Every cell contains hundreds of mitochondria, vital to cell function. These are the evolved descendants of ancient symbiotic bacteria, and contain a small remnant genome, the mitochondrial DNA. Most of the original mitochondrial DNA has migrated to the cell nucleus to be incorporated into the nuclear DNA, but a few genes remain. Unfortunately mitochondrial DNA is less well protected and more prone to damage than nuclear DNA, and loss of function can lead to malfunctioning mitochondria that harm the cell. This is thought to be an important contribution to age-related loss of mitochondrial function, one of the contributing causes of degenerative aging.
Scientists at the SENS Research Foundation, now the Longevity Research Institute since the merger with Lifespan.io, have long been working on allotopic expression of mitochondrial genes, meaning to place a copy of these genes into the nuclear DNA, suitably altered such that the proteins can find their way back to the mitochondria. A backup source of necessary proteins would render mitochondrial DNA damage irrelevant to cell function. This is a challenging project, and given the limited funds available to date, has been achieved for only a few of the thirteen remaining mitochondrial genes. But progress has been made, and today's research materials describe an important step forward. Researchers have created a genetically engineered mouse lineage in which allotopic expression of ATP8 clearly works to provide the ATP8 protein needed in mitochondria.
Nuclear Expression of a Mitochondrial Gene in Mice
Previously, the same team had achieved promising results in vitro, but finding a suitable animal model proved difficult: mitochondrial DNA (mtDNA) genes are so essential that mutations in them usually render mice non-viable. However, a particular strand of mice exists that harbors a relatively benign mutation in ATP8, a gene encoding a subunit of the ATP synthase complex, which causes only a mildly pathologic phenotype. Alongside those mutants, wild-type mice were used as controls.
The team synthesized a nuclear-compatible version of ATP8 and inserted it into the ROSA26 locus, a well-characterized "safe harbor" site in the mouse genome. This locus is widely used in genetic engineering because it allows stable organism-wide expression of inserted genes without interfering with other essential genomic functions. The researchers had to overcome significant technical challenges to achieve nuclear expression of a gene that is normally expressed in mitochondria (allotopic expression) and to make the protein transferrable to mitochondria. Eventually, their efforts paid off: allotopic ATP8 was able to compete with mitochondrial ATP8 even in wild-type mice and outperformed the mutant ATP8. The allotopic gene was expressed in all the tissues that the researchers tested, and the protein successfully integrated into the mitochondrial machinery.
Exogenous expression of ATP8, a mitochondrial encoded protein, from the nucleus in vivo
Replicative errors, inefficient repair, and proximity to sites of reactive oxygen species production make mitochondrial DNA (mtDNA) susceptible to damage with time. We explore in vivo allotopic expression (re-engineering mitochondrial genes and expressing them from the nucleus) as an approach to rescue defects arising from mtDNA mutations. We used a mouse strain C57BL/6J(mtFVB) with a natural polymorphism in the mitochondrial ATP8 gene that encodes a protein subunit of the ATP synthase. We generated a transgenic mouse with an epitope-tagged recoded mitochondrial-targeted ATP8 gene expressed from the ROSA26 locus in the nucleus and used the C57BL/6J(mtFVB) strain to verify successful incorporation.
The allotopically expressed ATP8 protein in transgenic mice was constitutively expressed across all tested tissues, successfully transported into the mitochondria, and incorporated into ATP synthase. The ATP synthase with transgene had similar activity to non-transgenic control, suggesting successful integration and function. Exogenous ATP8 protein had no negative impact on measured mitochondrial function, metabolism, or behavior. Successful allotopic expression of a mitochondrially encoded protein in vivo in a mammal is a step toward utilizing allotopic expression as a gene therapy in humans to repair physiological consequences of mtDNA defects that may accumulate in congenital mitochondrial diseases or with age.
View the full article at FightAging
