Label-free spatially maintained measurements of metabolic phenotypes in cells.

Metabolic reprogramming at a cellular level contributes to many diseases including cancer, yet few assays are capable of measuring metabolic pathway usage by individual cells within living samples. Here, autofluorescence lifetime imaging is combined with single-cell segmentation and machine-learning models to predict the metabolic pathway usage of cancer cells. The metabolic activities of MCF7 breast cancer cells and HepG2 liver cancer cells were controlled by growing the cells in culture media with specific substrates and metabolic inhibitors.

Cooperative sensing of mitochondrial DNA by ZBP1 and cGAS promotes cardiotoxicity.

Mitochondrial DNA (mtDNA) is a potent agonist of the innate immune system; however, the exact immunostimulatory features of mtDNA and the kinetics of detection by cytosolic nucleic acid sensors remain poorly defined. Here, we show that mitochondrial genome instability promotes Z-form DNA accumulation. Z-DNA binding protein 1 (ZBP1) stabilizes Z-form mtDNA and nucleates a cytosolic complex containing cGAS, RIPK1, and RIPK3 to sustain STAT1 phosphorylation and type I interferon (IFN-I) signaling.

Assessing Mitochondrial DNA Release into the Cytosol and Subsequent Activation of Innate Immune-related Pathways in Mammalian Cells.

Mitochondria have emerged as key drivers of mammalian innate immune responses, functioning as signaling hubs to trigger inflammation and orchestrating metabolic switches required for phagocyte activation. Mitochondria also contain damage-associated molecular patterns (DAMPs), molecules that share similarity with pathogen-associated molecular patterns (PAMPs) and can engage innate immune sensors to drive inflammation. The aberrant release of mitochondrial DAMPs during cellular stress and injury is an increasingly recognized trigger of inflammatory responses in human diseases.

Neuroimmune mechanisms of cognitive impairment in a mouse model of Gulf War illness.

Gulf War Illness (GWI) is a chronic, multi-symptom disorder affecting approximately 30 percent of the nearly 700,000 Veterans of the 1991 Persian Gulf War. GWI-related chemical (GWIC) exposure promotes immune activation that correlates with cognitive impairment and other symptoms of GWI. However, the molecular mechanisms and signaling pathways linking GWIC to inflammation and neurological symptoms remain unclear.

Elevated type I interferon responses potentiate metabolic dysfunction, inflammation, and accelerated aging in mtDNA mutator mice.

Mitochondrial dysfunction is a key driver of inflammatory responses in human disease. However, it remains unclear whether alterations in mitochondria-innate immune cross-talk contribute to the pathobiology of mitochondrial disorders and aging. Using the polymerase gamma (POLG) mutator model of mitochondrial DNA instability, we report that aberrant activation of the type I interferon (IFN-I) innate immune axis potentiates immunometabolic dysfunction, reduces health span, and accelerates aging in mutator mice.

Deletion of the neural tube defect-associated gene disrupts one-carbon and central energy metabolism in mouse embryos.

One-carbon (1C) metabolism is a universal folate-dependent pathway essential for purine and thymidylate synthesis, amino acid interconversion, universal methyl-donor production, and regeneration of redox cofactors. Homozygous deletion of the 1C pathway gene encoding methylenetetrahydrofolate dehydrogenase (NADP-dependent) 1-like, which catalyzes mitochondrial formate production from 10-formyltetrahydrofolate, results in 100% penetrant embryonic neural tube defects (NTDs), underscoring the central role of mitochondrially derived formate in embryonic development and providing a mechanistic link between folate and NTDs. However, the specific metabolic processes that are perturbed by deletion are not known.

Mitochondrial MTHFD2L is a dual redox cofactor-specific methylenetetrahydrofolate dehydrogenase/methenyltetrahydrofolate cyclohydrolase expressed in both adult and embryonic tissues.

Mammalian mitochondria are able to produce formate from one-carbon donors such as serine, glycine, and sarcosine. This pathway relies on the mitochondrial pool of tetrahydrofolate (THF) and several folate-interconverting enzymes in the mitochondrial matrix. We recently identified MTHFD2L as the enzyme that catalyzes the oxidation of 5,10-methylenetetrahydrofolate (CH2-THF) in adult mammalian mitochondria.

Deletion of Mthfd1l causes embryonic lethality and neural tube and craniofacial defects in mice.

Maternal supplementation with folic acid is known to reduce the incidence of neural tube defects (NTDs) by as much as 70%. Despite the strong clinical link between folate and NTDs, the biochemical mechanisms through which folic acid acts during neural tube development remain undefined. The Mthfd1l gene encodes a mitochondrial monofunctional 10-formyl-tetrahydrofolate synthetase, termed MTHFD1L.

Mammalian MTHFD2L encodes a mitochondrial methylenetetrahydrofolate dehydrogenase isozyme expressed in adult tissues.

Previous studies in our laboratory showed that isolated, intact adult rat liver mitochondria are able to oxidize the 3-carbon of serine and the N-methyl carbon of sarcosine to formate without the addition of any other cofactors or substrates. Conversion of these 1-carbon units to formate requires several folate-interconverting enzymes in mitochondria. The enzyme(s) responsible for conversion of 5,10-methylene-tetrahydrofolate (CH(2)-THF) to 10-formyl-THF in adult mammalian mitochondria are currently unknown.