Aging and severe mitochondrial disease are disparate, complex processes whose causes have yet to be fully understood. One protein involved in both is the mechanistic target of rapamycin (mTOR), which regulates cell growth and metabolism. mTOR protein is a component of two complexes, the mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Inhibiting mTORC1 with the drug rapamycin increases lifespan in model organisms and suppresses severe mitochondrial disease and shortened lifespan in the Ndufs4 knockout (KO) mouse model of Leigh Syndrome. In mouse models, KO of the S6K1 protein, a substrate of mTORC1, also increases lifespan. Therefore, we hypothesized that KO of S6K1 in the whole body and specific tissues (brain, liver, fat) would rescue the short lifespan of Ndufs4 KO mice. A breeding strategy utilizing Cre-recombinase mice, S6K1^Flox/Flox mice, and Ndufs4 +/- mice was employed to ultimately produce tissue-specific Cre +/- Ndufs4 -/- S6K1^Flox/Flox mice. PCR and Western blot analysis confirmed the whole body and tissue-specific deletion of S6K1 gene and depletion of the protein in each mouse. Our findings indicate that whole body and liver-specific S6K1 KO modestly increased lifespan and delayed the onset of neurological symptoms originating from the mitochondrial disease. However, S6K1 KO is still less effective in rescuing mitochondrial dysfunction than the optimal dose of rapamycin. We speculate that S6K1 partially contributes to the mitochondrial disease of Ndufs4 KO mice, and the liver may play an important role in regulating lifespan. In the long run, these findings may enable us to potentially produce drug treatments to extend the lifespan of healthy humans as well as Leigh Syndrome patients.

The Ndufs4 gene knockout (Ndufs4-/-) mice show the phenotype of severe neurodegenerative mitochondrial disease analogous to human Leigh syndrome. Phosphoproteomics is a large-scale study of phosphorylated proteins, which are post-translational modifications that can turn on and off biological signaling pathways. Phosphoproteomics and proteomics experiments using mass spectrometry can allow us to quantify protein levels in mouse tissues. The broad goal of my project is to define the underlying mechanism of mitochondrial dysfunction using a phosphoproteomics approach in the Ndufs4-/- mouse model and translate the findings to improve the health of patients with severe mitochondrial disorders. Our lab previously showed that rapamycin, a drug that inhibits mTOR kinase, partially rescued the disease phenotype of Ndufs4-/- mice. However, the downstream biological pathways affected by rapamycin in this setting are not understood yet. Those may be discovered by analyzing differences in phosphorylated proteins among brain tissues of wildtype, Ndufs4-/-, and rapamycin treated Ndufs4-/- mice. For the initial analysis, protein data sets with quantitative measurements of more than 4,000 proteins were analyzed. Proteins that may be important and most affected by mitochondrial disease and rescued by rapamycin were pinpointed. After the analysis of the data was complete, the proteins that passed the filtering thresholds were quantified with Western blots, a biochemical technique to quantify protein level individually. We have already identified two signaling pathways from the phosphoproteomics and will test the effect of inhibitors of these pathways in the mice. It is our hope that a better mechanistic understanding of disease progression in this mitochondrial disease mouse model will yield new therapies to treat mitochondrial disease in patients for whom there is not currently any effective treatment.
 

Rapamycin, an FDA approved drug that inhibits the mTOR pathway, is known to prolong lifespan in yeast, invertebrates, and vertebrates. In aged mice, reports of lifespan extension had been demonstrated using continuous treatment. More recently, our lab showed that a shorter three-month transient treatment of rapamycin sufficiently extends lifespan. However, some questions remain. Is there an optimal rapamycin dosage and treatment length that results in a robust effect on healthspan and lifespan? We had noted a sex-specific increase in cancer incidence and no lifespan extension in females that received daily intraperitoneal (IP) injections of rapamycin, the same regimen that robustly extended lifespan in male mice. Currently, we are testing the effects of daily, once weekly, and twice weekly rapamycin injections in female mice. We have already uncovered an increased incidence of eye inflammation in the animals that received daily drug treatment compared to all the other cohorts. Interestingly, this increase in eye inflammation also occurred in mice that were fed high-dose rapamycin. These data suggest that high dosages present unwanted side effects in female mice and it is crucial to find an optimal treatment regimen to minimize the undesirable effects of rapamycin. Additional assays to measure physiological parameters such as body fat mass, body temperature, muscle function, cognitive function, and lifespan will be done in our study. We hope to reveal the proper drug dose and intervention frequency that provides the best outcome, enhancing longevity while minimizing or even abolishing the detrimental outcomes associated with rapamycin.