Salk Institute scientists have developed a biological platform for studying mitochondrial DNA in physiology, adaptation, disease mechanisms, and therapeutic development. Headed by Ronald Evans, PhD, professor and director of the Gene Expression Laboratory and holder of the March of Dimes Chair in Molecular and Developmental Biology at Salk, the team has already used the platform to generate a library of 155 mitochondrial DNA mutant cell lines and reveal correlations between mouse development and mitochondrial function. They suggest that the platform, library, and findings will accelerate therapeutic development for mitochondrial disorders, as well as help scientists treat mitochondrial dysfunction in other diseases and conditions like cancer or aging.
“Mitochondrial DNA accumulates mutations at a high rate, and more than 260 inherited disease-causing mtDNA mutations have been identified in humans,” said Evans. “Until now, a lack of models representing this diversity has limited mechanistic insight and therapeutic development. Our new platform will allow scientists to investigate mitochondrial DNA variation in health, disease, and evolution, which will enable therapeutic innovation for mitochondrial disorders.”
Evans is co-corresponding author of the team’s published paper in PNAS, titled “A scalable embryonic stem cell–based platform for efficient generation of mitochondrial DNA mutant mice,” in which they concluded that their new platform, “… opens the door to mechanistic dissection of how mtDNA variation influences metabolism, adaptation, and disease, and provides a strategically valuable foundation for accelerating therapeutic development through genetically precise mitochondrial disease models.”
Some of your most important life partners are the mitochondria that power all your cells. You and these little cellular powerhouses are in a 1.5-billion-year-old evolutionary relationship—but mitochondria brought some baggage. Mitochondria brought their own DNA with them when they joined with the bigger, more complex cells so long ago, and today that mitochondrial DNA influences human health. Mitochondrial DNA does the extremely important job of creating the proteins needed for energy production—but it also has an especially high rate of mutation, and those mutations can accumulate thanks to inefficient repair mechanisms. Because mitochondria are essential parts of every cell, their dysfunction can lead to body-wide dysfunction, with especially devastating impact on high-energy organs like the brain and heart. Without enough power in your cells, symptoms like migraines, muscle weakness, and loss of hearing or sight can begin to manifest.
“Mitochondria are central to energy metabolism and cellular signaling, and mutations in mitochondrial DNA (mtDNA) can disrupt these processes and contribute to human disease,” the authors wrote. “Mitochondrial DNA (mtDNA) accumulates mutations at a high rate, and more than 260 pathogenic germline mtDNA mutations have been identified in humans, producing diverse and often tissue-specific disorders.”
The chronic and broad impact of mitochondrial dysfunction makes it especially important to study. However, trying to pinpoint the outcome of specific mitochondrial DNA mutations has for many years been a slow, arduous process for many years. “… progress in defining how mtDNA variation influences adaptation, pathophysiology, and disease susceptibility has been limited by the lack of suitable animal models,” the team continued. “Researchers would create mouse models one-by-one with different mitochondrial DNA mutations, with just one model sometimes taking years,” said Salk staff scientist Weiwei Fan, PhD. This was a problem that Fan had noted early in his scientific career and set his mind to as a PhD student.
The new Salk model is a scalable, embryonic stem-cell (ES)-based platform creating mice with mutations to their mitochondrial DNA. “This new work is all building off an original platform I generated during my PhD,” says Fan, first and co-corresponding author of the study. “That platform was inefficient—it took a long time to generate just one mitochondrial DNA mutant. With some technological improvements and modifications, this new platform is much more efficient and can create dozens of mutants with far greater ease.”
The authors explained, “… we developed a scalable ES cell–based platform that integrates mtDNA mutagenesis, cybrid technology, high-sensitivity mutation detection, and optimized mouse transgenesis.” The platform starts with a protein, called mitochondrial DNA polymerase, generating randomly mutated mitochondrial DNA. That mutated mitochondrial DNA is then transferred into stem cells, which can be integrated with mouse embryos to create mice for study. Once one of these mice is established, researchers can investigate the specific symptoms of their specific mitochondrial DNA mutation and the mechanisms by which those symptoms arise—insight that can be used to design targeted therapies down the line. “Optimized ES cell–embryo aggregation enables robust contribution of mtDNA mutant ES cells to host embryos, producing chimeric mice with germline transmission,” the investigators noted.
Using this platform, the Salk team generated a library of 155 mitochondrial DNA mutation cell lines, each with its own distinct impact on mitochondrial performance. “Using this platform, we generate a library of 155 donor fibroblast lines carrying distinct homoplasmic single-nucleotide mtDNA mutations that produce diverse mitochondrial phenotypes, including impaired oxidative phosphorylation, increased reactive oxygen species, and altered mitochondrial membrane potential,” they stated. They then used that library to validate that the cells could be used to generate mice with single mitochondrial DNA mutations. These mice allowed them to find a strong correlation between mitochondrial function and early embryonic development, suggesting a baseline energy level 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,” said Fan. “And 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 authors added, “Together, the advances outlined in this study establish a powerful and generalizable platform for systematically modeling the functional diversity of human mtDNA mutations and polymorphisms in vivo.”
The new platform and library are cracking open the world of mitochondrial DNA. With the ability to generate mitochondrial DNA mutants more rapidly, therapeutic development for mitochondrial disease and dysfunction will come more rapidly, too. The mouse models are already a huge step forward for the field, but the researchers are also eager to move into human models in a more human-relevant context.
“The majority of human diseases come with or cause mitochondrial dysfunction,” said Evans. “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.”
In their paper the team concluded, “The library provides a unique and comprehensive resource for modeling the diversity of human mtDNA variation in vitro and can also be used to generate in vivo models through ES-cybrid technology … By enabling the generation of both pathogenic and physiologically relevant mtDNA variants—including those resembling somatic mutations associated with aging and cancer—this platform substantially expands the toolkit available to mitochondrial researchers.”
