Mitochondrial DNA (mtDNA) polymorphisms have been associated with production traits in farm animals, including backfat thickness in pigs, yet direct in vivo evidence establishing a causal link between specific mtDNA haplotypes and fat deposition remains limited. In this study, we generated transmitochondrial pigs (mitopigs) by combining the Dapulian nuclear genome with Wuzhishan mtDNA via somatic cell nuclear transfer, introducing 23 mtDNA mutations relative to controls. Mitopigs exhibited significantly increased backfat thickness at 5 months, a difference that persisted in their offspring, without significant differences in body weight, body size, or litter size. Fibroblasts derived from mitopigs exhibited reduced mtDNA copy numbers, decreased expression of mitochondrial biogenesis genes (PPARA, PPARGC1A, RRM2B, and LRPPRC), impaired mitochondrial respiration, elevated reactive oxygen species (ROS), and upregulated adipogenic transcription factors (CEBPA, CEBPB, and PPARG). Consistent with these fibroblast findings, backfat tissue of mitopigs showed corresponding upregulation of adipogenic transcription factors and downregulation of mitochondrial biogenesis genes. Integrated transcriptomic and whole-genome bisulfite sequencing (WGBS) analyses revealed nuclear transcriptional reprogramming that was closely associated with differential DNA methylation, predominantly affecting mitochondrial function and lipid metabolism pathways. Mitopig fibroblasts also showed a pro-inflammatory response to lipopolysaccharide stimulation, with elevated expression of IL-12, NOS2, RELA, and TNF-α. Our findings provide direct in vivo evidence that mtDNA variants regulate adiposity in pigs through mitochondrial dysfunction, oxidative stress, and nuclear epigenetic modulation, highlighting the potential for incorporating mtDNA haplotype information into pig breeding programs as a complementary strategy to nuclear genomic selection.
Cerebral ischemia/reperfusion (I/R) injury refers to the exacerbation of tissue damage following the restoration of blood flow to ischemic brain regions. This condition remains a major challenge in the clinical management of ischemic stroke due to limited therapeutic options. At present, no approved drugs specifically target cerebral I/R injury. Multiple mechanisms contribute to its pathogenesis, with mitochondrial dysfunction playing a central role. During cerebral I/R injury, mitochondria generate excessive reactive oxygen species (ROS), leading to impaired mitochondrial function and further tissue damage. In addition, mitochondrial calcium overload triggers neuronal apoptosis, which promotes disease progression. Given the critical role of mitochondrial dysfunction, preservation of mitochondrial homeostasis may attenuate cerebral I/R injury. Mitophagy, a selective process that removes damaged mitochondria, has been shown to mitigate cerebral I/R injury by limiting the release of harmful mitochondrial-derived factors. Therefore, mitophagy represents a potential therapeutic target for maintaining mitochondrial homeostasis in cerebral I/R injury treatment. This review summarizes the molecular regulation of mitophagy, its role in cerebral I/R injury, and current therapeutic strategies aimed at modulating mitophagy.
Diabetes mellitus is a global health burden recognized by progressive microvascular complications, which comprise diabetic retinopathy, nephropathy, and neuropathy, often known as diabetic triopathy. Despite extensive research, the mechanistic convergence of multi-organ damage is not fully understood. Recent studies highlighted that ferroptosis, an iron-dependent form of regulated cell death mediated by lipid peroxidation, plays a crucial role in diabetic tissue damage. Importantly, mitochondria are key modulators of ferroptotic susceptibility because they regulate reactive oxygen species (ROS) production, iron homeostasis, and bioenergetic homeostasis. This review proposes mitochondrial-ferroptotic crosstalk as a unifying mechanistic axis associating retinal, renal, and neural complications in diabetes. We present a comprehensive overview of the molecular basis of ferroptosis through a mitochondria-centered perspective, covering key pathways such as iron homeostasis, the glutathione-GPX4 system, and emerging regulators of ferroptosis such as SLC7A11 and the FSP1-coenzyme Q axis. We also discuss shared vulnerabilities across microvascular tissues, such as mitochondrial dysfunction, iron overload, lipid peroxidation, and chronic inflammation. Tissue-specific evidence supporting ferroptosis in diabetic retinopathy, nephropathy, and neuropathy is discussed critically, with with mitochondrial impairment and redox imbalance emerging as prevalent drivers of the pathophysiology. Finally, we assess current and novel therapeutic approaches targeting the mitochondrial-ferroptotic axis, such as ferroptosis inhibitors, mitochondrial antioxidants, and iron-regulating approaches. Altogether, this integrative model identifies mitochondrial-ferroptotic crosstalk as a key pathogenic mechanism and therapeutic opportunity to mitigate multi-organ complications in diabetes.
Coa6 controls copper delivery into the Cox2 subunit of the mitochondrial respiratory complex IV. Mutations in COA6 lead to Cox2 degradation and cause cardiomyopathy in humans. We show that decreasing the level of the mobile electron transporter cytochrome c improves both Cox2 accumulation and complex IV assembly in the budding yeast coa6-null mutant. As the heme attachment to apo-cytochrome c and the copper delivery to apo-Cox2 both require cysteine reduction, we propose that Coa6 plays a role in coordinating the maturation of Cox2 and Cytc with their cofactors.
Traumatic brain injury (TBI) is a leading global cause of death and long-term disability, primarily due to secondary injury mechanisms such as mitochondrial dysfunction and impaired mitophagy，the selective degradation of damaged mitochondria. While the ACE2/Angiotensin-(1-7)/Mas receptor (MasR) axis is recognized for its neuroprotective effects in various neurological disorders, its role in regulating mitochondrial quality control after TBI remains unclear. In this study, we investigated the regulatory function of MasR in post-traumatic mitophagy using controlled cortical impact (CCI) mice and scratch-injured neuronal cultures. We employed MasR knockdown (MasR-KD) and pharmacological activation with the selective MasR agonist AVE0991 to assess neurobehavioral outcomes, neuronal survival, and mitophagy flux. Activation of MasR significantly improved motor coordination, cognitive performance, and reduced anxiety-like behaviors following TBI, whereas MasR deficiency exacerbated neurological deficits. Histologically, MasR-KD mice exhibited increased neuronal loss, dendritic degeneration, and oxidative stress. In contrast, AVE0991 treatment preserved neuronal integrity and mitochondrial ultrastructure, effects that were abolished in MasR-KD animals. Mechanistically, MasR activation promoted PINK1/Parkin-mediated mitophagy, enhanced TOMM20-LC3 colocalization, stabilized mitochondrial membrane potential, reduced mitochondrial ROS production, and improved respiratory capacity. In vitro, Ang-(1-7) restored mitophagy flux through MasR-dependent clearance of damaged mitochondria, as confirmed by mt-Keima assays. Collectively, these findings identify MasR as an endogenous regulator of PINK1/Parkin-mediated mitophagy and mitochondrial homeostasis following TBI and demonstrate that MasR signaling is required for preserving mitochondrial function and neurological outcomes after injury.
Mitochondria exhibit substantial organ-specific heterogeneity arising from the distinct physiological, metabolic, and functional demands of individual organs. Differences in bioenergetics, oxidative stress handling, calcium homeostasis, and metabolic adaptability may influence mitochondrial responses to pharmacological agents and contribute to organ-selective drug toxicity. However, contemporary preclinical drug toxicity screening continues to rely predominantly on generalized experimental systems that may incompletely capture these organ-specific mitochondrial vulnerabilities. This Perspective proposes integrating organ-adapted mitochondrial physiology into preclinical drug safety evaluation through tissue-relevant cellular models, functional mitochondrial assays, and emerging microphysiological platforms to improve predictive toxicology and strengthen translational relevance.
Primary mitochondrial diseases are a heterogeneous group of neurometabolic disorders recognized as the most common metabolic genetic diseases. They manifest at any age, affecting any tissue or organ, especially those with high energy demands, and are caused by pathogenic variants in both mitochondrial and nuclear genomes. Here, we aimed to describe the genetic spectrum of a Tunisian pediatric cohort with suspected mitochondrial diseases. We recruited 47 unrelated families who underwent exome sequencing as a first-tier test followed by whole mitochondrial genome sequencing for unsolved cases. Dedicated bioinformatic pipelines and prediction tools were used to determine the potential disease-causing variants. Sanger sequencing confirmed the presence and segregation within parents. For the newly identified variants, structural modeling was conducted to study the impact of these variants on protein structure and motions. Dual genome sequencing yielded a molecular diagnosis in 33/47 families (70%) and 18/47 (38%) showed disease-causing variants in genes encoding mitochondrial proteins. Among them, four families disclosed novel variants in FASTKD2, SERAC1 and GATB, which were supported by in-depth in silico and structural analyses demonstrating their deleterious effect. The remaining families (32%, 15/47) disclosed other metabolic and neurological disorders. An exome-first strategy delivers a high diagnostic yield in Tunisia, where consanguinity remains high and simultaneously captures mitochondrial and non-mitochondrial etiologies. Mitochondrial sequencing remains indispensable in the case of an inconclusive exome. Thus, our data expand the clinical and genetic spectrum of primary mitochondrial diseases in Tunisia, an underrepresented and admixed population.
Traumatic brain injury (TBI) often occurs alongside polytrauma, which involves injuries to vital organs, resulting in severe compounded effects to the body. Our research program has been involved in broadening the understanding of organ-specific cellular responses relevant to TBI exposures in the military. In the current study, we collected reference data from multiple organs and brain regions on mitochondrial functional parameters using adult naive control swine. With their large brain mass and gyrencephalic architecture, the swine model has been utilized to develop and replicate experimental TBI and polytrauma conditions. In the present study, mitochondria were freshly isolated from Yorkshire male swine (30-35 kg) vital organs (e.g., brain, heart, lung, liver, kidney, spleen, muscle and intestine) and brain regions (e.g., cortex, striatum, cerebellum and hippocampus). Real-time analyses of mitochondrial bioenergetics and calcium (Ca2+) buffering experiments were conducted within multi-organs, and brain regions samples. Remaining mitochondrial samples were immediately stored and later used to evaluate cell death responses by assessing mitochondrial membrane integrity and antioxidant marker protein expression. Overall, our findings revealed organ-specific, and brain region-specific patterns of mitochondrial functional outcomes in healthy control swine. Notably, brain and heart mitochondrial bioenergetics and Ca2+ buffering capacity exhibited higher compared to other organs. Organ-specific unique differences were also identified in mitochondrial membrane integrity and cell death markers (i.e., Complex IV, Cyt C, VDAC, and Bcl-2), and antioxidant protein markers (i.e., SOD, CAT, TRX, and PRX). These baseline reference data establish a foundation for future injury-model studies of mitochondrial responses in TBI and polytrauma.
Gills are multifunctional organs that integrate respiration with homeostasis, including energy demanding processes such as osmoregulation and excretion. In osmoregulating decapod crustaceans, two spatially segregated gill types differ in function, ultrastructure and membrane composition, as well as in their responses to environmental change. Yet mitochondrial function in crab gills remains poorly characterized. Here, for the first time, we used high-resolution respirometry with a substrate-uncoupler-inhibitor titration protocol to characterize the mitochondrial phenotypes in anterior (respiratory) and posterior (osmoregulatory) gills. Gill filaments of the shore crab Carcinus maenas were permeabilized with saponin. Anterior gills exhibited higher leak control ratios (L/P, L/E), consistent with a leak-dominated mitochondrial phenotype that may contribute to redox balance at expense of maximal ATP yield. In contrast, posterior gills, displayed a higher phosphorylation control ratio and tighter coupling (higher Net P), corresponding to a tightly-coupled, ATP-producing mitochondrial phenotype, in line with their role in sustaining ATP-intensive activities such as osmoregulation and excretion. Our data revealed that anterior and posterior gills operate as "two engines in one organ". By quantifying how each gill type partitions respiratory capacity between phosphorylation and leak pathways, this study provides a mechanistic framework for understanding how mitochondrial specialization supports a functional division of labor within a single organ. In other words, while the two gill types have the same sized mitochondrial "engines", they differ in how that engine is tuned: posterior gills exhibit a Production (ATP-biased) mitochondrial phenotype, whereas anterior gills display a Preservation (leak-biased) mitochondrial phenotype, reflecting contrasting strategies for allocating respiratory capacity. This study provides a mechanistic framework for understanding how mitochondrial plasticity/specialization can contribute to tissue-specific responses in dynamically fluctuating marine environments.
Mitochondrial transcription is gaining increasing attention as researchers seek to better understand the full coding potential of mitochondrial DNA (mtDNA). Emerging evidence suggests that mtDNA may encode additional elements beyond classical oxidative phosphorylation genes, pointing to a more complex transcriptional architecture than previously recognized. In this study, we explored the mitochondrial transcriptome of Apis mellifera (Insecta: Hymenoptera), with a particular focus on polyadenylation-associated features. Our analysis revealed that both sense and antisense transcripts undergo polyadenylation, although transcript abundance and poly(A) tail lengths varied markedly across mitochondrial genes. Several transcripts exhibited alternative isoforms, either extended or truncated, frequently including intergenic regions. These regions may represent functional non-coding elements or structural variants rather than conventional untranslated regions (UTRs). Interestingly, some transcripts also contained non-templated nucleotide additions particularly cytosine residues immediately upstream of the poly(A) tails. Monocistronic units that included portions of downstream intergenic regions were among the most abundantly represented, suggesting a possible regulatory role for these sequences. To experimentally validate our in silico findings, we performed RT-qPCR to assess relative gene expression and applied 3' RACE-PCR to define transcript boundaries. These approaches confirmed the presence of multiple transcript isoforms and supported the involvement of polyadenylation in shaping mitochondrial RNA diversity. Together, our findings reveal a previously underappreciated level of complexity in the A. mellifera mitochondrial transcriptome and highlight the potential regulatory significance of polyadenylation dynamics and intergenic region transcription.
Mito-interpreter is an online tool developed to implement the ACMG/AMP standards and guidelines for mitochondrial DNA (mtDNA) variant interpretation. Manually applying these specialized guidelines is time-consuming and error-prone due to mtDNA complexities. This tool streamlines the process by systematically organizing evidence categories and integrating key data sources, including population frequencies from MITOMAP and Helix, as well as pre-computed scores from the APOGEE and expert-validated evidence from ClinGen. By automating data aggregation and rule-based logic, Mito-interpreter enables clinicians and researchers to focus on evidence evaluation, thereby enhancing efficiency and accuracy in clinical diagnostics and research. The tool is freely available at https://www.mtdna-interpreter.com.
Acute brain injury is difficult to evaluate in veterinary medicine and tools to investigate the potential involvement of mitochondrial involvement are limited. The brain is highly enriched in mitochondria and contains thousands of copies of mitochondrial DNA (mtDNA) per cell, but robust methods for quantifying mitochondrial DNA copy number (mtDNA-CN) in canine tissues are lacking. We describe the development of a quantitative real-time PCR assay for absolute measurement of mtDNA-CN which was validated in canine blood and brain tissue. To minimize amplification of nuclear mitochondrial insertion sequences (NumtS) and repetitive regions, species-specific oligonucleotide primers were designed following in silico genomic filtering. The assay was applied to a small pilot cohort comprising blood samples from dogs with and without acute brain injury (n = 4-6 per group) and cerebral cortex samples (n = 1 per group) to assess feasibility and biological plausibility. In non-brain injury dogs, blood mtDNA-CN ranged from 98 to 288 copies per nuclear genome (mean 193 ± 72), while values in brain-injured cases ranged from 163 to 228 copies per genome (mean 200 ± 33). Cerebral cortex samples exhibited higher mtDNA-CN than blood, consistent with known tissue-specific mitochondrial enrichment. In a single brain-injured case with serial sampling, mtDNA-CN increased over five days. This study presents a validated assay and pilot data for mtDNA-CN quantification in canine samples. While not powered for biomarker evaluation, this method may enable future studies of mitochondrial dynamics in canine brain injury and metabolic disease.
ATP synthase inhibitory factor 1 (ATPIF1) is a critical regulator of the activity of F1F0-ATPase, a central enzyme in mitochondrial ATP production. Accumulating evidence highlights ATPIF1 as a master modulator of mitochondrial morphology, function, cellular metabolism, and stress responses in diverse physiological and pathological conditions. In this review, we first provide a brief overview of mitochondrial structure and ATP production. We then focus on the cellular and molecular mechanisms of mitochondrial bioenergetics regulated by ATPIF1 and emphasize the role of ATPIF1 in energy preservation, mitophagy and redox balance. Furthermore, we comprehensively summarize recent advances about the pathological function of ATPIF1 in various mitochondrial dysfunction related diseases, including ischemia/reperfusion injury, aging, cancer, sepsis and chronic inflammation, and neurodegenerative disorders. ATP1IF1, the mitochondrial rheostat, emerges as a novel therapeutic target to combat mitochondrial dysfunction across multiple organ systems.
Circadian rhythms orchestrate a wide array of behavioral and physiological functions, coordinating cellular and organismal processes on an approximately 24-h cycle through an intrinsic timekeeping system. Among the many processes subject to this temporal regulation, mitochondrial function has emerged as a critical and dynamic target of circadian control. Mitochondria, far from being static organelles, undergo continuous morphological remodeling through cycles of fusion and fission, collectively termed mitochondrial dynamics, that are essential for maintaining metabolic homeostasis, energy production, and cellular quality control. Disruptions in circadian rhythmicity, such as those arising from sleep disturbances or irregular feeding patterns, have been associated with impaired glucose tolerance, insulin resistance, and increased risk of metabolic syndrome, diabetes, and cardiovascular disease. Emerging evidence suggests that the circadian clock and mitochondrial dynamics are engaged in a bidirectional interplay, whereby clock-controlled gene expression shapes mitochondrial morphology and function, while mitochondrial metabolic states in turn feedback to influence circadian timing. This review explores the evolutionary origins of mitochondrial rhythmicity, synthesizes current evidence on how the circadian clock regulates mitochondrial dynamics, and examines the physiological and pathological implications of their interconnection. A particular focus is placed on how disruptions in this circadian-mitochondrial axis may contribute to the development of common diseases, including neurodegenerative disorders, metabolic diseases, and cancer, highlighting novel avenues for chronobiologically informed therapeutic strategies.
Large-scale mitochondrial DNA (mtDNA) deletions can result in deficiency of oxidative phosphorylation and subsequent mitochondrial dysfunction, ultimately leading to mitochondrial disease. To investigate effective treatments, we report a characterised heteroplasmic iPSC-derived neuronal model with a single, large scale ∼6 kb mtDNA deletion. While mtDNA heteroplasmy remains stable during iNGN2-induced neuronal differentiation from iPSCs, the presence of this mtDNA deletion results in an upregulation of mtDNA copy number and compensatory adaptation of oxidative phosphorylation (OXPHOS) machinery. Despite this increase, mitochondrial dysfunction and reduced oxygen consumption is prevalent. Furthermore, as differentiated neurons mature over time, mitochondrial supercomplexes and isolated complex II diminish, suggesting an increase of severity of the mitochondrial dysfunction. In summary, this study provides insight into a novel compensatory mechanism during iPSC differentiation to bypass mitochondrial dysfunction, and how this response exacerbates dysfunction during culture of mature neurons.
Erythroid differentiation requires a metabolic shift to oxidative phosphorylation (OXPHOS). We investigated the effects of Spatholobi Caulis (SC) and its active flavonoid, epicatechin (EC), on erythropoiesis. Both SC and EC significantly amplified erythroid differentiation in vitro and in vivo. Mechanistically, SC and EC further triggered differentiation-induced AKT activation and its mitochondrial translocation, leading to upregulation of mitochondrial DNA-encoded respiratory chain genes and enhanced OXPHOS capacity. Notably, this functional enhancement occurred without changes in mitochondrial mass or mtDNA copy number, indicating a biogenesisindependent mechanism. Both PI3K/AKT signaling and intact OXPHOS function were essential, as pharmacological inhibition of either pathway abolished SC's pro-erythropoietic activity. Our findings establish an AKTmitochondrial axis that couples proliferative signaling to bioenergetics, offering a therapeutic strategy for anemias involving mitochondrial dysfunction.
Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disease that occurs primarily in the elderly. Although senescence of lung fibroblasts (LFs) contributes to IPF development, the potential mechanisms underlying LF senescence are not fully understood. This study aimed to delineate the role and underlying mechanisms of miR-205-5p in regulating LF senescence in patients with IPF. The LFs from IPF patients (IPF-LFs) and age-matched controls (Control-LFs) were isolated and cultured. Senescence of LFs was determined by senescence-associated β-galactosidase (SA-β-gal) staining. Mitochondrial morphology of LFs was evaluated by MitoTracker staining and transmission electron microscope. The expression of miR-205-5p was examined by RT-PCR. Compared with Control-LFs, IPF-LFs exhibited increased cellular senescence with higher expression of SA-β-gal, p21 and p16 as well as decreased proliferative capacity. Importantly, IPF-LFs had decreased mitochondrial fission, evidenced by elongated mitochondria and downregulation of mitochondrial fission regulator 1-like protein (MTFR1L). The expression of miR-205-5p was much higher in IPF-LFs than Control-LFs. Notably, upregulation of miR-205-5p in Control-LFs led to increased cellular senescence, whereas downregulation rescued IPF-LF senescence. Mechanistically, miR-205-5p downregulated mitochondrial fission in LFs via MTFR1L, leading to mitochondrial dysfunction and cellular senescence. Taken together, our study illustrated that miR-205-5p serves as a critical regulator of cellular senescence of LFs isolated from IPF patients via mediation of mitochondrial dynamics.
Nucleotide composition bias in mitochondrial DNA (mtDNA) makes the heavy strand prone to form a DNA secondary structure called a guanine quadruplex (G4). This secondary structure has been shown to inhibit polymerase processivity in vitro. We previously identified pathogenic mtDNA variants that lead to increased G4-forming propensity, including a T to C mutation at m.10191 (m.10191 T > C) that causes Leigh syndrome. Cells treated with G4 binding agent (G4BA) berberine show a reduction in m.10191C pathogenic heteroplasmy levels. To help better understand the underlying mechanism behind berberine-induced heteroplasmy shift, we examined the relationship between mitochondrial fission and berberine-mediated shift. Here we show that knockdown of the fission factor DNM1L leads to an accelerated heteroplasmy shift towards the healthy mtDNA allele, lowering m.10191C by 10% in 3 weeks, compared to the 5 weeks required for berberine alone. The specific mechanism involves ATG7, as knockdown of ATG7 is able to partially delay this accelerated heteroplasmy shift. Taken together, we show that DNM1L knockdown is able to accelerate berberine-induced m.10191C heteroplasmy shifting through an autophagy-related mechanism.
