Xutong Zhao1, Meng Yu1, Yawen Zhao1, Yiming Zheng1, Lingchao Meng1, Kang Du1, Zhiying Xie1, He Lv1, Wei Zhang1, Jing Liu1, Qingqing Wang1, Yun Yuan1, Zhaoxia Wang2,3, Jianwen Deng4,5. 1. Department of Neurology, Peking University First Hospital, #8 Xishiku St, Xicheng District, Beijing, 100034, China. 2. Department of Neurology, Peking University First Hospital, #8 Xishiku St, Xicheng District, Beijing, 100034, China. drwangzx@163.com. 3. Beijing Key Laboratory of Neurovascular Disease Discovery, Beijing, 100034, China. drwangzx@163.com. 4. Department of Neurology, Peking University First Hospital, #8 Xishiku St, Xicheng District, Beijing, 100034, China. jianwendeng@pkufh.com. 5. Beijing Key Laboratory of Neurovascular Disease Discovery, Beijing, 100034, China. jianwendeng@pkufh.com.
Abstract
BACKGROUND: There is increasing evidence for the role of inflammation in the pathogenesis of mitochondrial diseases (MDs). However, the mechanisms underlying mutation-induced inflammation in MD remain elusive. Our previous study suggested that mitophagy is impaired in the skeletal muscle of those with MD, likely causing mitochondrial DNA (mtDNA) release and thereby triggering inflammation. We here aimed to decipher the role of the cGAS-STING pathway in inflammatory process in MDs. METHODS: We investigated the levels of circulating cell-free mtDNA (ccf-mtDNA) in the serum of 104 patients with MDs. Immunofluorescence was performed in skeletal muscles in MDs and control. Biochemical analysis of muscle biopsies was conducted with western blot to detect cGAS, STING, TBK1, IRF3 and phosphorylated IRF3 (p-IRF3). RT-qPCR was performed to detect the downstream genes of type I interferon in skeletal muscles. Furthermore, a protein microarray was used to examine the cytokine levels in the serum of patients with MDs. RESULTS: We found that ccf-mtDNA levels were significantly increased in those with MDs compared to the controls. Consistently, the immunofluorescent results showed that cytosolic dsDNA levels were increased in the muscle samples of MD patients. Biochemical analysis of muscle biopsies showed that cGAS, IRF3, and TBK1 protein levels were significantly increased in those with MDs, indicating that there was activation of the cGAS-STING pathway. RT-qPCR showed that downstream genes of type I interferon were upregulated in muscle samples of MDs. Protein microarray results showed that a total of six cytokines associated with the cGAS-STING pathway were significantly increased in MD patients (fold change > 1.2, p value < 0.05). CONCLUSIONS: These findings suggest that increases in ccf-mtDNA levels is associated with the activation of the cGAS-STING pathway, thereby triggering inflammation in MDs.
BACKGROUND: There is increasing evidence for the role of inflammation in the pathogenesis of mitochondrial diseases (MDs). However, the mechanisms underlying mutation-induced inflammation in MD remain elusive. Our previous study suggested that mitophagy is impaired in the skeletal muscle of those with MD, likely causing mitochondrial DNA (mtDNA) release and thereby triggering inflammation. We here aimed to decipher the role of the cGAS-STING pathway in inflammatory process in MDs. METHODS: We investigated the levels of circulating cell-free mtDNA (ccf-mtDNA) in the serum of 104 patients with MDs. Immunofluorescence was performed in skeletal muscles in MDs and control. Biochemical analysis of muscle biopsies was conducted with western blot to detect cGAS, STING, TBK1, IRF3 and phosphorylated IRF3 (p-IRF3). RT-qPCR was performed to detect the downstream genes of type I interferon in skeletal muscles. Furthermore, a protein microarray was used to examine the cytokine levels in the serum of patients with MDs. RESULTS: We found that ccf-mtDNA levels were significantly increased in those with MDs compared to the controls. Consistently, the immunofluorescent results showed that cytosolic dsDNA levels were increased in the muscle samples of MD patients. Biochemical analysis of muscle biopsies showed that cGAS, IRF3, and TBK1 protein levels were significantly increased in those with MDs, indicating that there was activation of the cGAS-STING pathway. RT-qPCR showed that downstream genes of type I interferon were upregulated in muscle samples of MDs. Protein microarray results showed that a total of six cytokines associated with the cGAS-STING pathway were significantly increased in MD patients (fold change > 1.2, p value < 0.05). CONCLUSIONS: These findings suggest that increases in ccf-mtDNA levels is associated with the activation of the cGAS-STING pathway, thereby triggering inflammation in MDs.
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