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Molecular Characteristics of Cybrids with Severe Mitochondrial Dysfunctions Associated with Aging
Shen Luxi1, Yuan Weihua2, Cao Ying2, Huang Rong2, Xiong Xusheng2, Li Hongzhi2*
1Xuanwu Hospital, Capital Medical University, Beijing 100053, China;
2Key Laboratory of Laboratory Medicine, Ministry of Education, Zhejiang Provincial Key Laboratory of Medical Genetics, College of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou 325035, China
Abstract: In our previous study， we generated cybrids with identical background of nuclear genome containing mitochondria of synaptosomes from mice at different ages， and found that the overall mitochondrial function in old group was significantly decreased compared with that in young group. To explore the molecular characteristics of cybrids with severe mitochondrial dysfunction associated with aging， we tested three cybrids from old group with severe overall mitochondrial dysfunction and one cybrid from young group with normal function as control. After the levers of oxygen consumption and ATP synthesis were ensured with significant decreases (P<0.05 or P<0.01)， high-throughput sequencing was used to detect mitochondrial DNA (mtDNA) mutations， especially detecting heteroplasmic mutations with low abundance， and further， the respiratory chain complex-dependent respiration was examined. The sequencing results showed that point mutations in mtDNA were significantly accumulated in aging cybrids. These mutations included three variations in non-coding region， two (heteroplasmy m.15849G>T m.16289A>T) of them were likely to be deleterious based on the analysis of DNA conservation; four variations in coding region， two (homoplasmy m.12496T>C in ND5， heteroplasmy m.15199A>T in Cyt b) of them were likely to be damaging based on the analysis of DNA and amino acid conservation， and the prediction of protein function. Further results showed that cybrids with mutations in subunit ND5 of complex I (or subunit Cyt b of complex III) together with two mutations in control region showed significant decreases in the complex I (or complex III) dependent respiration (P<0.05 or P<0.01). In conclusion， the overall mitochondrial dysfunction occurred in aging cells probably resulted from heteroplasmic and even homoplasmic mutations in the regulatory region and coding region of mtDNA. Accumulation of multiple mtDNA mutations could result in impairments in the function of mitochondrial respiratory chain complexes， and thus contributed to overall mitochondrial dysfunction and led to aging.
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