Huntington's disease (HD) is a neurodegenerative disorder characterized by motor, cognitive, and behavioral impairments, as well as a peripheral phenotype involving skeletal muscle. Mitochondrial dysfunction has been observed in HD tissues, including skeletal muscle, and is linked to the expression of mutant huntingtin (mutHTT) protein, which impairs mitochondrial quality control and accelerates mitochondrial aging. This study investigated whether mitochondrial dysfunction and mtDNA mutations occur in human HD skeletal muscle. Using ultra-deep mtDNA sequencing, the researchers found an accumulation of mtDNA mutations affecting oxidative phosphorylation, with impaired mtDNA maintenance and increased mitochondrial biogenesis of less efficient oxidative phosphorylation. In primary human cell lines expressing full-length mutHTT, mitochondrial stress induced mitochondrial fission but normal mitophagy. However, high levels of N-terminal mutHTT fragments promoted mitochondrial fission and reduced mitophagy, leading to pathogenic mtDNA mutations. The study showed that lifelong expression of mutant HTT causes mtDNA instability in post-mitotic human skeletal muscle, suggesting that genomic instability may not be limited to nuclear DNA. Promoting mitochondrial health could be a complementary strategy in treating HD. The findings highlight the role of mitochondrial dysfunction in HD pathogenesis and the importance of mitochondrial quality control in maintaining mtDNA stability. The study used a combination of mtDNA sequencing, proteomics, and mitochondrial network analysis to demonstrate the impact of mutHTT on mitochondrial dynamics and mtDNA mutations in HD. The results suggest that impaired mitochondrial quality control in HD leads to chronic failure in dealing with mutagenic stress, resulting in higher levels of mutant mtDNA copies. The study also found that mtDNA mutations in HD patients were more frequent and pathogenic compared to healthy controls, with a significant association with early-stage HD. The findings provide new insights into the molecular mechanisms underlying HD and highlight the importance of mitochondrial health in the disease's progression.Huntington's disease (HD) is a neurodegenerative disorder characterized by motor, cognitive, and behavioral impairments, as well as a peripheral phenotype involving skeletal muscle. Mitochondrial dysfunction has been observed in HD tissues, including skeletal muscle, and is linked to the expression of mutant huntingtin (mutHTT) protein, which impairs mitochondrial quality control and accelerates mitochondrial aging. This study investigated whether mitochondrial dysfunction and mtDNA mutations occur in human HD skeletal muscle. Using ultra-deep mtDNA sequencing, the researchers found an accumulation of mtDNA mutations affecting oxidative phosphorylation, with impaired mtDNA maintenance and increased mitochondrial biogenesis of less efficient oxidative phosphorylation. In primary human cell lines expressing full-length mutHTT, mitochondrial stress induced mitochondrial fission but normal mitophagy. However, high levels of N-terminal mutHTT fragments promoted mitochondrial fission and reduced mitophagy, leading to pathogenic mtDNA mutations. The study showed that lifelong expression of mutant HTT causes mtDNA instability in post-mitotic human skeletal muscle, suggesting that genomic instability may not be limited to nuclear DNA. Promoting mitochondrial health could be a complementary strategy in treating HD. The findings highlight the role of mitochondrial dysfunction in HD pathogenesis and the importance of mitochondrial quality control in maintaining mtDNA stability. The study used a combination of mtDNA sequencing, proteomics, and mitochondrial network analysis to demonstrate the impact of mutHTT on mitochondrial dynamics and mtDNA mutations in HD. The results suggest that impaired mitochondrial quality control in HD leads to chronic failure in dealing with mutagenic stress, resulting in higher levels of mutant mtDNA copies. The study also found that mtDNA mutations in HD patients were more frequent and pathogenic compared to healthy controls, with a significant association with early-stage HD. The findings provide new insights into the molecular mechanisms underlying HD and highlight the importance of mitochondrial health in the disease's progression.