Person:

Sharma, Rohit

Mitochondrial dysfunction is associated with a spectrum of human disorders, ranging from rare, inborn errors of metabolism to common, age-associated diseases such as neurodegeneration. How these lesions give rise to diverse pathology is not well understood, partly because their proximal consequences have not been well-studied in mammalian cells. Here we provide two lines of evidence that mitochondrial respiratory chain dysfunction leads to alterations in one-carbon metabolism pathways. First, using hypothesis-generating metabolic, proteomic, and transcriptional profiling, followed by confirmatory experiments, we report that mitochondrial DNA depletion leads to an ATF4-mediated increase in serine biosynthesis and transsulfuration. Second, we show that lesioning the respiratory chain impairs mitochondrial production of formate from serine, and that in some cells, respiratory chain inhibition leads to growth defects upon serine withdrawal that are rescuable with purine or formate supplementation. Our work underscores the connection between the respiratory chain and one-carbon metabolism with implications for understanding mitochondrial pathogenesis. DOI: http://dx.doi.org/10.7554/eLife.10575.001

SUMMARY A decline in mitochondrial respiration represents the root cause of a large number of inborn errors of metabolism. It is also associated with common age-associated diseases and the aging process. To gain insight into the systemic, biochemical consequences of respiratory chain dysfunction, we performed a case-control, prospective metabolic profiling study in a genetically homogenous cohort of patients with Leigh syndrome French Canadian variant, a mitochondrial respiratory chain disease due to loss-of-function mutations in LRPPRC. We discovered 45 plasma and urinary analytes discriminating patients from controls, including classic markers of mitochondrial metabolic dysfunction (lactate and acylcarnitines), as well as unexpected markers of cardiometabolic risk (insulin and adiponectin), amino acid catabolism linked to NADH status (α-hydroxybutyrate), and NAD+ biosynthesis (kynurenine and 3-hydroxyanthranilic acid). Our study identifies systemic, metabolic pathway derangements that can lie downstream of primary mitochondrial lesions, with implications for understanding how the organelle contributes to rare and common diseases.

An elevated intracellular NADH/NAD+ ratio, or “reductive stress,” has been associated with multiple diseases, including disorders of the mitochondrial electron transport chain (ETC). As the intracellular NADH/NAD+ ratio can be in near-equilibrium with the circulating lactate/pyruvate ratio, we hypothesized that reductive stress could be alleviated by oxidizing extracellular lactate into pyruvate. We engineered LOXCAT, a fusion of bacterial lactate oxidase (LOX) and catalase (CAT), which irreversibly converts lactate and oxygen to pyruvate and water. Addition of recombinant LOXCAT to the media of cultured human cells with a defective ETC was able to decrease the extracellular lactate/pyruvate ratio, normalize the intracellular NADH/NAD+ ratio, upregulate glycolytic ATP production, and restore cellular proliferation. In mice, tail-vein injected LOXCAT reduced circulating lactate/pyruvate ratio, blunted a metformin-induced rise in blood lactate/pyruvate, and improved NADH/NAD+ balance in heart and brain. Our study lays the groundwork for a class of injectable therapeutic enzymes that alleviate intracellular redox imbalances by directly targeting circulating redox-coupled metabolites.