Mapping mitochondrial NADPH utilization by exploiting hypomorphic variants of NADK2

Mitochondria are multifaceted organelles responsible for cellular energy production and nutrient catabolism, efficiently performing these tasks as distinct metabolic compartments. Such compartmentalization requires fine- tuned regulation of not only catabolic enzymes but also their associated co-factors. One such compartmentalized co-factor is nucleotide adenosine diphosphate phosphate (NAPDH), which enables numerous metabolic pathways within mitochondria including unsaturated fatty acid oxidation, lysine catabolism, type II fatty acid synthesis, and proline biogenesis, amongst others. Due to its broad utilization, perturbation of NADPH synthesis would have widespread, detrimental effects on mitochondrial metabolism. Indeed, human mutations in a key NADPH-producing enzyme, NADK2, can lead to Progressive encephalopathy with leukodystrophy due to DECR deficiency, an ultra-rare disease presenting with failure to thrive, pronounced hypotonia, and microcephaly. However, a recently identified patient harboring hypomorphic NADK2 alleles presented with a much milder disease profile and only select metabolic defects, suggesting that full loss of NADK2 broadly compromises mitochondrial metabolism, while partial loss of NADK2 affects only select pathways. We hypothesize that mild- to-intermediate loss of NADPH production disproportionately affects select pathways to spare more “essential” pathways, leading to a “priority code” of mitochondrial co-factor usage. As NADK2 represents a central NADPH- producer within mitochondria, we propose that an in-depth functional assessment of disease-associated NADK2 variants will illuminate a “priority code” for mitochondrial NADPH allocation, while simultaneously providing a mechanistic framework to understand disease heterogeneity. In this proposal, we will generate a series of cell lines that harbor NADK2 variants of uncertain significance with different predicted mutational burdens: low, moderate, or high. We will first establish and characterize these mutants by expressing each variant in NADK2 knockout HAP1 cells and measuring the extent to which these mutants can rescue mitochondrial respiration as well as organellar and cellular NADPH levels. We will then profile these cell lines with multiomic analyses of lipids and metabolites to understand the extent to which metabolic pathways become compromised upon limiting NADK2-produced mitochondrial NADPH. Completion of the proposed work will, as a proof of principle, demonstrate the power of cellular models of rare disease, as well as the breadth of dysfunction associated with specific mutational burdens, which could inform accurate diagnoses or therapeutic options for patients with NADK2 or DECR deficiency. Furthermore, these cells will provide a comprehensive toolkit to study how NADPH perturbations affect mitochondrial metabolism, providing a rich dataset of the dynamic nature of organelle-wide NADPH-consuming pathways. Project Number: 1R03NS149107-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of Neurological Disorders and Stroke (NINDS) | Principal Investigator: Natalie Niemi | Institution: WASHINGTON UNIVERSITY, SAINT LOUIS, MO | Award Amount: $155,500 | Activity Code: R03 | Study Section: Therapeutic Approaches to Genetic Diseases Study Section[TAG] View on NIH RePORTER: https://reporter.nih.gov/project-details/11354342