Mutations in Complex I (NADH:Ubiquinone Oxidoreductase) of the mitochondrial electron transport chain have been reported in up to 30% of pediatric mitochondrial diseases (MDs) and affect 1 in 5000 live births. One of such pathologies, Leigh Syndrome, is often fatal in the first three years of life and has no known cure. Knock-out (KO) of Ndufs4 in mice recapitulates several aspects of the disease, including lethargy, encephalopathy and retarded growth. Acarbose delays the onset of neurological symptoms and prolongs the lifespan of both Ndufs4−/− mice and heterogeneous wild type mice. Acarbose is a type 2 diabetes drug that inhibits alpha glucosidases, resulting in delayed absorption of complex carbohydrates and increased activity of the intestinal flora, including increased levels of short-chain fatty acids (SCFAs) and other fermentation products. No formal study has been conducted to determine whether increased SCFA concentration mediates the effects of acarbose on longevity and MD suppression. Our goal is to elucidate the mechanistic basis of acarbose. We hypothesize that acarbose delays MD progression in Nduf4-/- mice by increasing circulating levels of SCFAs. Thus, SCFAs supplementation should recapitulate the effects of acarbose. We will feed chow containing tributyrin to control and mutant mice from weaning until humane endpoint. We will measure body weight, observe the incidence of forelimb clasping behavior (a widely tested neurological symptom), and record percent survival daily. We expect to observe a delay in clasping behavior and prolonged survival in acarbose-treated KO mice compared to that of untreated KO mice, and that these effects are recapitulated more pronouncedly with higher tributyrin doses. SCFAs are promising therapeutics because they are natural metabolites that are inexpensive and can be manufactured into easy consumable pills. A successful outcome of this study will help progress therapies for patients with MDs and for anti-aging purposes.

Much of aging and longevity research is linked to the mitochondria; specifically mitochondrial dysfunction are an important factor in the etiology of age-related diseases. An important metabolic pathway for mitochondrial function is the Nicotinamide Adenine Dinucleotide (NAD+) biosynthesis pathway. NAD+ is a coenzyme found in hundreds of metabolic pathways within the body, and many are specific to high energy metabolic transformations within the mitochondria. This research introduces a multistep procedure which aims to quantify and identify specific metabolites within the NAD+ biosynthesis pathway. The procedure uses Liquid Chromatography-coupled Mass Spectrometry to detect and quantify fourteen metabolites in the NAD+ metabolome. This method can be used to analyze how specific mitochondrial dysfunction, for example deletion of the NADH Ubiquinone Oxidoreductase subunit 4 (Ndufs4), affects the NAD+ biosynthetic pathway, and to explore potential drug treatments for the improvement of such disorders.

Mitochondrial disease refers to a group of disorders that affects the mitochondria and therefore influences energy production and metabolism. The main purpose of this study is to determine the impact of pharmacological interventions with known age-delaying activity on neurological mitochondrial disease. In order to achieve this, a mouse model of mitochondrial disease lacking a subunit of the NADH-Ubiquinone Oxidoreductase Complex (Ndufs4-/-) was used to conduct experiments. This model recapitulates Leigh Syndrome, a childhood mitochondrial disease characterized by progressive loss of psychomotor activity, retarded growth, and death within the first three years of life. Inhibition of mTOR (mechanistic Target of Rapamycin) with rapamycin increases lifespan across multiple model organisms. Rapamycin also increases lifespan in Ndufs4-/- mice. In this study, we tested whether acarbose, another drug that extends lifespan in mice, could also extend lifespan in Ndufs4-/- mice. Mice treated with acarbose had longer lifespan compared to untreated animals, and a significant delay in the onset of neurological symptoms. We also obtained brain tissue from these mice to determine whether rapamycin and acarbose are acting on the same biochemical pathways to rescue disease in these animals. Western blot analysis of brain protein extract from rapamycin treated mice showed no phosphorylation of S6 ribosomal protein, a marker of mTOR activity. Conversely, mice treated with acarbose showed phosphorylation of S6 ribosomal protein in the brain, suggesting that acarbose does not inhibit mTOR. Although both drugs prolonged lifespan in this model, these results suggest that they do not act on the same biochemical mechanisms. However, both rapamycin and acarbose appear to restore the NAD+/NADH ratio, reduce accumulation of glycolytic intermediates, and reduce acetylation of mitochondrial proteins in the brains of Ndufs4-/- mice, suggesting that the two drugs may have convergent effects on disease suppression.

Knock out of Ndufs4, a gene that encodes a nuclear-encoded subunit of complex 1, models neurological mitochondrial disease in mice. Ndufs4^-/- mice are shorter lived, show fur loss around 21 days of age, and begin to show neurological symptoms around day 35. Acarbose is a FDA-approved anti-diabetic drug used to manage post-prandial glucose spikes. Administration of acarbose increases lifespan in Ndufs4^-/- mice and delays the onset of neurological symptoms. Importantly, acarbose does not appear to restore mitochondrial respiration; rather it decreases protein acetylation in the mitochondria of Ndufs4^-/- mice and restores the NAD+ /NADH ratio in the brain. Due to this observation, we wanted to look into how Ndufs4^-/-Sirt3^-/- mice responded to acarbose treatment because Sirt3 is a NAD-dependent deacetylase responsible for the deacetylation of proteins in the mitochondria. To test this, we crossed Ndufs4^+/- mice into a Sirt3^-/- strain of mice to determine whether Sirt3 is necessary to extend lifespan in response to acarbose. We set up 4 experimental groups consisting of Sirt3^+/+Ndufs4^-/- controls, Sirt3^-/-Ndufs4^-/- controls, Sirt3^+/+Ndufs4^-/- on continual 0.1% acarbose chow from day 21, and Sirt3^-/-Ndufs4^-/- on continual 0.1% acarbose chow from day 21. All mice were housed with companion mice that were heterozygous for the Ndufs4 gene to help with thermal regulation and prevent premature death. Mice were monitored and weighed daily and fed bi-weekly with 0.1% acarbose chow. Knock out of Sirt3 did not affect lifespan in Ndufs4^-/- mice. Furthermore, we measured extended lifespan in the mice treated with acarbose in both the Sirt3^+/+ and Sirt3^-/- genotypes indicating that Sirt3 is not required for lifespan extension from acarbose in this disease model. We are planning to collect brains from Ndufs4^-/- Sirt3^-/- animals to determine whether acarbose reduces acetylation in the mitochondria through a different mechanism, not the upregulation of Sirt3 deacetylase.