How does mitochondrial DNA affect health? Investigating ‘powerhouse’ mutations with novel platform

A new platform for studying mitochondrial DNA mutations implicated in human disease could revolutionize research in health, disease and evolution, and accelerate efforts to develop treatments for mitochondrial disorders.

Salk Institute (CA, USA) researchers have developed a scalable embryonic stem cell-based platform for more efficient production of mitochondrial DNA (mtDNA) mutant mice, paving the way for therapeutic development, as well as research investigating the role of mtDNA in physiology, adaptation and disease mechanisms. Already, the technology has been used to generate a library of 155 mtDNA mutant cell lines and reveal links between mouse development and mitochondrial function that could herald new treatments for mitochondrial disorders and perhaps other conditions like cancer or aging.

Famed in science class as ‘the powerhouse of the cell’, mitochondria are central to energy metabolism and cellular signaling. Mutations in mtDNA, therefore, can be incredibly disruptive to these processes and may contribute to human disease. To date, more than 260 pathogenic germline mtDNA mutations have been identified in humans, leading to diverse and often tissue-specific disorders. Moreover, population-specific mtDNA variants arise through evolution and may influence adaptation and disease susceptibility.

To fully understand the impact of this variation, researchers require animal models that represent the diversity of human mtDNA mutations, but these are unfortunately lacking. Despite recent base-editing approaches that enable direct mtDNA modification, this roadblock continues to limit mechanistic insight and therapeutic development.

With their stem cell-based platform, the researchers behind the new study are hoping to change that. They performed random mutagenesis using an error-prone mtDNA polymerase to produce a broad spectrum of mtDNA mutations. These were then transferred into embryonic stem cells via a multiplexed cybrid fusion strategy coupled with sensitive mutation detection. The stem cells can then be integrated into mouse embryos to create mtDNA mutant mice.

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Using their platform, the team generated a library of 155 donor fibroblast cell lines, each carrying distinct mtDNA mutations that produce diverse mitochondrial phenotypes, including impaired oxidative phosphorylation, increased reactive oxygen species and altered mitochondrial membrane potential.

They then generated 34 female C57BL/6 embryonic stem cell lines harboring 18 mtDNA mutations, which were used to create multiple chimeric mice and enable germline transmission for one mutation.

These findings reveal a strong correlation between mitochondrial function and early embryonic development, suggesting a minimal energetic threshold is required for normal development.

“Our library is a huge milestone and is very diverse, with a scale of diversity similar to the known human disease-causing mutation diversity of around 260,” noted first author Weiwei Fan. “With this collection of mutant cells, we can not only look at inherited mutations but also at ones that occur based on other stresses like environmental cues or aging.”

The platform is capable of creating dozens of mtDNA mutant mice much more easily than traditional methods, and as such, should help advance research and therapeutic development for mitochondrial disease and dysfunction.

“The majority of human diseases come with or cause mitochondrial dysfunction,” senior author Ronald Evans added. “Progress in this field has been limited, but this new platform is going to fuel so much important research that points to therapeutic approaches to combat mitochondrial diseases, as well as diseases or conditions associated with mitochondrial dysfunction like cancer or aging.”