Targeting NADH-Reductive Stress in Mitochondrial Disease

Project Summary Mitochondria are essential organelles for maintaining cellular redox, energy, and metabolic homeostasis. The hub of mitochondrial function is its electron transport chain (ETC) which is necessary for numerous functions, including ATP generation. There are over 300 inherited mutations that can impair ETC function and lead to

devastating neuromuscular disorders (e.g., Leigh syndrome) for which there are no FDA-approved therapies. This is in large part because of our incomplete understanding of the disease pathophysiology. Recent studies have demonstrated that an elevated tissue NADH/NAD+ ratio, also known as NADH-reductive stress, caused by

ETC dysfunction is a major contributor to ETC diseases. NADH-reductive stress inhibits numerous crucial biochemical reactions to cause tissue dysfunction and disease. However, the potential of targeting NADH- reductive stress in ETC dysfunction for therapeutic benefits has not been extensively explored, primarily due to

the absence of tools to simultaneously mitigate NADH-reductive stress in all affected tissues. To this end, we recently developed a proof-of-concept enzyme tool called LOXCAT to alleviate NADH-reductive stress in ETC dysfunctions. LOXCAT is an engineered fusion of enzymes lactate oxidase (LOX) and catalase (CAT) that is

designed to irreversibly convert lactate into pyruvate. Intravenous injection of LOXCAT into mice with ETC dysfunction lowers the blood lactate/pyruvate ratio and simultaneously decreases the NADH/NAD+ ratio in key organs like the heart and the brain. This is because the extracellular lactate/pyruvate ratio is in near equilibrium

with the intracellular NADH/NAD+ ratio via the cytosolic lactate dehydrogenase (LDH) reaction. In cell culture models of ETC dysfunction, supplemented extracellular LOXCAT can restore cellular redox and energy homeostasis as well as rescue cell proliferation by alleviating NADH-reductive stress. This proposal aims to

utilize LOXCAT as a tool to further understand its mechanism of action and explore the therapeutic potential of targeting NADH-reductive stress in ETC dysfunction. We will investigate: (a) how LOXCAT impacts global metabolism and mitochondrial function in primary cortical neurons and fibroblasts of NDUFS4-null mice that

recapitulate complex I dysfunction in Leigh Syndrome, (b) how brain-specific vs systemic effects of LOXCAT impacts survival, neurological symptoms, and tissue metabolism of the NDUFS4-null mice, and (c) re-engineer LOXCAT to evade immunogenicity, if necessary. Leigh syndrome, a condition leading to progressive

neurodegeneration, is the most common pediatric manifestation of an ETC dysfunction. Since LOXCAT alleviates NADH-reductive stress, a common outcome of the majority of ETC dysfunctions, it holds the potential to be a therapy for various mitochondrial diseases, regardless of their underlying mutations. Overall, our proposal

will provide unprecedented opportunities to further evaluate NADH-reductive stress in mitochondrial disease and open up the potential to develop the first therapy for neuromuscular diseases caused by mitochondrial dysfunction.