Abstract

Accumulation of mutations in mitochondrial DNA (mtDNA) is thought to be responsible for mitochondrial, and other, diseases and biological phenomena, such as diabetes, cancer, neurodegenerative diseases, and aging. Mouse models may elucidate the relationship between mutations in mtDNA and these abnormalities. However, because of the difficulty of mtDNA manipulation, generation of mouse models has not sufficiently progressed to enable such studies. To overcome this difficulty and to establish a source of diverse mtDNA mutations, we here generated cultured mouse cells containing mtDNA derived from an mtDNA mutator mouse that accumulates random mtDNA mutations with age. Mutation analysis of the obtained transmitochondrial cytoplasmic hybrid cells (cybrids) revealed that the cells harbored diverse mtDNA mutations occurring at a higher frequency than in mouse tissues, and exhibited severe respiration defects that would be lethal in tissues or organs. Abnormal respiratory complex formation and high stress on the mitochondrial protein quality control system appeared to be involved in these severe respiration defects. The mutation rates of the majority of highly accumulated mutations converged to either approximately 5%, 10%, or 40%, suggesting that these mutations are linked on the respective mtDNA molecules, and mtDNA in cybrid cells likely consisted of mtDNA molecules clonally expanded from the small population of introduced mtDNAs. Thus, the linked mutations in these cybrid cells cannot be evaluated individually. In addition, mtDNA mutations homologous to confirmed pathogenic mutations in human were rarely observed in our generated cybrids. However, the transmitochondrial cybrids constitute a useful tool for concentrating pathogenic mtDNA mutations and as a source of diverse mtDNA mutations to elucidate the relationship between mtDNA mutations and diseases.

Highlights

  • Mitochondria produce the majority of ATP required by the body by oxidative phosphorylation (OXPHOS)

  • Establishment of transmitochondrial cybrid cells harboring random Mitochondrial DNA (mtDNA) mutations Previously, based on the original papers by Trifunovic et al [7] and Kujoth et al [8], we have introduced the D257A mutation into the PolG gene of C57BL/6J mouse and generated mtDNA mutator mouse [14]

  • Deep-sequence analysis of the generated cybrid cells revealed that they harbored various mtDNA mutations at a higher frequency

Read more

Summary

Introduction

Mitochondria produce the majority of ATP required by the body by oxidative phosphorylation (OXPHOS). Mitochondrial DNA (mtDNA), the unique mitochondrial genome, encodes 13 polypeptides—the subunits of respiratory complexes, and rRNA and tRNA molecules needed for translation of these polypeptides. Mutations in mtDNA result in reduced ATP production because they lead to abnormal structure of respiratory chain subunits or reduced translation of mitochondrial proteins, and underlie various disorders, termed mitochondrial diseases [1]. All pathogenic mtDNA mutations result in reduced ATP production as a primary phenotype. The most effective approach to elucidate these mechanisms is generation and analysis of animal models of disease, in which an animal harbors a mutation that corresponds to a disease of interest. Because techniques for artificial manipulation and mutagenesis of mtDNA are not well established, generating animal models of disease corresponding to each human mitochondrial disease phenotype is very difficult

Methods
Results
Conclusion
Full Text
Published version (Free)

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call