Pathogenic mutations in mitochondrial (mt) tRNA genes that compromise oxidative phosphorylation (OXPHOS) exhibit heteroplasmy and cause a range of multisyndromic conditions. Although mitochondrial disease patients are known to suffer from abnormal immune responses, how heteroplasmic mtDNA mutations affect the immune system at the molecular level is largely unknown. Here, in mice carrying pathogenic C5024T in mt-tRNA ^Ala and in patients with mitochondrial encephalomyopathy, lactic acidosis, stroke-like episodes (MELAS) syndrome carrying A3243G in mt-tRNA ^Leu , we found memory T and B cells to have lower pathogenic mtDNA mutation burdens than their antigen-inexperienced naive counterparts, including after vaccination. Pathogenic burden reduction was less pronounced in myeloid compared with lymphoid lineages, despite C5024T compromising macrophage OXPHOS capacity. Rapid dilution of the C5024T mutation in T and B cell cultures could be induced by antigen receptor–triggered proliferation and was accelerated by metabolic stress conditions. Furthermore, we found C5024T to dysregulate CD8 ⁺ T cell metabolic remodeling and IFN-γ production after activation. Together, our data illustrate that the generation of memory lymphocytes shapes the mtDNA landscape, wherein pathogenic variants dysregulate the immune response.

BACKGROUND: Mitochondrial diseases belong to the group of inborn errors of metabolism (IEM), with a prevalence of 1 in 2,000–5,000 individuals. They are the most common form of IEM, but, despite advances in next-generation sequencing technologies, almost half of the patients are left genetically undiagnosed. METHODS: We investigated a cohort of 61 patients with defined mitochondrial disease to improve diagnostics, identify biomarkers, and correlate metabolic pathways to specific disease groups. Clinical presentations were structured using human phenotype ontology terms, and mass spectrometry–based proteomics was performed on primary fibroblasts. Additionally, we integrated 6 patients carrying variants of uncertain significance (VUS) to test proteomics as a diagnostic expansion. RESULTS: Proteomic profiles from patient samples could be classified according to their biochemical and genetic characteristics, with the expression of 5 proteins (GPX4, MORF4L1, MOXD1, MSRA, and TMED9) correlating with the disease cohort, thus acting as putative biomarkers. Pathway analysis showed a deregulation of inflammatory and mitochondrial stress responses. This included the upregulation of glycosphingolipid metabolism and mitochondrial protein import, as well as the downregulation of arachidonic acid metabolism. Furthermore, we could assign pathogenicity to a VUS in MRPS23 by demonstrating the loss of associated mitochondrial ribosome subunits. CONCLUSION: We established mass spectrometry–based proteomics on patient fibroblasts as a viable and versatile tool for diagnosing patients with mitochondrial disease. FUNDING: The NovoNordisk Foundation, Knut and Alice Wallenberg Foundation, Wellcome Centre for Mitochondrial Research, UK Medical Research Council, and the UK NHS Highly Specialised Service for Rare Mitochondrial Disorders of Adults and Children.

SPNS1 is a lysosomal transporter that mediates the salvage of lysoglycerophospholipids, the degradative products of lysosomal phospholipid catabolism. However, an understanding of the role of lysolipid transport and salvage in regulating cellular lipid homeostasis and in disease is lacking. Here, we identified members of 2 families with biallelic SPNS1 loss-of-function variants, who presented primarily with progressive liver and striated muscle injury. Patients’ fibroblasts accumulated lysophospholipids including lysoplasmalogens and cholesterol in lysosomes with reduced cellular plasmalogens. Notably, SPNS1 deficiency resulted in reduced biogenesis of cytosolic lipid droplets containing triglycerides and cholesteryl esters. Mechanistically, we found that lysophospholipids transported by SPNS1 into the cytosol quantitatively contributed to triglyceride synthesis, whereas lysosomal buildup of lyso-ether-phospholipid inhibited lysosomal cholesterol egress, effects that were enhanced with inhibition of mTOR. These findings support a gene-disease association and reveal connectivity between lysosomal transport of lysophospholipids and storage of reserve cellular energy as triglycerides and the regulation of cholesterol homeostasis, processes that become important under nutrient limitation.

Background: Recent studies describe an association between sertraline and acquired late‐onset multiple acyl coenzyme A dehydrogenase deficiency (MADD), a rare disorder affecting fatty acid metabolism. MADD was previously considered solely a genetic disorder caused by recessive pathogenic variants mainly in the ETFDH gene. Methods: A nationwide population‐based registry search was conducted to identify adult patients with sertraline‐associated MADD in Sweden 2014–2024. Patients with muscle biopsy reports of lipid storage myopathy or biochemical investigations showing acylcarnitine profile suggesting MADD were included. Medical records were retrospectively reviewed. Results: We identified 40 (30/10 female/male) patients using sertraline when diagnosed with MADD . Median age at symptom onset was 42 years and muscle weakness was the most common first symptom. All patients reported muscle symptoms during the disease course. Extra‐muscular symptoms, including sensory disturbance (29 patients) and fatigue (18 patients), were common. Most had lipid accumulation in muscle fibers, an abnormal acylcarnitine profile, a myopathic electromyography pattern, and elevated creatine kinase. Cyanocobalamin, folic acid, and proton‐pump inhibitors were the most frequent other medications at diagnosis. Five patients carried a heterozygous ETFDH variant. All patients were treated with riboflavin. Almost all (38/39) experienced clinical improvement. Acylcarnitine profile improved in all patients (31/31) with available data. The mean incidence was 1.24 patients per 100,000 adults receiving sertraline treatment per year. Conclusions: Acquired MADD associated with sertraline is the most common type of adult‐onset MADD in Sweden. The disease is generally responsive to riboflavin treatment. Patients presenting with muscle symptoms during sertraline therapy should undergo adequate evaluation to exclude MADD.

We present an optimised luminometric method for measuring muscle mitochondrial ATP production rate (MAPR), adapted to a 96-well microplate format. The enhanced assay enables quantification of ATP production from 12 or more substrate combinations within 15 min, using only 10 μL of isolated mitochondria. The method demonstrates high accuracy and precision, with a validated measurement range of 0.3-70 nmol/min/L. To support clinical interpretation, a reference dataset was established from 92 individuals aged seven months to 79 years. All these individuals were referred for muscle biopsy but were subsequently deemed unlikely to have a mitochondrial disorder following comprehensive clinical evaluation. An overview of the current version of our assays for oxidative phosphorylation (OXPHOS) enzymes is also provided. As proof of concept, we present three patients carrying pathogenic variants in mitochondrial DNA (ATP6 and MT-TL1) and the nuclear PDHA1 gene. All exhibited decreased MAPR with one or more substrates, along with additional clinical, biochemical, and morphological features consistent with mitochondrial disease. Furthermore, we illustrate the age-dependent development of MAPR in muscle across the human lifespan, demonstrating a 60-80 % higher maximal capacity for oxidative ATP production in adults compared with young children. In contrast, MAPR supported by fatty acid-derived substrates remains unchanged over the same period. In conclusion, the improved MAPR assay offers a robust and efficient tool for assessing mitochondrial function in both clinical diagnostics and research. Its high-throughput format and reliable performance make it particularly well-suited for the investigation of suspected mitochondrial disorders.

Pathogenic mitochondrial (mt)DNA molecules can exhibit heteroplasmy in single cells and cause a range of clinical phenotypes, although their contribution to immunity is poorly understood. Here, in mice carrying heteroplasmic C5024T in mt-tRNA ^Ala – that impairs oxidative phosphorylation – we found a reduced mutation burden in peripheral T and B memory lymphocyte subsets, compared to their naïve counterparts. Furthermore, selection diluting the mutation was induced in vitro by triggering T and B cell antigen receptors. While C5024T dysregulated naïve CD8 ⁺ T cell respiration and metabolic remodeling post-activation, these phenotypes were partially ameliorated by selection. Analogous to mice, peripheral blood memory T and B lymphocyte subsets from human MELAS (Mitochondrial Encephalomyopathy with Lactic Acidosis and Stroke-like episodes) patients – carrying heteroplasmic A3243G in mt-tRNA ^Leu – displayed a reduced mutation burden, compared to naïve cells. In both humans and mice, mtDNA selection was observed in IgG ⁺ antigen-specific B cells after SARS-CoV-2 Spike vaccination, illustrating an on-going process in vivo . Taken together, these data illustrate purifying selection of pathogenic mtDNA variants during the oxidative phosphorylation checkpoints of the naïve-memory lymphocyte transition. Highlights: In human MELAS patients (A3243G in mt-tRNA ^Leu ) and a related mouse model (C5024T in mt-tRNA ^Ala ), T and B memory subsets displayed a reduced mtDNA mutation burden compared to their naïve counterparts. Selection was observed in antigen-specific IgG ⁺ B cells after SARS-CoV-2 Spike protein vaccination. T and B cell antigen receptor stimulation triggered purifying selection in vitro , facilitating mechanistic studies of mtDNA selection. Heteroplasmic pathogenic mutations in mtDNA dysregulated metabolic remodeling after lymphocyte activation and reduced macrophage OXPHOS capacity.