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Browsing by Author "Zhuang, Yuan"

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  • Zhuang, Yuan (2018)
    Mitochondrial dysfunction is involved in many neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS) and Huntington's disease (HD). Most of the mitochondrial disorders lack effective treatments so far. One of the important defects of mitochondrial dysfunction is the blockade of mitochondrial respiratory chain. An alternative oxidase (AOX), originated from Ciona intestinalis, has been found to counteract this defect by providing a bypath of the mitochondrial respiratory chain in plants, drosophila, mice and human cultured cells. Therefore, we suggest that AOX might be a promising tool for curing neurodegenerative diseases from the aspect of alleviating mitochondrial defects. An engineered mouse model has been created to express the Ciona intestinalis AOX ubiquitously in the whole animal. Our study aims at investigating the expression and function of AOX in the transgenic mouse brain and exploring therapeutic potential of AOX in human diseases. AFG3l2 gene encodes a subunit of m-AAA metalloprotease, which is involved in mitochondrial protein quality control process. Mutations in the AFG3L2 is known to be related to spinocerebellar ataxias 28 (SCA28), a neurodegenerative disease resulting from cerebellar damage. Loss of AFG3L2 gene causes OPA1 protein cleavage and mitochondrial fragmentation which are easily detected. Thus, we propose an in vitro neurodegenerative disease model generated by knocking down of AFG3L2 in target cells. We used western blotting to detect the expression level of AOX in the brain and primary neuro-glia cells of the transgenic mice, finding that the AOX protein reduces in the brain along with age. Antimycin A treatment on neuro-glia cells demonstrated the function of AOX in transgenic pups. However, the respiratory activity caused by AOX also decreases with the mice age. In addition, we constructed a lentiviral vector with DNA recombinant technology to express AOX in U2OS cells. To examine the function of AOX under disease condition, we established an in vitro neurodegenerative disease model by knocking down the AFG3L2 gene of U2OS cells with siRNA, and infected the cells with AOX lentivirus. By comparing the immunostaining images of wild-type and AOX infected cells, we saw a significant difference in the degree of mitochondrial fragmentation between these cells. Nevertheless, the cleavage pattern of OPA1 protein, which is mainly responsible for mitochondrial fragmentation, remains unchanged whether AOX is present in the cell or not. In conclusion, our study first investigated the expression and function of AOX in the brain of this transgenic mouse model. And we established a lentivirus vector to apply AOX gene to human cells, testing the therapeutic function of AOX in a neurodegenerative disease model in vitro. Moreover, the mitochondrial fragmentation of the disease model is alleviated by AOX despite of OPA1 cleavage, indicating that the mitochondrial phenotype caused by loss of AFG3L2 is not only due to OPA1 cleavage, but also related to respiratory defects. This highlights the possibility to rescue the mitochondrial morphological changes from new aspects.