure and analysis of the proportion of abnormal structure in inner ear HCs, SGN cells and SV cells of Timm8a-/- mice and WT mice

参考文献 18

1	KANG Y L, ANDERSON A J, JACKSON T D, et al. Function of hTim8a in complex Ⅳ assembly in neuronal cells provides insight into pathomechanism underlying Mohr-Tranebjærg syndrome[J]. Elife, 2019, 8: e48828.
2	SONG P P, GUAN Y Q, CHEN X, et al. Frameshift mutation of Timm8a1 gene in mouse leads to an abnormal mitochondrial structure in the brain, correlating with hearing and memory impairment[J]. J Med Genet, 2021, 58(9): 619-627.
3	HEINEMEYER T, STEMMET M, BARDIEN S, et al. Underappreciated roles of the translocase of the outer and inner mitochondrial membrane protein complexes in human disease[J]. DNA Cell Biol, 2019, 38(1): 23-40.
4	NEIGHBORS A, MOSS T, HOLLOWAY L, et al. Functional analysis of a novel mutation in the TIMM8A gene that causes deafness-dystonia-optic neuronopathy syndrome[J]. Mol Genet Genomic Med, 2020, 8(3): e1121.
5	HA A D, PARRATT K L, RENDTORFF N D, et al. The phenotypic spectrum of dystonia in Mohr-Tranebjaerg syndrome[J]. Mov Disord, 2012, 27(8): 1034-1040.
6	RUSSELL O M, GORMAN G S, LIGHTOWLERS R N, et al. Mitochondrial diseases: hope for the future[J]. Cell, 2020, 181(1): 168-188.
7	STENTON S L, PROKISCH H. Genetics of mitochondrial diseases: identifying mutations to help diagnosis[J]. EBioMedicine, 2020, 56: 102784.
8	GUSIC M, PROKISCH H. Genetic basis of mitochondrial diseases[J]. FEBS Lett, 2021, 595(8): 1132-1158.
9	MATRICARDI S, CANAFOGLIA L, ARDISSONE A, et al. Epileptic phenotypes in children with early-onset mitochondrial diseases[J]. Acta Neurol Scand, 2019, 140(3): 184-193.
10	GORMAN G S, CHINNERY P F, DIMAURO S, et al. Mitochondrial diseases[J]. Nat Rev Dis Primers, 2016, 2: 16080.
11	NG Y S, BINDOFF L A, GORMAN G S, et al. Mitochondrial disease in adults: recent advances and future promise[J]. Lancet Neurol, 2021, 20(7): 573-584.
12	ROESCH S, O'SULLIVAN A, ZIMMERMANN G, et al. Mitochondrial disease and hearing loss in children: a systematic review[J]. Laryngoscope, 2022, 132(12): 2459-2472.
13	LI J N, LIU C, MÜLLER U, et al. Autophagy proteins are essential for aminoglycoside-induced hearing loss[J]. Autophagy, 2022: 1-2.
14	GAO Y, KAMOGASHIRA T, FUJIMOTO C, et al. Effects of pyrroloquinoline quinone on noise-induced and age-related hearing loss in mice[J]. Sci Rep, 2022, 12(1): 15911.
15	KISHIMOTO-URATA M, URATA S, FUJIMOTO C, et al. Role of oxidative stress and antioxidants in acquired inner ear disorders[J]. Antioxidants (Basel), 2022, 11(8): 1469.
16	QUAN Y, XIN Y G, TIAN G E, et al. Mitochondrial ROS-modulated mtDNA: a potential target for cardiac aging[J]. Oxid Med Cell Longev, 2020, 2020: 9423593.
17	FETONI A R, PACIELLO F, ROLESI R, et al. Targeting dysregulation of redox homeostasis in noise-induced hearing loss: oxidative stress and ROS signaling[J]. Free Radic Biol Med, 2019, 135: 46-59.
18	PACIELLO F, PISANI A, ROLESI R, et al. Noise-induced cochlear damage involves PPAR down-regulation through the interplay between oxidative stress and inflammation[J]. Antioxidants (Basel), 2021, 10(8): 1188.

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线粒体内膜转位酶8A基因敲除小鼠的构建及其内耳功能研究

Construction of translocase of inner mitochondrial membrane 8A gene knockout mice and study of its inner ear function

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