Deoxyribonucleoside triphosphates (dNTPs) are vital for the biosynthesis and repair of DNA. Their cellular concentration peaks during the S phase of the cell cycle. In non-proliferating cells, dNTP concentrations are low, making their reliable quantification from tissue samples of heterogeneous cellular composition challenging. Partly because of this, the current knowledge related to the regulation of and disturbances in cellular dNTP concentrations derive mostly from cell culture experiments with little corroboration at the tissue or organismal level. Here, we fill the methodological gap by presenting a simple non-radioactive microplate assay for the quantification of dNTPs with a minimum requirement of 4–12 mg of biopsy material. In contrast to published assays, this assay is based on long synthetic single-stranded DNA templates (50–200 nucleotides), an inhibitor-resistant high-fidelity DNA polymerase, and the double-stranded-DNA-binding EvaGreen dye. The assay quantified reliably less than 50 fmol of each of the four dNTPs and discriminated well against ribonucleotides. Additionally, thermostable RNAse HII-mediated nicking of the reaction products and a subsequent shift in their melting temperature allowed near-complete elimination of the interfering ribonucleotide signal, if present. Importantly, the assay allowed measurement of minute dNTP concentrations in mouse liver, heart and skeletal muscle.

Alternative oxidase (AOX) is a non‐mammalian enzyme that can bypass blockade of the complex III‐IV segment of the respiratory chain (RC). We crossed a Ciona intestinalis AOX transgene into RC complex III (cIII)‐deficient Bcs1l ^p.S78G knock‐in mice, displaying multiple visceral manifestations and premature death. The homozygotes expressing AOX were viable, and their median survival was extended from 210 to 590 days due to permanent prevention of lethal cardiomyopathy. AOX also prevented renal tubular atrophy and cerebral astrogliosis, but not liver disease, growth restriction, or lipodystrophy, suggesting distinct tissue‐specific pathogenetic mechanisms. Assessment of reactive oxygen species (ROS) production and damage suggested that ROS were not instrumental in the rescue. Cardiac mitochondrial ultrastructure, mitochondrial respiration, and pathological transcriptome and metabolome alterations were essentially normalized by AOX, showing that the restored electron flow upstream of cIII was sufficient to prevent cardiac energetic crisis and detrimental decompensation. These findings demonstrate the value of AOX, both as a mechanistic tool and a potential therapeutic strategy, for cIII deficiencies.

Mitochondrial disorders are among the most prevalent inborn errors of metabolism but largely lack treatments and have poor outcomes. High-fat, low-carbohydrate ketogenic diets (KDs) have shown beneficial effects in mouse models of mitochondrial myopathies, with induction of mitochondrial biogenesis as the suggested main mechanism. We fed KD to mice with respiratory chain complex III (CIII) deficiency and progressive hepatopathy due to mutated BCS1L, a CIII assembly factor. The mutant mice became persistently ketotic and tolerated the KD for up to 11 weeks. Liver disease progression was attenuated by KD as shown by delayed fibrosis, reduced cell death, inhibition of hepatic progenitor cell response and stellate cell activation, and normalization of liver enzyme activities. Despite no clear signs of increased mitochondrial biogenesis in the liver, CIII assembly and activity were improved and mitochondrial morphology in hepatocytes normalized. Induction of hepatic glutathione transferase genes and elevated total glutathione level were normalized by KD. Histological findings and transcriptome changes indicated modulation of liver macrophage populations by the mutation and the diet. These results reveal a striking beneficial hepatic response to KD in mice with mitochondrial hepatopathy and warrant further investigations of dietary modification in the management of these conditions in patients.

Mitochondrial diseases due to defective respiratory chain complex III (CIII) are relatively uncommon. The assembly of the eleven-subunit CIII is completed by the insertion of the Rieske iron-sulfur protein, a process for which BCS1L protein is indispensable. Mutations in the BCS1L gene constitute the most common diagnosed cause of CIII deficiency, and the phenotypic spectrum arising from mutations in this gene is wide. A case of CIII deficiency was investigated in depth to assess respiratory chain function and assembly, and brain, skeletal muscle and liver histology. Exome sequencing was performed to search for the causative mutation(s). The patient’s platelets and muscle mitochondria showed respiration defects and defective assembly of CIII was detected in fibroblast mitochondria. The patient was compound heterozygous for two novel mutations in BCS1L, c.306A > T and c.399delA. In the cerebral cortex a specific pattern of astrogliosis and widespread loss of microglia was observed. Further analysis showed loss of Kupffer cells in the liver. These changes were not found in infants suffering from GRACILE syndrome, the most severe BCS1L-related disorder causing early postnatal mortality, but were partially corroborated in a knock-in mouse model of BCS1L deficiency. We describe two novel compound heterozygous mutations in BCS1L causing CIII deficiency. The pathogenicity of one of the mutations was unexpected and points to the importance of combining next generation sequencing with a biochemical approach when investigating these patients. We further show novel manifestations in brain, skeletal muscle and liver, including abnormality in specialized resident macrophages (microglia and Kupffer cells). These novel phenotypes forward our understanding of CIII deficiencies caused by BCS1L mutations.

We previously observed an unexpected fivefold (35 vs. 200 days) difference in the survival of respiratory chain complex III (CIII) deficient Bcs1l^p.S78G mice between two congenic backgrounds. Here, we identify a spontaneous homoplasmic mtDNA variant (m.G14904A, mt-Cyb^p.D254N), affecting the CIII subunit cytochrome b (MT-CYB), in the background with short survival. We utilize maternal inheritance of mtDNA to confirm this as the causative variant and show that it further decreases the low CIII activity in Bcs1l^p.S78G tissues to below survival threshold by 35 days of age. Molecular dynamics simulations predict D254N to restrict the flexibility of MT-CYB ef loop, potentially affecting RISP dynamics. In Rhodobacter cytochrome bc₁ complex the equivalent substitution causes a kinetics defect with longer occupancy of RISP head domain towards the quinol oxidation site. These findings represent a unique case of spontaneous mitonuclear epistasis and highlight the role of mtDNA variation as modifier of mitochondrial disease phenotypes.

Biosynthetic precursors of NAD ⁺ can replenish a decreased cellular NAD ⁺ pool and, supposedly via sirtuin (SIRT) deacetylases, improve mitochondrial function. We found decreased hepatic NAD ⁺ concentration and downregulated biosynthesis in Bcs1l ^pS78G knock‐in mice with respiratory chain complex III deficiency and mito chondrial hepatopathy. Aiming at ameliorating disease progression via NAD ⁺ repletion and improved mitochon drial function, we fed these mice nicotinamide riboside (NR), a NAD ⁺ precursor. A targeted metabolomics verified successful administration and suggested enhanced NAD ⁺ biosynthesis in the treated mice, although hepatic NAD ⁺ concentration was unchanged at the end point. In contrast to our expectations, NR did not improve the hepatopathy, hepatic mitochondrial respiration, or survival of Bcs1l ^pS78G mice. We linked this lack of therapeutic effect to NAD ⁺ ‐independent activation of SIRT‐1 and ‐3 via AMPK and cAMP signaling related to the starvation‐like metabolic state of Bcs1l ^p.S78G mice. In summary, we describe an unusual metabolic state with NAD ⁺ depletion accompanied by energy deprivation signals, uncompromised SIRT function, and upregulated oxidative me tabolism. Our study highlights that the knowledge of the underlying complex metabolic alterations is critical when designing therapies for mitochondrial dysfunction.—Purhonen, J., Rajendran, J., Tegelberg, S., Smolander, O.‐P., Pirinen, E., Kallijärvi, J., Fellman, V. NAD ⁺ repletion produces no therapeutic effect in mice with respiratory chain complex III deficiency and chronic energy deprivation. FASEB J. 32, 5913–5926 (2018). www.fasebj.org