Wellcome Centre for Mitochondrial Research

NDUFAF6 encodes a mitochondrial complex I assembly factor essential for the proper biogenesis and stability of the nicotinamide adenine dinucleotide (NAD) + hydrogen (H) (NADH)–ubiquinone oxidoreductase complex. Pathogenic variants in NDUFAF6 have been increasingly recognized as a cause of mitochondrial disease, particularly Leigh syndrome, a severe neurodegenerative disorder characterized by bilateral symmetrical lesions in the central nervous system. To date, fewer than 50 patients with NDUFAF6-related mitochondrial disease have been reported, displaying a broad phenotypic spectrum ranging from early-onset neurodevelopmental regression to milder, more chronic presentations. The molecular mechanisms underlying these phenotypes are linked to impaired complex I assembly and reduced enzymatic activity, highlighting the critical role of NDUFAF6 in mitochondrial function.Here we present a cohort of 27 patients (14 males and 13 females) from 18 families harbouring biallelic variants in the NDUFAF6 gene. The patient’s mean age was 9.15 ± 8.30 years (range: 4 weeks to 25 years); 12 patients (37%) had died by the time the data were collected for this article. The clinical presentation showed wide phenotypic variability, from mild to severe psychomotor regression (74%) most commonly before the age of 5 years, hypotonia (22%), movement disorders (30%), and hypertonia (15%). Bilateral striatal necrosis lesions were the most characteristic features on cranial MRI (67%) although white matter abnormalities were also noted (15%), occasionally accompanied by cystic formations, suggestive of early neurodevelopmental anomalies.Genomic sequencing was applied, leading to the identification of 19 distinct variants in the NDUFAF6 gene, including nine novel variants not previously reported and either absent or extremely rare in public population databases. Functional studies confirmed the pathogenicity of these variants, demonstrating a deleterious effect on NDUFAF6 protein expression and a consequent impairment in complex I assembly and stability.To date, this represents the largest reported cohort of patients with NDUFAF6-associated mitochondrial disease. Our findings provide a comprehensive overview of clinical characteristics—including age of symptom onset, phenotypic variability, and patient outcomes—aiming to improve prognostic information and facilitate genetic counselling in clinical practice.

Biallelic hypomorphic variants in PRORP cause the rare autosomal recessive disorder combined oxidative phosphorylation deficiency type 54 (COXPD54). COXPD54 encompasses a clinical spectrum of sensorineural hearing loss and ovarian insufficiency (Perrault syndrome) to leukodystrophy with developmental delay and epilepsy. Here, we report two new affected individuals with biallelic PRORP variants with clinical features consistent with COXPD54. One individual was homozygous for c.1505G > A p.Arg502Gln, whereas the other was compound heterozygous for c.1510C > T, p.His504Tyr and c.893C > A, p.Ser298Ter (NM_014672.4). In vitro tRNA processing assays revealed decreased mitochondrial 5′ tRNA leader cleavage by human RNase P complex with the two novel missense PRORP metallonuclease domain variants. These data provide further evidence that biallelic PRORP variants disrupt 5’ tRNA leader cleavage and are associated with a pleiotropic phenotype of COXPD54.

Myopathy is a prevalent and disabling feature of mitochondrial disease, in which skeletal muscle accumulates fibres with mitochondrial dysfunction in a variable mosaic pattern. This intra-individual spatial heterogeneity, a key consideration in longitudinal assessments, remains largely uncharacterised, hindering mechanistic studies and clinical trials by obscuring or confounding findings. We quantified this variability in m.3243 A > G-related myopathy, a leading cause of adult mitochondrial disease. Post-mortem biopsies from quadriceps femoris and tibialis anterior muscles of four patients were analysed for single-fibre deficiency in oxidative phosphorylation (OXPHOS) complex I and IV, while homogenate mitochondrial DNA (mtDNA) copy number and m.3243 A > G heteroplasmy were respectively determined by quantitative PCR and pyrosequencing. Bootstrapped combinatorial analyses established thresholds for minimum meaningful change above the 97.5th percentile, while accounting for anatomical biopsy distancing. Spatial variability in the proportion of OXPHOS-deficient fibres increased with distancing; within the same muscle, this threshold was 13.8% for NDUFB8 and 9.8% for MT-CO1. Variability in mtDNA copy number modestly increased with distance, while m.3243 A > G heteroplasmy remained largely stable, with within-muscle thresholds of 1,136 copies per nucleus and 8.2%, respectively. These findings provide assay-specific thresholds and offer mechanistic and translational insights for trial design, patient monitoring, and reliable detection of disease progression or therapeutic response.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-33106-3.

Despite suppressive anti-retroviral therapy (ART), some older people with HIV show adverse ageing phenotypes, and the underlying mechanisms remain incompletely understood. We recruited 30 men with HIV aged ≥ 50 years and 15 well-matched men without HIV and performed histological analyses on skeletal muscle biopsies and plasma biomarker measurement, in combination with clinical and functional assessments. Men with HIV showed higher frequencies of frailty, pre-frailty, sarcopenia, and pre-sarcopenia when compared to men without HIV. When assessing skeletal muscle, men with HIV had decreased mitochondrial complex I and IV protein abundance and myofibre regeneration, whilst fibrosis was increased, and plasma TNFα and MCP-4 levels were elevated. Spearman correlation analyses suggested that inflammation and mitochondrial respiratory chain deficiency may result in a damage response in skeletal muscle with resolution by fibrosis rather than regeneration. These findings thus provide plausible rationales for adverse ageing phenotypes in older men with HIV, including frailty and sarcopenia.

Idiopathic pulmonary fibrosis (IPF) is a devastating interstitial lung disease characterized by the excessive accumulation of activated myofibroblasts that deposit extracellular matrix (ECM) protein, leading to progressive scar formation and mechanical stress. However, the cellular origin and fate of myofibroblasts remain controversial, and the mechanisms by which myofibroblasts sense mechanical cues in the lung are unclear. Here, we report that periostin (Postn) is a reliable and distinctive marker for pulmonary myofibroblasts, while ablation of Postn+ myofibroblasts after injury ameliorated lung fibrosis. PIEZO1 was highly expressed in Postn+ myofibroblast and played a vital role in mechanoactivation of Postn+ myofibroblast and development of lung fibrosis. Conditional deletion of Piezo1 in Postn+ myofibroblasts significantly inhibited lung fibrosis by suppressing myofibroblast activation and proliferation. Loss of Piezo1 led to disruption of actin organization and prevention of Yap/Taz nuclear localization, thus shifting the myofibroblasts from a proliferative state into a stressed and apoptotic state. Furthermore, myofibroblast-specific Yap/Taz deletion fully recapitulated the protective phenotypes of myofibroblast-Piezo1–KO mice. These findings show that periostin marks pulmonary myofibroblasts, and that PIEZO1-mediated mechanosensation is essential for myofibroblast activation in the lung. Targeting PIEZO1 in the periostin-expressing cells is a novel therapeutic option to interfere with fibrotic diseases such as IPF .

Homeostasis in the kidney proximal tubule (PT) requires coordination between metabolism and differentiation, yet the mechanisms governing this balance remain elusive. Here, we integrate model organisms, multiomics profiling, and human genetics to identify the autophagy regulator ATG7 as a key determinant of cell-fate decisions, sustaining PT specialization in health and contributing to dysfunction in disease. In mice, PT-specific deletion of ATG7 reprograms differentiated cells into anabolic, proliferative states, impairing their specialized function and causing kidney tubulopathy. Mechanistically, loss of ATG7-dependent autophagy hinders lipid droplet clearance and restricts fatty-acid oxidation (FAO), leading to energy depletion and functional decline. In zebrafish pronephros, re-expression of wild-type ATG7 restores homeostasis in atg7 mutants, while pharmacological FAO inhibition triggers dysfunction. In humans, ATG7 variants associate with cardio-renal-metabolic traits and increased disease risk, whereas low ATG7 expression correlates with transcriptional signatures of metabolic reprogramming, loss of epithelial markers, and poor prognosis in renal cell carcinoma. These findings establish a conserved genetic paradigm that links autophagy to kidney epithelial cell-fate specialization, with implications for disease, cancer, and metabolic health.

Mitochondria are organelles in most human cells which release the energy required for cells to function. Oxidative phosphorylation (OXPHOS) is a key biochemical process within mitochondria required for energy production and requires a range of proteins and protein complexes. Mitochondria contain multiple copies of their own genome (mtDNA), which codes for some of the proteins and ribonucleic acids required for mitochondrial function and assembly. Pathology arises from genetic defects in mtDNA and can reduce cellular abundance of OXPHOS proteins, affecting mitochondrial function. Due to the continuous turn-over of mtDNA, pathology is random and neighbouring cells can possess different OXPHOS protein abundance. Estimating the proportion of cells where OXPHOS protein abundance is too low to maintain normal function is critical to understanding disease severity and predicting disease progression. Currently, one method to classify single cells as being OXPHOS deficient is prevalent in the literature. The method compares a patient's OXPHOS protein abundance to that of a small number of healthy control subjects. If the patient's cell displays an abundance which differs from the abundance of the controls then it is deemed deficient. However, due to the natural variation between subjects and the low number of control subjects typically available, this method is inflexible and often results in a large proportion of patient cells being misclassified. These misclassifications have significant consequences for the clinical interpretation of these data. We propose a single-cell classification method using a Bayesian hierarchical mixture model, which allows for inter-subject OXPHOS protein abundance variation. The model accurately classifies an example dataset of OXPHOS protein abundances in skeletal muscle fibres (myofibres). When comparing the proposed and existing model classifications to manual classifications performed by experts, the proposed model results in estimates of the proportion of deficient myofibres that are consistent with expert manual classifications.

Dominant optic atrophy (DOA) is the most common inherited optic neuropathy, characterised by the selective loss of retinal ganglion cells (RGCs). Over 60% of DOA cases are caused by pathogenic variants in the OPA1 gene, which encodes a dynamin-related GTPase protein. OPA1 plays a key role in the maintenance of the mitochondrial network, mitochondrial DNA integrity and bioenergetic function. However, our current understanding of how OPA1 dysfunction contributes to vision loss in DOA patients has been limited by access to patient-derived RGCs. Here, we used induced pluripotent stem cell (iPSC)-RGCs to study how OPA1 dysfunction affects cellular homeostasis in human RGCs. iPSCs derived from a DOA+ patient with the OPA1 R445H variant and isogenic CRISPR-Cas9-corrected iPSCs were differentiated to iPSC-RGCs. Defects in mitochondrial networks and increased levels of reactive oxygen species were observed in iPSC-RGCs carrying OPA1 R445H. Ultrastructural analyses also revealed changes in mitochondrial shape and cristae structure, with decreased endoplasmic reticulum (ER): mitochondrial contact length in DOA iPSC-RGCs. Mitochondrial membrane potential was reduced and its maintenance was also impaired following inhibition of the F1Fo-ATP synthase with oligomycin, suggesting that mitochondrial membrane potential is maintained in DOA iPSC-RGCs through reversal of the ATP synthase and ATP hydrolysis. These impairments in mitochondrial structure and function were associated with defects in cytosolic calcium buffering following ER calcium release and store-operated calcium entry, and following stimulation with the excitatory neurotransmitter glutamate. In response to mitochondrial calcium overload, DOA iPSC-RGCs exhibited increased sensitivity to opening of the mitochondrial permeability transition pore. These data reveal novel aspects of DOA pathogenesis in R445H patient-derived RGCs. The findings suggest a mechanism in which primary defects in mitochondrial network dynamics disrupt core mitochondrial functions, including bioenergetics, calcium homeostasis, and opening of the permeability transition pore, which may contribute to vision loss in DOA patients.