bims-mitdis Biomed News
on Mitochondrial disorders
Issue of 2025–03–16
fifty papers selected by
Catalina Vasilescu, Helmholz Munich
  1. Mitochondrial genetics, signalling and stress responses.
  2. Mitochondrial damage in muscle specific PolG mutant mice activates the integrated stress response and disrupts the mitochondrial folate cycle.
  3. Mitochondria are positioned at dendritic branch induction sites, a process requiring rhotekin2 and syndapin I.
  4. Identification of determinants for variability in mitochondrial biochemical complex activities.
  5. Engineering mtDNA deletions by reconstituting end joining in human mitochondria.
  6. Structural basis for intrinsic strand displacement activity of mitochondrial DNA polymerase.
  7. Structure of human PINK1 at a mitochondrial TOM-VDAC array.
  8. Electron Microscopy to Visualize and Quantify Mitochondrial Structure and Organellar Interactions in Cultured Cells During Senescence.
  9. Shape Matters: The Utility and Analysis of Altered Yeast Mitochondrial Morphology in Health, Disease, and Biotechnology.
  10. Ectopic protein lysine methacrylation contributes to defects caused by loss of HIBCH or ECHS1.
  11. Mitochondrial glutaredoxin Grx5 functions as a central hub for cellular iron-sulfur cluster assembly.
  12. A dual-purification system to isolate mitochondrial subpopulations.
  13. Isolation and Characterization of EVs Containing Mitochondria from OIS Cells.
  14. Quality control of mitochondrial nucleoids.
  15. Labeling of mitochondria for detection of intercellular mitochondrial transfer.
  16. CHCHD2 rescues the mitochondrial dysfunction in iPSC-derived neurons from patient with Mohr-Tranebjaerg syndrome.
  17. The Nuclear-Mitochondrial Crosstalk in Aging: From Mechanisms to Therapeutics.
  18. MIA40 suppresses cell death induced by apoptosis-inducing factor 1.
  19. The Mitochondria-Targeted Peptide Therapeutic Elamipretide Improves Cardiac and Skeletal Muscle Function During Aging Without Detectable Changes in Tissue Epigenetic or Transcriptomic Age.
  20. GSK3 coordinately regulates mitochondrial activity and nucleotide metabolism in quiescent oocytes.
  21. Expanding the Dimensions of Single-Cell Multi-Omics To Include Mitochondrial DNA through Fixation and Permeabilization.
  22. Mitochondrial dysfunction drives a neuronal exhaustion phenotype in methylmalonic aciduria.
  23. Mitochondria-targeted nanovesicles for ursodeoxycholic acid delivery to combat neurodegeneration by ameliorating mitochondrial dysfunction.
  24. Puromycin Proximity Ligation Assay (Puro-PLA) to Assess Local Translation in Axons From Human Neurons.
  25. Diabetes, macrocytosis, and skin changes in large-scale mtDNA deletion.
  26. A Rigorous Nuclear DNA-Excluding Mass Spectrometry Assay for Accurate Identification of 5-Methylcytosine in Mitochondrial DNA.
  27. Measurement of Mitochondrial Membrane Potential In Vivo using a Genetically Encoded Voltage Indicator.
  28. Mitochondrial fatty acid oxidation regulates adult muscle stem cell function through modulating metabolic flux and protein acetylation.
  29. Succinate dehydrogenase activity supports de novo purine synthesis.
  30. Novel Presentation of McLeod Syndrome With Muscle Weakness and Biopsy Findings Indicative of Mitochondrial Dysfunction.
  31. A novel c.59 C > T variant of the HSD17B10 gene as a possible cause of the neonatal form of HSD10 mitochondrial disease with hepatic dysfunction: a case report and review of the literature.
  32. Mitochondrial respiratory complex IV deficiency recapitulates amyotrophic lateral sclerosis.
  33. Genetic and reproductive strategies to prevent mitochondrial diseases.
  34. Mitochondria transfer for myelin repair.
  35. PHEMI-Phenylbutyrate in Patients With Lactic Acidosis: A Pilot, Single Arm, Phase I/II, Open-Label Trial.
  36. Time Series Imaging the Mitochondrial Microenvironment and Its Interactions with Lysosomes during Ferroptosis.
  37. Fatty Acid Trafficking Between Lipid Droplets and Mitochondria: An Emerging Perspective.
  38. Aggregatin is a mitochondrial regulator of MAVS activation to drive innate immunity.
  39. Mitochondria‑derived peptides: Promising microproteins in cardiovascular diseases (Review).
  40. Skeletal muscle mitochondrial dysfunction is associated with increased Gdf15 expression and circulating GDF15 levels in aged mice.
  41. Adapting systems biology to address the complexity of human disease in the single-cell era.
  42. Data-driven insights to inform splice-altering variant assessment.
  43. Amplification-Free Quantification of Endogenous Mitochondrial DNA Copy Number Using Solid-State Nanopores.
  44. A steric gate prevents mutagenic dATP incorporation opposite 8-oxo-deoxyguanosine in mitochondrial DNA polymerases.
  45. Functional Nucleic Acid Nanostructures for Mitochondrial Targeting: The Basis of Customized Treatment Strategies.
  46. The E3 ubiquitin ligase RNF126 facilitates quality control of unimported mitochondrial membrane proteins.
  47. A positive feedback loop between SMAD3 and PINK1 in regulation of mitophagy.
  48. Retinopathy associated with MELAS syndrome. A case report.
  49. Neurofilament accumulation disrupts autophagy in giant axonal neuropathy.
  50. Metabolic remodeling in hiPSC-derived myofibers carrying the m.3243A>G mutation.
  1. Nat Cell Biol. 2025 Mar;27(3): 393-407
    Mitochondrial genetics, signalling and stress responses.
    Yasmine J Liu, Jonathan Sulc, Johan Auwerx.
      Mitochondria are multifaceted organelles with crucial roles in energy generation, cellular signalling and a range of synthesis pathways. The study of mitochondrial biology is complicated by its own small genome, which is matrilineally inherited and not subject to recombination, and present in multiple, possibly different, copies. Recent methodological developments have enabled the analysis of mitochondrial DNA (mtDNA) in large-scale cohorts and highlight the far-reaching impact of mitochondrial genetic variation. Genome-editing techniques have been adapted to target mtDNA, further propelling the functional analysis of mitochondrial genes. Mitochondria are finely tuned signalling hubs, a concept that has been expanded by advances in methodologies for studying the function of mitochondrial proteins and protein complexes. Mitochondrial respiratory complexes are of dual genetic origin, requiring close coordination between mitochondrial and nuclear gene-expression systems (transcription and translation) for proper assembly and function, and recent findings highlight the importance of the mitochondria in this bidirectional signalling.
    DOI:  https://doi.org/10.1038/s41556-025-01625-w
  2. Nat Commun. 2025 Mar 08. 16(1): 2338
    Mitochondrial damage in muscle specific PolG mutant mice activates the integrated stress response and disrupts the mitochondrial folate cycle.
    Simon T Bond, Emily J King, Shannen M Walker, Christine Yang, Yingying Liu, Kevin H Liu, Aowen Zhuang, Aaron W Jurrjens, Haoyun A Fang, Luke E Formosa, Artika P Nath, Sergio Ruiz Carmona, Michael Inouye, Thy Duong, Kevin Huynh, Peter J Meikle, Simon Crawford, Georg Ramm, Sheik Nadeem Elahee Doomun, David P de Souza, Danielle L Rudler, Anna C Calkin, Aleksandra Filipovska, David W Greening, Darren C Henstridge, Brian G Drew.
      During mitochondrial damage, information is relayed between the mitochondria and nucleus to coordinate precise responses to preserve cellular health. One such pathway is the mitochondrial integrated stress response (mtISR), which is known to be activated by mitochondrial DNA (mtDNA) damage. However, the causal molecular signals responsible for activation of the mtISR remain mostly unknown. A gene often associated with mtDNA mutations/deletions is Polg1, which encodes the mitochondrial DNA Polymerase γ (PolG). Here, we describe an inducible, tissue specific model of PolG mutation, which in muscle specific animals leads to rapid development of mitochondrial dysfunction and muscular degeneration in male animals from ~5 months of age. Detailed molecular profiling demonstrated robust activation of the mtISR in muscles from these animals. This was accompanied by striking alterations to enzymes in the mitochondrial folate cycle that was likely driven by a specific depletion in the folate cycle metabolite 5,10 methenyl-THF, strongly implying imbalanced folate intermediates as a previously unrecognised pathology linking the mtISR and mitochondrial disease.
    DOI:  https://doi.org/10.1038/s41467-025-57299-3
  3. Nat Commun. 2025 Mar 10. 16(1): 2353
    Mitochondria are positioned at dendritic branch induction sites, a process requiring rhotekin2 and syndapin I.
    Jessica Tröger, Regina Dahlhaus, Anne Bayrhammer, Dennis Koch, Michael M Kessels, Britta Qualmann.
      Proper neuronal development, function and survival critically rely on mitochondrial functions. Yet, how developing neurons ensure spatiotemporal distribution of mitochondria during expansion of their dendritic arbor remained unclear. We demonstrate the existence of effective mitochondrial positioning and tethering mechanisms during dendritic arborization. We identify rhotekin2 as outer mitochondrial membrane-associated protein that tethers mitochondria to dendritic branch induction sites. Rhotekin2-deficient neurons failed to correctly position mitochondria at these sites and also lacked the reduction in mitochondrial dynamics observed at wild-type nascent dendritic branch sites. Rhotekin2 hereby serves as important anchor for the plasma membrane-binding and membrane curvature-inducing F-BAR protein syndapin I (PACSIN1). Consistently, syndapin I loss-of-function phenocopied the rhotekin2 loss-of-function phenotype in mitochondrial positioning at dendritic branch induction sites. The finding that rhotekin2 deficiency impaired dendritic branch induction and that a syndapin binding-deficient rhotekin2 mutant failed to rescue this phenotype highlighted the physiological importance of rhotekin2 functions for neuronal network formation.
    DOI:  https://doi.org/10.1038/s41467-025-57399-0
  4. Biochim Biophys Acta Bioenerg. 2025 Mar 09. pii: S0005-2728(25)00019-2. [Epub ahead of print]1866(2): 149553
    Identification of determinants for variability in mitochondrial biochemical complex activities.
    Sandra Monica Bach de Courtade, Marte Eikenes, Ying Sheng, Tuula A Nyman, Yngve Thomas Bliksrud, Katja Scheffler, Lars Eide.
      Diagnostics of mitochondrial disease requires a combination of clinical evaluations and biochemical characterization. However, the large normal variation in mitochondrial complex activity limits the precision of biochemical diagnostics. Thus, identifying factors that contribute to such variations could enhance diagnostic accuracy. In comparison, inbred mice demonstrate much less variations in brain mitochondrial activity, but a clear reduction with age. Interestingly, pretreatment of mouse brain mitochondria with the detergent dodecyl maltoside abolishes the reduction. We therefore postulated that DDM pretreatment could be valuable tool for distinguishing between variations caused by posttranslational modifications and those caused by genetic heterogeneity. In this study, we evaluated the effects of age, DDM sensitivity, oxidative damage and single nucleotide polymorphism on biochemical complex activity and the proteome of human muscle mitochondria, which serve as reference standards for mitochondrial diagnostics. Our results indicate that mtDNA variants are the primary contributors to the diversity in biochemical activity in human muscle mitochondria from healthy individuals.
    Keywords:  Dodecyl maltoside; ETC; Heteroplasmy; Mitochondrial function; mtDNA damage
    DOI:  https://doi.org/10.1016/j.bbabio.2025.149553
  5. Cell. 2025 Mar 05. pii: S0092-8674(25)00194-1. [Epub ahead of print]
    Engineering mtDNA deletions by reconstituting end joining in human mitochondria.
    Yi Fu, Max Land, Tamar Kavlashvili, Ruobing Cui, Minsoo Kim, Emily DeBitetto, Toby Lieber, Keun Woo Ryu, Elim Choi, Ignas Masilionis, Rahul Saha, Meril Takizawa, Daphne Baker, Marco Tigano, Caleb A Lareau, Ed Reznik, Roshan Sharma, Ronan Chaligne, Craig B Thompson, Dana Pe'er, Agnel Sfeir.
      Recent breakthroughs in the genetic manipulation of mitochondrial DNA (mtDNA) have enabled precise base substitutions and the efficient elimination of genomes carrying pathogenic mutations. However, reconstituting mtDNA deletions linked to mitochondrial myopathies remains challenging. Here, we engineered mtDNA deletions in human cells by co-expressing end-joining (EJ) machinery and targeted endonucleases. Using mitochondrial EJ (mito-EJ) and mito-ScaI, we generated a panel of clonal cell lines harboring a ∼3.5 kb mtDNA deletion across the full spectrum of heteroplasmy. Investigating these cells revealed a critical threshold of ∼75% deleted genomes, beyond which oxidative phosphorylation (OXPHOS) protein depletion, metabolic disruption, and impaired growth in galactose-containing media were observed. Single-cell multiomic profiling identified two distinct nuclear gene deregulation responses: one triggered at the deletion threshold and another progressively responding to heteroplasmy. Ultimately, we show that our method enables the modeling of disease-associated mtDNA deletions across cell types and could inform the development of targeted therapies.
    Keywords:  DOGMA-seq; end joining; mitochondrial pathologies; mtDNA; mtDNA deletion
    DOI:  https://doi.org/10.1016/j.cell.2025.02.009
  6. Nat Commun. 2025 Mar 11. 16(1): 2417
    Structural basis for intrinsic strand displacement activity of mitochondrial DNA polymerase.
    Ashok R Nayak, Viktoriia Sokolova, Sirelin Sillamaa, Karl Herbine, Juhan Sedman, Dmitry Temiakov.
      Members of the Pol A family of DNA polymerases, found across all domains of life, utilize various strategies for DNA strand separation during replication. In higher eukaryotes, mitochondrial DNA polymerase γ relies on the replicative helicase TWINKLE, whereas the yeast ortholog, Mip1, can unwind DNA independently. Using Mip1 as a model, we present a series of high-resolution cryo-EM structures that capture the process of DNA strand displacement. Our data reveal previously unidentified structural elements that facilitate the unwinding of the downstream DNA duplex. Yeast cells harboring Mip1 variants defective in strand displacement exhibit impaired oxidative phosphorylation and loss of mtDNA, corroborating the structural observations. This study provides a molecular basis for the intrinsic strand displacement activity of Mip1 and illuminates the distinct unwinding mechanisms utilized by Pol A family DNA polymerases.
    DOI:  https://doi.org/10.1038/s41467-025-57594-z
  7. Science. 2025 Mar 13. eadu6445
    Structure of human PINK1 at a mitochondrial TOM-VDAC array.
    Sylvie Callegari, Nicholas S Kirk, Zhong Yan Gan, Toby Dite, Simon A Cobbold, Andrew Leis, Laura F Dagley, Alisa Glukhova, David Komander.
      Mutations in the ubiquitin kinase PINK1 cause early onset Parkinson's Disease, but how PINK1 is stabilized at depolarized mitochondrial translocase complexes has remained poorly understood. We determined a 3.1-Å resolution cryo-electron microscopy structure of dimeric human PINK1 stabilized at an endogenous array of mitochondrial TOM and VDAC complexes. Symmetric arrangement of two TOM core complexes around a central VDAC2 dimer is facilitated by TOM5 and TOM20, both of which also bind PINK1 kinase C-lobes. PINK1 enters mitochondria through the proximal TOM40 barrel of the TOM core complex, guided by TOM7 and TOM22. Our structure explains how human PINK1 is stabilized at the TOM complex and regulated by oxidation, uncovers a previously unknown TOM-VDAC assembly, and reveals how a physiological substrate traverses TOM40 during translocation.
    DOI:  https://doi.org/10.1126/science.adu6445
  8. Methods Mol Biol. 2025 ;2906 229-242
    Electron Microscopy to Visualize and Quantify Mitochondrial Structure and Organellar Interactions in Cultured Cells During Senescence.
    Christina Glytsou.
      Mitochondria are multifunctional organelles that play a crucial role in numerous cellular processes, including oncogene-induced senescence. Recent studies have demonstrated that mitochondria undergo notable morphological and functional changes during senescence, with mitochondria dysregulation being a critical factor contributing to the induction of this state. To elucidate the intricate and dynamic structure of these organelles, high-resolution visualization techniques are imperative. Electron microscopy offers nanometer-scale resolution images, enabling the comprehensive study of organelles' architecture. This chapter provides a detailed guide for preparing fixed samples from cultured cells for electron microscopy imaging. It also describes various quantification methods to accurately assess organellar parameters, including morphometric measurements of mitochondrial shape, cristae structure, and mitochondria-endoplasmic reticulum contact sites. These analyses yield valuable insights into the status of subcellular organelles, advancing our understanding of their involvement in cellular senescence and disease.
    Keywords:  EM sample preparation; Electron microscopy; MERCs; Mitochondria visualization; Mitochondrial structure
    DOI:  https://doi.org/10.1007/978-1-0716-4426-3_13
  9. Int J Mol Sci. 2025 Feb 27. pii: 2152. [Epub ahead of print]26(5):
    Shape Matters: The Utility and Analysis of Altered Yeast Mitochondrial Morphology in Health, Disease, and Biotechnology.
    Therese Kichuk, José L Avalos.
      Mitochondria are involved in a wide array of critical cellular processes from energy production to cell death. The morphology (size and shape) of mitochondrial compartments is highly responsive to both intracellular and extracellular conditions, making these organelles highly dynamic. Nutrient levels and stressors both inside and outside the cell inform the balance of mitochondrial fission and fusion and the recycling of mitochondrial components known as mitophagy. The study of mitochondrial morphology and its implications in human disease and microbial engineering have gained significant attention over the past decade. The yeast Saccharomyces cerevisiae offers a valuable model system for studying mitochondria due to its ability to survive without respiring, its genetic tractability, and the high degree of mitochondrial similarity across eukaryotic species. Here, we review how the interplay between mitochondrial fission, fusion, biogenesis, and mitophagy regulates the dynamic nature of mitochondrial networks in both yeast and mammalian systems with an emphasis on yeast as a model organism. Additionally, we examine the crucial role of inter-organelle interactions, particularly between mitochondria and the endoplasmic reticulum, in regulating mitochondrial dynamics. The dysregulation of any of these processes gives rise to abnormal mitochondrial morphologies, which serve as the distinguishing features of numerous diseases, including Parkinson's disease, Alzheimer's disease, and cancer. Notably, yeast models have contributed to revealing the underlying mechanisms driving these human disease states. In addition to furthering our understanding of pathologic processes, aberrant yeast mitochondrial morphologies are of increasing interest to the seemingly distant field of metabolic engineering, following the discovery that compartmentalization of certain biosynthetic pathways within mitochondria can significantly improve chemical production. In this review, we examine the utility of yeast as a model organism to study mitochondrial morphology in both healthy and pathologic states, explore the nascent field of mitochondrial morphology engineering, and discuss the methods available for the quantification and classification of these key mitochondrial morphologies.
    Keywords:  analysis; biofuel; cancer; contact sites; engineering; fission; fusion; imaging; mitochondria; morphology; neurodegenerative; pathology
    DOI:  https://doi.org/10.3390/ijms26052152
  10. Cell Rep. 2025 Mar 06. pii: S2211-1247(25)00150-0. [Epub ahead of print]44(3): 115379
    Ectopic protein lysine methacrylation contributes to defects caused by loss of HIBCH or ECHS1.
    Yawen Li, Ting Wu, Yaoyao Li, Chaolong Xu, Caixia Zhou, Zhirong Li, Weina Shang, Liquan Wang, Zhimei Liu, Junling Wang, Yang Liu, Fang Fang, Bing Yang, Chao Tong.
      The absence of HIBCH or ECHS1, two Leigh syndrome genes, in cultured cells results in abnormal mitochondrial morphology and respiratory defects. Fly eyes lacking either protein exhibit age-dependent degeneration. Elevated lysine methacrylation (Kmea) is observed in both HIBCH- and ECHS1-deficient cells and fly tissues. Quantitative mass spectrometry reveals that many proteins are ectopically modified by Kmea in these cells. Mimicking Kmea in proteins like CH60, FKBP4, BIP, LDHB, or DHRS2 replicates the mitochondrial morphology changes seen in HIBCH- or ECHS1-deficient cells. Reducing Kmea modification partially rescues mitochondrial morphology changes in cells and eye degeneration in flies. Fibroblasts from patients with HIBCH or ECHS1 mutations show similar mitochondrial changes and elevated Kmea, which are significantly reversed by administering N-acetyl-L-cysteine to reduce Kmea levels. We propose that ectopic Kmea modification mediates the defects caused by HIBCH- or ECHS1-deficiency. Reducing Kmea modification provides a new approach for treating HIBCH- or ECHS1-related Leigh syndrome.
    Keywords:  CP: Molecular biology; CP: Neuroscience; Drosophila; ECHS1; HIBCH; Leigh syndrome; lysine methacrylation; mitochondria; neurodegeneration
    DOI:  https://doi.org/10.1016/j.celrep.2025.115379
  11. J Biol Chem. 2025 Mar 10. pii: S0021-9258(25)00240-6. [Epub ahead of print] 108391
    Mitochondrial glutaredoxin Grx5 functions as a central hub for cellular iron-sulfur cluster assembly.
    Ashutosh K Pandey, Jayashree Pain, Pratibha Singh, Andrew Dancis, Debkumar Pain.
      Iron-sulfur (FeS) protein biogenesis in eukaryotes is mediated by two different machineries - one in the mitochondria and another in the cytoplasm. Glutaredoxin 5 (Grx5) is a component of the mitochondrial iron-sulfur cluster (ISC) machinery. Here we define the roles of Grx5 in maintaining overall mitochondrial/cellular FeS protein biogenesis, utilizing mitochondria and cytoplasm isolated from Saccharomyces cerevisiae cells. We previously demonstrated that isolated wild-type (WT) mitochondria themselves can synthesize new FeS clusters, but isolated WT cytoplasm alone cannot do so unless it is mixed with WT mitochondria. WT mitochondria generate an intermediate, called (Fe-S)int, that is exported to the cytoplasm and utilized for cytoplasmic FeS cluster assembly. We here show that mitochondria lacking endogenous Grx5 (Grx5↓) failed to synthesize FeS clusters for proteins within the organelle. Similarly, Grx5↓ mitochondria were unable to synthesize (Fe-S)int, as judged by their inability to promote FeS cluster biosynthesis in WT cytoplasm. Most importantly, purified Grx5 precursor protein, imported into isolated Grx5↓ mitochondria, rescued these FeS cluster synthesis/trafficking defects. Notably, mitochondria lacking immediate downstream components of the ISC machinery (Isa1 or Isa2) could synthesize [2Fe-2S] but not [4Fe-4S] clusters within the organelle. Isa1↓ (or Isa2↓) mitochondria could still support FeS cluster biosynthesis in WT cytoplasm. These results provide evidence for Grx5 serving as a central hub for FeS cluster intermediate trafficking within mitochondria and export to the cytoplasm. Grx5 is conserved from yeast to humans, and deficiency or mutation causes fatal human diseases. Data as presented here will be informative for human physiology.
    Keywords:  Yeast; cytoplasm; export; iron; iron-sulfur protein; metal cofactor; mitochondria; sulfur; tRNA thiolation
    DOI:  https://doi.org/10.1016/j.jbc.2025.108391
  12. J Cell Sci. 2025 Mar 13. pii: jcs.263693. [Epub ahead of print]
    A dual-purification system to isolate mitochondrial subpopulations.
    Corey N Cunningham, Jonathan G Van Vranken, Jakeline Larios, Katarina Heyden, Steven P Gygi, Jared Rutter.
      Mitochondria perform diverse functions, such as producing ATP through oxidative phosphorylation, synthesizing macromolecule precursors, maintaining redox balance, and many others. Given this diversity of functions, we and others have hypothesized that cells maintain specialized subpopulations of mitochondria. To begin addressing this hypothesis, we developed a new dual-purification system to isolate subpopulations of mitochondria for chemical and biochemical analyses. We used APEX2 proximity labeling such that mitochondria were biotinylated based on proximity to another organelle. All mitochondria were isolated by an elutable MitoTag-based affinity precipitation system. Biotinylated mitochondria were then purified using immobilized avidin. We used this system to compare the proteomes of endosome- and lipid droplet-associated mitochondria in U-2 OS cells, which demonstrated that these subpopulations were indistinguishable from one another but were distinct from the global mitochondria proteome. Our results suggest that this purification system could aid in describing subpopulations that contribute to intracellular mitochondrial heterogeneity, and that this heterogeneity might be more substantial than previously imagined.
    Keywords:  Biochemistry; Mitochondria; Proximity Labeling; Purification
    DOI:  https://doi.org/10.1242/jcs.263693
  13. Methods Mol Biol. 2025 ;2906 243-254
    Isolation and Characterization of EVs Containing Mitochondria from OIS Cells.
    Ines Martic, David Feldmann, Maria Cavinato.
      Oncogene activation triggers oncogene-induced senescence (OIS), a tumor-suppression mechanism characterized by mitochondrial dysfunction and the secretion of various factors collectively known as the senescence-associated secretory phenotype (SASP). Recent evidence highlights that extracellular vesicles (EVs), nanosized membrane-bound particles, are part of the SASP and act as a novel intercellular communication pathway. Additionally, EVs containing mitochondrial compartments are hypothesized to function as a mitochondrial quality control mechanism, eliminating damaged mitochondria in senescent cells. However, the exact role of mitochondria-enriched vesicles in OIS remains elucidated. The diversity of protocols for isolating and characterizing EVs complicates the unification of research findings. Here, we provide a concise overview of current protocols for investigating vesicles-containing mitochondria in oncogene-induced senescent cells including size exclusion chromatography, NTA analysis, flow cytometry, confocal microscopy, and Western blot.
    Keywords:  Extracellular vesicles; Flow cytometry; Mitochondria; NTA; Senescence
    DOI:  https://doi.org/10.1007/978-1-0716-4426-3_14
  14. Trends Cell Biol. 2025 Mar 07. pii: S0962-8924(25)00039-X. [Epub ahead of print]
    Quality control of mitochondrial nucleoids.
    Hao Liu, Haixia Zhuang, Du Feng.
      Mitochondrial nucleoids, organized complexes that house and protect mitochondrial DNA (mtDNA), are normally confined within the mitochondrial double-membrane system. Under cellular stress conditions, particularly oxidative and inflammatory stress, these nucleoids can undergo structural alterations that lead to their aberrant release into the cytoplasm. This mislocalization of nucleoid components, especially mtDNA, can trigger inflammatory responses and cell death pathways, highlighting the critical importance of nucleoid quality control mechanisms. The release of mitochondrial nucleoids occurs through specific membrane channels and transport pathways, fundamentally disrupting cellular homeostasis. Cells have evolved multiple clearance mechanisms to manage cytoplasmic nucleoids, including nuclease-mediated degradation, lysosomal elimination, and cellular excretion. This review examines the molecular mechanisms governing nucleoid quality control and explores the delicate balance between mitochondrial biology and cellular immunity. Our analysis provides insights that could inform therapeutic strategies for mtDNA-associated diseases and inflammatory disorders.
    Keywords:  mitochondria; mitophagy; mtDNA; nucleoid-phagy; nucleoids
    DOI:  https://doi.org/10.1016/j.tcb.2025.02.005
  15. Methods Cell Biol. 2025 ;pii: S0091-679X(24)00143-2. [Epub ahead of print]194 1-17
    Labeling of mitochondria for detection of intercellular mitochondrial transfer.
    Isamu Taiko, Chika Takano, Shingo Hayashida, Kazunori Kanemaru, Toshio Miki.
      The phenomenon of intercellular transfer of mitochondria has been reported and has attracted significant interest in recent years. The phenomena involve a range of physiological and pathological conditions, such as tumor growth, immunoregulation, and tissue regeneration. There is speculation on the potential restoration of cellular energy status through the transfer of healthy mitochondria from donor cells to cells with impaired mitochondria. Multiple mechanisms and routes of mitochondria transfer have been suggested, including direct cell-to-cell connections, extracellular vesicles, and cell fusion. However, there is limited understanding regarding the precise mechanisms behind mitochondrial transfer, particularly the initiation signals and the associated processes. In order to explore these fundamental mechanisms of mitochondrial transfer, it is imperative to employ techniques that enable direct labeling of mitochondria. Here, we present a detailed methodology utilizing fluorescent protein tagging to visualize mitochondria. The molecular biological techniques applied in this study entail the precise localization of mitochondria with reduced cytotoxicity. This approach facilitates the direct observation of transferred mitochondria through fluorescent and confocal microscopy. The described method can be readily implemented in other mammalian cell types with few modifications, enabling the continuous monitoring of mitochondrial trafficking processes over an extended period.
    Keywords:  Amniotic epithelial cells; Mitochondria; Mitochondrial transfer
    DOI:  https://doi.org/10.1016/bs.mcb.2024.05.001
  16. Cell Death Dis. 2025 Mar 12. 16(1): 173
    CHCHD2 rescues the mitochondrial dysfunction in iPSC-derived neurons from patient with Mohr-Tranebjaerg syndrome.
    Yihua Huang, Zirui Chen, Weiling Deng, Yawei Jiang, Yue Pan, Zhirong Yuan, Hailiang Hu, Yongming Wu, Yafang Hu.
      Mohr-Tranebjaerg syndrome (MTS) is a rare X-linked recessive neurodegenerative disorder caused by mutations in the Translocase of Inner Mitochondrial Membrane 8A (TIMM8A) gene, which encodes TIMM8a, a protein localized to the mitochondrial intermembrane space (IMS). The pathophysiology of MTS remains poorly understood. To investigate the molecular mechanisms underlying MTS, we established induced pluripotent stem cells (iPSCs) from a male MTS patient carrying a novel TIMM8A mutation (c.225-229del, p.Q75fs95*), referred to as MTS-iPSCs. To generate an isogenic control, we introduced the same mutation into healthy control iPSCs (CTRL-iPSCs) using the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9), resulting in mutant iPSCs (MUT-iPSCs). We differentiated the three iPSC lines into neurons and evaluated their mitochondrial function and neuronal development. Both MTS- and MUT-iPSCs exhibited impaired neuronal differentiation, characterized by smaller somata, fewer branches, and shorter neurites in iPSC-derived neurons. Additionally, these neurons showed increased susceptibility to apoptosis under stress conditions, as indicated by elevated levels of cytochrome c and cleaved caspase-3. Mitochondrial function analysis revealed reduced protein levels and activity of complex IV, diminished ATP synthesis, and increased reactive oxygen species (ROS) generation in MTS- and MUT-neurons. Furthermore, transmission electron microscopy revealed mitochondrial fragmentation in MTS-neurons. RNA sequencing identified differentially expressed genes (DEGs) involved in axonogenesis, synaptic activity, and apoptosis-related pathways. Among these DEGs, coiled-coil-helix-coiled-coil-helix domain-containing 2 (CHCHD2), which encodes a mitochondrial IMS protein essential for mitochondrial homeostasis, was significantly downregulated in MTS-neurons. Western blot analysis confirmed decreased CHCHD2 protein levels in both MTS- and MUT-neurons. Overexpression of CHCHD2 rescued mitochondrial dysfunction and promoted neurite elongation in MTS-neurons, suggesting that CHCHD2 acts as a downstream effector of TIMM8a in the pathogenesis of MTS. In summary, loss-of-function of TIMM8a leads to a downstream reduction in CHCHD2 levels, collectively impairing neurogenesis by disrupting mitochondrial homeostasis. TIMM8a mutation (p.Q75fs95*) leads to mitochondrial dysfunction and neuronal defects in iPSC-derived neurons from patient with Mohr-Tranebjaerg syndrome, which are rescued by overexpression of CHCHD2. TIMM8a translocase of inner mitochondrial membrane 8a, CHCHD2 coiled-coil-helix-coiled-coil-helix domain-containing protein 2, MTS Mohr-Tranebjaerg syndrome, I mitochondrial complex I, II mitochondrial complex II, III mitochondrial complex III, IV mitochondrial complex IV, Q coenzyme Q10, Cyt c cytochrome c.
    DOI:  https://doi.org/10.1038/s41419-025-07472-9
  17. Free Radic Biol Med. 2025 Mar 12. pii: S0891-5849(25)00162-5. [Epub ahead of print]
    The Nuclear-Mitochondrial Crosstalk in Aging: From Mechanisms to Therapeutics.
    Yifei Feng, Yan Lu.
      Aging is a complex physiological process characterized by an irreversible decline in tissue and cellular functions, accompanied by an increased risk of age-related diseases, including neurodegenerative, cardiovascular, and metabolic disorders. Central to this process are epigenetic modifications, particularly DNA methylation, which regulate gene expression and contribute to aging-related epigenetic drift. This drift is characterized by global hypomethylation and localized hypermethylation, impacting genomic stability and cellular homeostasis. Simultaneously, mitochondrial dysfunction, a hallmark of aging, manifests as impaired oxidative phosphorylation, excessive reactive oxygen species production, and mitochondrial DNA mutations, driving oxidative stress and cellular senescence. Emerging evidence highlights a bidirectional interplay between epigenetics and mitochondrial function. DNA methylation modulates the expression of nuclear genes governing mitochondrial biogenesis and quality control, while mitochondrial metabolites, such as acetyl-CoA and S-adenosylmethionine, reciprocally influence epigenetic landscapes. This review delves into the intricate nuclear-mitochondrial crosstalk, emphasizing its role in aging-related diseases and exploring therapeutic avenues targeting these interconnected pathways to counteract aging and promote health span extension.
    Keywords:  Aging; DNA Methylation; Epigenetics; Mitochondrial Dysfunction
    DOI:  https://doi.org/10.1016/j.freeradbiomed.2025.03.012
  18. EMBO Rep. 2025 Mar 07.
    MIA40 suppresses cell death induced by apoptosis-inducing factor 1.
    Ben Hur Marins Mussulini, Klaudia K Maruszczak, Piotr Draczkowski, Mayra A Borrero-Landazabal, Selvaraj Ayyamperumal, Artur Wnorowski, Michal Wasilewski, Agnieszka Chacinska.
      Mitochondria harbor respiratory complexes that perform oxidative phosphorylation. Complex I is the first enzyme of the respiratory chain that oxidizes NADH. A dysfunction in complex I can result in higher cellular levels of NADH, which in turn strengthens the interaction between apoptosis-inducing factor 1 (AIFM1) and Mitochondrial intermembrane space import and assembly protein 40 (MIA40) in the mitochondrial intermembrane space. We investigated whether MIA40 modulates the activity of AIFM1 upon increased NADH/NAD+ balance. We found that in model cells characterized by an increase in NADH the AIFM1-MIA40 interaction is strengthened and these cells demonstrate resistance to AIFM1-induced cell death. Either silencing of MIA40, rescue of complex I, or depletion of NADH through the expression of yeast NADH-ubiquinone oxidoreductase-2 sensitized NDUFA13-KO cells to AIFM1-induced cell death. These findings indicate that the complex of MIA40 and AIFM1 suppresses AIFM1-induced cell death in a NADH-dependent manner. This study identifies an effector complex involved in regulating the programmed cell death that accommodates the metabolic changes in the cell and provides a molecular explanation for AIFM1-mediated chemoresistance of cancer cells.
    Keywords:  Cancer; Metabolism; Mitochondria; Programmed Cell Death; Protein Import
    DOI:  https://doi.org/10.1038/s44319-025-00406-8
  19. Aging Cell. 2025 Mar 13. e70026
    The Mitochondria-Targeted Peptide Therapeutic Elamipretide Improves Cardiac and Skeletal Muscle Function During Aging Without Detectable Changes in Tissue Epigenetic or Transcriptomic Age.
    Wayne Mitchell, Gavin Pharaoh, Alexander Tyshkovskiy, Matthew Campbell, David J Marcinek, Vadim N Gladyshev.
      Aging-related decreases in cardiac and skeletal muscle function are strongly associated with various comorbidities. Elamipretide (ELAM), a novel mitochondria-targeted peptide, has demonstrated broad therapeutic efficacy in ameliorating disease conditions associated with mitochondrial dysfunction across both clinical and pre-clinical models. Herein, we investigated the impact of 8-week ELAM treatment on pre- and post-measures of C57BL/6J mice frailty, skeletal muscle, and cardiac muscle function, coupled with post-treatment assessments of biological age and affected molecular pathways. We found that health status, as measured by frailty index, cardiac strain, diastolic function, and skeletal muscle force, is significantly diminished with age, with skeletal muscle force changing in a sex-dependent manner. Conversely, ELAM mitigated frailty accumulation and was able to partially reverse these declines, as evidenced by treatment-induced increases in cardiac strain and muscle fatigue resistance. Despite these improvements, we did not detect statistically significant changes in gene expression or DNA methylation profiles indicative of molecular reorganization or reduced biological age in most ELAM-treated groups. However, pathway analyses revealed that ELAM treatment showed pro-longevity shifts in gene expression, such as upregulation of genes involved in fatty acid metabolism, mitochondrial translation, and oxidative phosphorylation, and downregulation of inflammation. Together, these results indicate that ELAM treatment is effective at mitigating signs of sarcopenia and cardiac dysfunction in an aging mouse model, but that these functional improvements occur independently of detectable changes in epigenetic and transcriptomic age. Thus, some age-related changes in function may be uncoupled from changes in molecular biological age.
    Keywords:  aging; aging biomarkers; cardiac dysfunction; elamipretide; epigenetic clocks; mitochondria; transcriptomic clocks
    DOI:  https://doi.org/10.1111/acel.70026
  20. Biol Open. 2025 Mar 06. pii: bio.061815. [Epub ahead of print]
    GSK3 coordinately regulates mitochondrial activity and nucleotide metabolism in quiescent oocytes.
    Leah Eller, Lei Wang, Mehmet Oguz Gok, Helin Hocaoglu, Shenlu Qin, Parul Gupta, Matthew H Sieber.
      As cells transition between periods of growth and quiescence, their metabolic demands change. During this transition, cells must coordinate changes in mitochondrial function with the induction of biosynthetic processes. Mitochondrial metabolism and nucleotide biosynthesis are key rate-limiting factors in regulating early growth. However, it remains unclear what coordinates these mechanisms in developmental systems. Here, we show that during quiescence, as mitochondrial activity drops, nucleotide breakdown increases. However, at fertilization, mitochondrial oxidative metabolism and nucleotide biosynthesis are coordinately activated to support early embryogenesis. We have found that the serine/threonine kinase GSK3 is a key factor in coordinating mitochondrial metabolism with nucleotide biosynthesis during transitions between quiescence and growth. Silencing GSK3 in quiescent oocytes causes increased levels of mitochondrial activity and a shift in the levels of several redox metabolites. Interestingly, silencing GSK3 in quiescent oocytes also leads to a precocious induction of nucleotide biosynthesis in quiescent oocytes. Taken together, these data indicate that GSK3 functions to suppress mitochondrial oxidative metabolism and prevent the premature onset of nucleotide biosynthesis in quiescent eggs. These data reveal a key mechanism that coordinates mitochondrial function and nucleotide synthesis with fertilization.
    Keywords:  Drosophila; Embryo; Metabolism; Mitochondria; Oocyte
    DOI:  https://doi.org/10.1242/bio.061815
  21. Anal Chem. 2025 Mar 13.
    Expanding the Dimensions of Single-Cell Multi-Omics To Include Mitochondrial DNA through Fixation and Permeabilization.
    Ting Li, Zhenglong Gu, Guoqiang Zhou.
      Single-cell multi-omics has transformed our understanding of cellular heterogeneity, yet incorporating mitochondrial DNA (mtDNA) remains challenging. Recent studies underscore the critical role of fixation and permeabilization techniques in preserving sample integrity, optimizing Tn5 tagmentation, and retaining mtDNA. In this Perspective, we review the chemical principles underlying fixation and permeabilization methods and highlight new single-cell multiomics technologies leveraging these approaches. We also explore future directions, particularly workflows designed to incorporate mtDNA mutations, enabling simultaneous analysis of mitochondrial genotypes and cellular states. These advances promise to deepen insights derived from single-cell multi-omics, broadening its impact on biological research and clinical applications.
    DOI:  https://doi.org/10.1021/acs.analchem.4c06777
  22. Commun Biol. 2025 Mar 11. 8(1): 410
    Mitochondrial dysfunction drives a neuronal exhaustion phenotype in methylmalonic aciduria.
    Matthew C S Denley, Monique S Straub, Giulio Marcionelli, Miriam A Güra, David Penton, Igor Delvendahl, Martin Poms, Beata Vekeriotaite, Sarah Cherkaoui, Federica Conte, Ferdinand von Meyenn, D Sean Froese, Matthias R Baumgartner.
      Methylmalonic aciduria (MMA) is an inborn error of metabolism resulting in loss of function of the enzyme methylmalonyl-CoA mutase (MMUT). Despite acute and persistent neurological symptoms, the pathogenesis of MMA in the central nervous system is poorly understood, which has contributed to a dearth of effective brain specific treatments. Here we utilised patient-derived induced pluripotent stem cells and in vitro differentiation to generate a human neuronal model of MMA. We reveal strong evidence of mitochondrial dysfunction caused by deficiency of MMUT in patient neurons. By employing patch-clamp electrophysiology, targeted metabolomics, and bulk transcriptomics, we expose an altered state of excitability, which is exacerbated by application of dimethyl-2-oxoglutarate, and we suggest may be connected to metabolic rewiring. Our work provides first evidence of mitochondrial driven neuronal dysfunction in MMA, which through our comprehensive characterisation of this paradigmatic model, enables first steps to identifying effective therapies.
    DOI:  https://doi.org/10.1038/s42003-025-07828-z
  23. J Nanobiotechnology. 2025 Mar 11. 23(1): 202
    Mitochondria-targeted nanovesicles for ursodeoxycholic acid delivery to combat neurodegeneration by ameliorating mitochondrial dysfunction.
    Shizheng Zhang, Mengmeng Li, Yuan Li, Shike Yang, Jian Wang, Xiaoxiang Ren, Xiuhui Wang, Long Bai, Jianping Huang, Zhen Geng, Guosheng Han, Yibin Fang, Jiacan Su.
      Mitochondria are pivotal in sustaining oxidative balance and metabolic activity within neurons. It is well-established that mitochondrial dysfunction constitutes a fundamental pathogenic mechanism in neurodegeneration, especially in the context of Parkinson's disease (PD), this represents a promising target for therapeutic intervention. Ursodeoxycholic acid (UDCA), a clinical drug used for liver disease, possesses antioxidant and mitochondrial repair properties. Recently, it has gained attention as a potential therapeutic option for treating various neurodegenerative diseases. However, multiple barriers, including the blood-brain barrier (BBB) and cellular/mitochondrial membranes, significantly hinder the efficient delivery of therapeutic agents to the damaged neuronal mitochondria. Macrophage-derived nanovesicles (NVs), which can traverse the BBB in response to brain inflammation signals, have demonstrated promising tools for brain drug delivery. Nevertheless, natural nanovesicles inherently lack the ability to specifically target mitochondria. Herein, artificial NVs are loaded with UDCA and then functionalized with triphenylphosphonium (TPP) molecules, denoted as UDCA-NVs-TPP. These nanovesicles specifically accumulate in damaged neuronal mitochondria, reduce oxidative stress, and enhance ATP production by 42.62%, thereby alleviating neurotoxicity induced by 1-methyl-4-phenylpyridinium (MPP+). Furthermore, UDCA-loaded NVs modified with TPP successfully cross the BBB and accumulate in the striatum of PD mice. These nanoparticles significantly improve PD symptoms, as demonstrated by a 48.56% reduction in pole climb time, a 59.09% increase in hanging ability, and the restoration of tyrosine hydroxylase levels to normal, achieving remarkable therapeutic efficacy. Our work highlights the immense potential of these potent UDCA-loaded, mitochondria-targeting nanovesicles for efficient treatment of PD and other central neurodegenerative diseases.
    Keywords:  BBB; MNVs; Mitochondrial dysfunction; Mitochondrial targeting; Neurodegenerative diseases; UDCA
    DOI:  https://doi.org/10.1186/s12951-025-03258-5
  24. Bio Protoc. 2025 Mar 05. 15(5): e5224
    Puromycin Proximity Ligation Assay (Puro-PLA) to Assess Local Translation in Axons From Human Neurons.
    Raffaella De Pace, Juan S Bonifacino, Saikat Ghosh.
      Local mRNA translation in axons is crucial for the maintenance of neuronal function and homeostasis, particularly in processes such as axon guidance and synaptic plasticity, due to the long distance from axon terminals to the soma. Recent studies have shown that RNA granules can hitchhike on the surface of motile lysosomal vesicles, facilitating their transport within the axon. Accordingly, disruption of lysosomal vesicle trafficking in the axon, achieved by knocking out the lysosome-kinesin adaptor BLOC-one-related complex (BORC), decreases the levels of a subset of mRNAs in the axon. This depletion impairs the local translation of mitochondrial and ribosomal proteins, leading to mitochondrial dysfunction and axonal degeneration. Various techniques have been developed to visualize translation in cells, including translating RNA imaging by coat protein knock-off (TRICK), SunTag, and metabolic labeling using the fluorescent non-canonical amino acid tagging (FUNCAT) systems. Here, we describe a sensitive technique to detect newly synthesized proteins at subcellular resolution, the puromycin proximity ligation assay (Puro-PLA). Puromycin, a tRNA analog, incorporates into nascent polypeptide chains and can be detected with an anti-puromycin antibody. Coupling this method with the proximity ligation assay (PLA) allows for precise visualization of newly synthesized target proteins. In this article, we describe a step-by-step protocol for performing Puro-PLA in human induced pluripotent stem cell (iPSC)-derived neuronal cultures (i3Neurons), offering a powerful tool to study local protein synthesis in the axon. This tool can also be applied to rodent neurons in primary culture, enabling the investigation of axonal protein synthesis across species and disease models. Key features • Establishment of quantitative local translation assay in axons of human iPSC-derived neurons. • Microscopy-based direct visualization of local translation events in neurons. • Puro-PLA is a sensitive method for detecting new protein synthesis occurring within minutes in neurons, enabling precise temporal analysis of translation dynamics.
    Keywords:  Axon; Human i3Neurons; Local translation; Lysosome; Proximity ligation assay (PLA); Puromycin
    DOI:  https://doi.org/10.21769/BioProtoc.5224
  25. J Pediatr Endocrinol Metab. 2025 Mar 10.
    Diabetes, macrocytosis, and skin changes in large-scale mtDNA deletion.
    Nevena Krnic, Duje Braovac, Maja Vinkovic, Jelena Petrinovic Doresic, Katja Dumic Kubat.
       OBJECTIVES: To present a patient diagnosed with single, large-scale mitochondrial DNA (mtDNA) deletion (SLSMD), a rare and progressive multisystem disorder. Diverse initial symptoms, evolving and overlapping phenotypes, along with genetic heterogeneity present significant challenges for diagnosis.
    CASE PRESENTATION: A 3.2-year-old girl presented with seronegative insulin-dependent diabetes, short stature, skin pigmentation anomalies, and macrocytic anemia. The anemia resolved spontaneously, but the macrocytosis persisted. Over time, diagnosis of corneal dystrophy and sensorineural hearing loss were established. Although no classical biochemical features of mitochondrial disease were present, comprehensive molecular mtDNA analysis was performed from peripheral blood. The results revealed a single mtDNA deletion of 7.423 bp, with 37 % of heteroplasmy, confirming the diagnosis of SLSMDs.
    CONCLUSIONS: The occurrence of diabetes mellitus as presenting endocrine manifestation of SLSMDs at an early age is uncommon. Macrocytosis, as well as hair and skin pigmentation changes, may be the early indicators of mitochondrial diseases. A cluster of symptoms including antibody-negative diabetes, short stature, and signs of sporadic dysfunction of organs with high energy demand, suggest a distinct pattern commonly observed in mitochondrial disorders.
    Keywords:  SLSMD; macrocytosis; mitochondrial diabetes; skin pigmentation disorder
    DOI:  https://doi.org/10.1515/jpem-2025-0016
  26. Anal Chem. 2025 Mar 13.
    A Rigorous Nuclear DNA-Excluding Mass Spectrometry Assay for Accurate Identification of 5-Methylcytosine in Mitochondrial DNA.
    Jing Zheng, Yiran Liu, Ziyu Liang, Yu Ang Wang, Wenqiang Yu, Baoquan Ding, Hailin Wang.
      5-Methylcytosine (5mC) functions as a well-characterized epigenetic DNA mark in nuclear DNA, but its presence in mitochondrial DNA (mtDNA) remains elusive. Here, we report a new and rigorous nuclear DNA (nDNA)-excluding mass spectrometry assay enabling the reliable and accurate identification of 5mC in mtDNA for the first time. First, circular mtDNA is enriched over 809-946-fold by combining alkaline lysis and linear DNA-specific RecBCD cutting; nDNA accounts for ∼12-19% of the DNA remaining after this step. Second, assisted by the restrictive endonucleases BbsI (for human mtDNA) and EcoRV (for mouse mtDNA), circular mtDNA was cut into only one or two linearized mtDNA fragments, while the residual nuclear DNA was efficiently degraded into shorter fragments; thus, the linearized mtDNA fragment(s) could be well isolated from the residual degraded nDNA via gel electrophoresis. Finally, the linearized mtDNA bands are excised and subjected to in-gel digestion followed by precise stable isotope-diluted LC-MS/MS analysis. With this sensitive and accurate method, we demonstrated that mtDNA is hypomethylated in a normal mouse cell line, which is rationally attributed to de novo methylation. Overall, we provide a powerful, gold-standard mass spectrometry assay for screening and identifying mtDNA 5mC in diverse scenarios.
    DOI:  https://doi.org/10.1021/acs.analchem.4c06090
  27. J Vis Exp. 2025 Feb 21.
    Measurement of Mitochondrial Membrane Potential In Vivo using a Genetically Encoded Voltage Indicator.
    Run-Zhou Yang, Dian-Dian Wang, Sen-Miao Li, Pei-Pei Liu, Jian-Sheng Kang.
      Mitochondrial membrane potential (MMP, ΔΨm) is critical for mitochondrial functions, including ATP synthesis, ion transport, reactive oxygen species (ROS) generation, and the import of proteins encoded by the nucleus. Existing methods for measuring ΔΨm typically use lipophilic cation dyes, such as Rhodamine 800 and tetramethylrhodamine methyl ester (TMRM), but these are limited by low specificity and are not well-suited for in vivo applications. To address these limitations, we have developed a novel protocol utilizing genetically encoded voltage indicators (GEVIs). Genetically encoded voltage indicators (GEVIs), which generate fluorescent signals in response to membrane potential changes, have demonstrated significant potential for monitoring plasma membrane and neuronal potentials. However, their application to mitochondrial membranes remains unexplored. Here, we developed protein-based mitochondrial-targeted GEVIs capable of detecting ΔΨm fluctuations in cells and the motor cortex of living animals. The mitochondrial potential indicator (MPI)offers a non-invasive approach to study ΔΨm dynamics in real-time, providing a method to investigate mitochondrial function under both normal and pathological conditions.
    DOI:  https://doi.org/10.3791/67911
  28. EMBO J. 2025 Mar 10.
    Mitochondrial fatty acid oxidation regulates adult muscle stem cell function through modulating metabolic flux and protein acetylation.
    Feng Yue, Lijie Gu, Jiamin Qiu, Stephanie N Oprescu, Linda M Beckett, Jessica M Ellis, Shawn S Donkin, Shihuan Kuang.
      During homeostasis and regeneration, satellite cells, the resident stem cells of skeletal muscle, have distinct metabolic requirements for fate transitions between quiescence, proliferation and differentiation. However, the contribution of distinct energy sources to satellite cell metabolism and function remains largely unexplored. Here, we uncover a role of mitochondrial fatty acid oxidation (FAO) in satellite cell integrity and function. Single-cell RNA sequencing revealed progressive enrichment of mitochondrial FAO and downstream pathways during activation, proliferation and myogenic commitment of satellite cells. Deletion of Carnitine palmitoyltransferase 2 (Cpt2), the rate-limiting enzyme in FAO, hampered muscle stem cell expansion and differentiation upon acute muscle injury, markedly delaying regeneration. Cpt2 deficiency reduces acetyl-CoA levels in satellite cells, impeding the metabolic flux and acetylation of selective proteins including Pax7, the central transcriptional regulator of satellite cells. Notably, acetate supplementation restored cellular metabolic flux and partially rescued the regenerative defects of Cpt2-null satellite cells. These findings highlight an essential role of fatty acid oxidation in controlling satellite cell function and suggest an integration of lipid metabolism and protein acetylation in adult stem cells.
    Keywords:  CPT2; Fatty Acid Oxidation; Muscle Regeneration; Muscle Satellite Cell; Protein Acetylation
    DOI:  https://doi.org/10.1038/s44318-025-00397-1
  29. bioRxiv. 2025 Mar 01. pii: 2025.02.26.640389. [Epub ahead of print]
    Succinate dehydrogenase activity supports de novo purine synthesis.
    Mushtaq A Nengroo, Austin T Klein, Heather S Carr, Olivia Vidal-Cruchez, Umakant Sahu, Daniel J McGrail, Nidhi Sahni, Peng Gao, John M Asara, Hardik Shah, Marc L Mendillo, Issam Ben-Sahra.
      The de novo purine synthesis pathway is fundamental for nucleic acid production and cellular energetics, yet the role of mitochondrial metabolism in modulating this process remains underexplored. In many cancers, metabolic reprogramming supports rapid proliferation and survival, but the specific contributions of the tricarboxylic acid (TCA) cycle enzymes to nucleotide biosynthesis are not fully understood. Here, we demonstrate that the TCA cycle enzyme succinate dehydrogenase (SDH) is essential for maintaining optimal de novo purine synthesis in normal and cancer cells. Genetic or pharmacological inhibition of SDH markedly attenuates purine synthesis, leading to a significant reduction in cell proliferation. Mechanistically, SDH inhibition causes an accumulation of succinate, which directly impairs the purine biosynthetic pathway. In response, cancer cells compensate by upregulating the purine salvage pathway, a metabolic adaptation that represents a potential therapeutic vulnerability. Notably, co-inhibition of SDH and the purine salvage pathway induces pronounced antiproliferative and antitumoral effects in preclinical models. These findings not only reveal a signaling role for mitochondrial succinate in regulating nucleotide metabolism but also provide a promising therapeutic strategy for targeting metabolic dependencies in cancer.
    DOI:  https://doi.org/10.1101/2025.02.26.640389
  30. J Clin Neurol. 2025 Mar;21(2): 150-152
    Novel Presentation of McLeod Syndrome With Muscle Weakness and Biopsy Findings Indicative of Mitochondrial Dysfunction.
    Zhihong Xu, Ying Zhao, YuYing Zhao, Chuanzhu Yan, Kunqian Ji.
      
    DOI:  https://doi.org/10.3988/jcn.2023.0253
  31. Orphanet J Rare Dis. 2025 Mar 07. 20(1): 108
    A novel c.59 C > T variant of the HSD17B10 gene as a possible cause of the neonatal form of HSD10 mitochondrial disease with hepatic dysfunction: a case report and review of the literature.
    Tao Jiang, Wenxian Ouyang, Haiyan Yang, Shuangjie Li.
       BACKGROUND: Pathogenic HSD17B10 gene variants cause HSD10 mitochondrial disease (HSD10 MD), which results in a wide spectrum of symptoms ranging from mild to severe. Typical symptoms include intellectual disability, choreoathetosis, cardiomyopathy, neurodegeneration, and abnormal behavior. This study investigated a novel c.59 C > T variant of the HSD17B10 gene and the clinical phenotypic features of HSD10 MD (neonatal form) patients.
    RESULTS: We describe a Chinese boy 2 months and 12 days old with intellectual disability, metabolic acidosis, hyperlactatemia, hypoglycemia, cholestatic hepatitis and myocardial enzyme levels, slightly elevated 2-methyl-3-hydroxybutyric acid (2M3HBA) levels and early death. Although full-length sequencing of the mitochondrial genome was normal, whole-exome sequencing of the proband and his parents revealed a novel de novo hemizygous variant, c.59 C > T (p.S20L), of the HSD17B10 gene. Molecular dynamics simulation analysis and protein structural analysis suggested that the c.59 C > T (p.S20L) variant may disrupt the conformational stability of the protein. On the basis of the combined results of phenotypic analysis, molecular genetic analysis, protein structural analysis and molecular dynamics simulation analysis, this novel variant is currently considered a likely pathogenic variant. HSD10 MD (neonatal form) can lead to hepatic dysfunction.
    CONCLUSIONS: HSD10 MD (neonatal form) can lead to hepatic dysfunction. The de novo c.59 C > T HSD17B10 variant suggested a neonatal form of the HSD10 mitochondrial disease phenotype in a patient 2 months and 12 days old, broadening the variant spectrum of HSD17B10-related disease.
    Keywords:   HSD17B10 gene; Cholestatic hepatitis; HSD10 mitochondrial disease; Metabolic disorder; Variant
    DOI:  https://doi.org/10.1186/s13023-024-03513-2
  32. Nat Neurosci. 2025 Mar 11.
    Mitochondrial respiratory complex IV deficiency recapitulates amyotrophic lateral sclerosis.
    Man Cheng, Dan Lu, Kexin Li, Yan Wang, Xiwen Tong, Xiaolong Qi, Chuanzhu Yan, Kunqian Ji, Junlin Wang, Wei Wang, Huijiao Lv, Xu Zhang, Weining Kong, Jian Zhang, Jiaxin Ma, Keru Li, Yaheng Wang, Jingyu Feng, Panpan Wei, Qiushuang Li, Chengyong Shen, Xiang-Dong Fu, Yuanwu Ma, Xiaorong Zhang.
      Amyotrophic lateral sclerosis (ALS) is categorized into ~10% familial and ~90% sporadic cases. While familial ALS is caused by mutations in many genes of diverse functions, the underlying pathogenic mechanisms of ALS, especially in sporadic ALS (sALS), are largely unknown. Notably, about half of the cases with sALS showed defects in mitochondrial respiratory complex IV (CIV). To determine the causal role of this defect in ALS, we used transcription activator-like effector-based mitochondrial genome editing to introduce mutations in CIV subunits in rat neurons. Our results demonstrate that neuronal CIV deficiency is sufficient to cause a number of ALS-like phenotypes, including cytosolic TAR DNA-binding protein 43 redistribution, selective motor neuron loss and paralysis. These results highlight CIV deficiency as a potential cause of sALS and shed light on the specific vulnerability of motor neurons, marking an important advance in understanding and therapeutic development of sALS.
    DOI:  https://doi.org/10.1038/s41593-025-01896-4
  33. Hum Reprod Update. 2025 Mar 14. pii: dmaf004. [Epub ahead of print]
    Genetic and reproductive strategies to prevent mitochondrial diseases.
    Noemi Castelluccio, Katharina Spath, Danyang Li, Irenaeus F M De Coo, Lyndsey Butterworth, Dagan Wells, Heidi Mertes, Joanna Poulton, Björn Heindryckx.
      Mitochondrial DNA (mtDNA) diseases pose unique challenges for genetic counselling and require tailored approaches to address recurrence risks and reproductive options. The intricate dynamics of mtDNA segregation and heteroplasmy shift significantly impact the chances of having affected children. In addition to natural pregnancy, oocyte donation, and adoption, IVF-based approaches can reduce the risk of disease transmission. Prenatal diagnosis (PND) and preimplantation genetic testing (PGT) remain the standard methods for women carrying pathogenic mtDNA mutations; nevertheless, they are not suitable for every patient. Germline nuclear transfer (NT) has emerged as a novel therapeutic strategy, while mitochondrial gene editing has increasingly become a promising research area in the field. However, challenges and safety concerns associated with all these techniques remain, highlighting the need for long-term follow-up studies, an improved understanding of disease mechanisms, and personalized approaches to diagnosis and treatment. Given the inherent risks of adverse maternal and child outcomes, careful consideration of the balance between potential benefits and drawbacks is also warranted. This review will provide critical insights, identify knowledge gaps, and underscore the importance of advancing mitochondrial disease research in reproductive health.
    Keywords:  germline nuclear transfer (NT); mitochondrial DNA (mtDNA); mitochondrial disease; mitochondrial gene editing; preimplantation genetic testing (PGT); prenatal diagnosis (PND)
    DOI:  https://doi.org/10.1093/humupd/dmaf004
  34. J Cereb Blood Flow Metab. 2025 Mar 13. 271678X251325805
    Mitochondria transfer for myelin repair.
    Sabah Mozafari, Luca Peruzzotti-Jametti, Stefano Pluchino.
      Demyelination is a common feature of neuroinflammatory and degenerative diseases of the central nervous system (CNS), such as multiple sclerosis (MS). It is often linked to disruptions in intercellular communication, bioenergetics and metabolic balance accompanied by mitochondrial dysfunction in cells such as oligodendrocytes, neurons, astrocytes, and microglia. Although current MS treatments focus on immunomodulation, they fail to stop or reverse demyelination's progression. Recent advancements highlight intercellular mitochondrial exchange as a promising therapeutic target, with potential to restore metabolic homeostasis, enhance immunomodulation, and promote myelin repair. With this review we will provide insights into the CNS intercellular metabolic decoupling, focusing on the role of mitochondrial dysfunction in neuroinflammatory demyelinating conditions. We will then discuss emerging cell-free biotherapies exploring the therapeutic potential of transferring mitochondria via biogenic carriers like extracellular vesicles (EVs) or synthetic liposomes, aimed at enhancing mitochondrial function and metabolic support for CNS and myelin repair. Lastly, we address the key challenges for the clinical application of these strategies and discuss future directions to optimize mitochondrial biotherapies. The advancements in this field hold promise for restoring metabolic homeostasis, and enhancing myelin repair, potentially transforming the therapeutic landscape for neuroinflammatory and demyelinating diseases.
    Keywords:  Extracellular vesicles (EVs); cell-free biotherapy; demyelination; mitochondria transfer; neuroinflammation
    DOI:  https://doi.org/10.1177/0271678X251325805
  35. Clin Ther. 2025 Mar 13. pii: S0149-2918(25)00047-5. [Epub ahead of print]
    PHEMI-Phenylbutyrate in Patients With Lactic Acidosis: A Pilot, Single Arm, Phase I/II, Open-Label Trial.
    Silvia Marchet, Alessia Catania, Anna Ardissone, Vincenzo Montano, Krisztina Einvag, Maria Pia Iermito, Daniele Sala, Manuela Spagnolo, Elena Mauro, Eleonora Lamantea, Giulia Cecchi, Piervito Lopriore, Michelangelo Mancuso, Costanza Lamperti.
       PURPOSE: The 6 months pilot, single arm, phase I/II, open-label clinical trial PHEMI investigated the safety and efficacy of daily administration of phenylbutyrate in reducing lactic acidosis by at least 20% in 3 children (ages 7-10 yrs) with pyruvate dehydrogenase deficiency and 6 adults with mitochondrial myopathy encephalopathy lactic acidosis and stroke-like episodes. As a side study, we investigated the response to phenylbutyrate treatment in skin fibroblasts and cybrids derived from PHEMI patients with the aim of unraveling a possible in vivo-in vitro correlation.
    METHODS: Safety was assessed through the collection of vital signs, clinical evaluations, blood samples, and reported adverse events. Efficacy was evaluated on biochemical and clinical endpoints. In vitro analysis explored the effects of phenylbutyrate in patients' fibroblasts and cybrids.
    FINDINGS: At the starting dosage regimen of 10 g/m2/day, phenylbutyrate was effective in reducing lactic acidosis (by a mean of 13%), but lead to the development of adverse events in all adults. The reduced dose of 5 g/m²/day was well tolerated but did not meet the study's primary outcome. In parallel, the in vitro analyses confirmed that phenylbutyrate led to a reduction in lactate measured in culture medium, an increase in cellular respiration, and a slight increase in the activity of the Respiratory Chain Complexes.
    IMPLICATIONS: Our study fosters further research on phenylbutyrate in individuals with primary mitochondrial disease suffering from lactic acidosis. Future investigation should focus on a highly bioavailable, easier-to-administer drug formulation that allows the administration of a lower dosage regimen.
    Keywords:  MELAS; PDH deficiency; lactic acidosis; mitochondrial diseases; pilot clinical trial; sodium phenylbutyrate
    DOI:  https://doi.org/10.1016/j.clinthera.2025.02.004
  36. Anal Chem. 2025 Mar 13.
    Time Series Imaging the Mitochondrial Microenvironment and Its Interactions with Lysosomes during Ferroptosis.
    Xinyu Shao, Xusheng Ren, Tianshuo Xing, Xueying Zheng, Cuimin Feng, Tian Cheng, Junling Yin.
      In the realm of cutting-edge scientific inquiry, the development and application of integrated optical molecular probes for the simultaneous detection and tracing of mitochondrial microenvironments during ferroptosis, as well as the visualization of their interactions with lysosomes, stands as a pivotal advancement. In this work, we developed a probe, IMT, that integrates viscosity sensing with mitochondrial targeting, and used it in conjunction with commercial lysosome green tracers (LGT) to investigate mitochondrial-lysosome interactions (MLIs). This approach avoids the uneven labeling caused by subcellular microenvironment differences when using single-molecule dual-targeting probes. Using the developed IMT, we observed an increase in mitochondrial viscosity during erastin-induced ferroptosis and a decrease during ferrostatin-1-inhibited ferroptosis. Moreover, the time series imaging of the mitochondrial profile lighted by the IMT showed that the mitochondrial area, perimeter, aspect ratio, and mitochondrial form factor changed significantly as ferroptosis progressed. In addition, combined with LGT, we visualized the dynamic process of first contact and then separation between lysosomes and mitochondria during ferroptosis, confirming the complexity and variability of MLIs. This work not only enhances our understanding of the complex biochemical processes underlying ferroptosis but also opens new avenues for therapeutic intervention in diseases characterized by this form of cell death.
    DOI:  https://doi.org/10.1021/acs.analchem.4c06840
  37. Int J Biol Sci. 2025 ;21(5): 1863-1873
    Fatty Acid Trafficking Between Lipid Droplets and Mitochondria: An Emerging Perspective.
    Katarína Smolková, Klára Gotvaldová.
      The current understanding of lipid droplets (LDs) in cell biology has evolved from being viewed merely as storage compartments. LDs are now recognized as metabolic hubs that act as cytosolic buffers against the detrimental effects of free fatty acids (FAs). Upon activation, FAs traverse various cellular pathways, including oxidation in mitochondria, integration into complex lipids, or storage in triacylglycerols (TGs). Maintaining a balance among these processes is crucial in cellular FA trafficking, and under metabolically challenging circumstances the routes of FA metabolism adapt to meet the current cellular needs. This typically involves an increased demand for anabolic intermediates or energy and the prevention of redox stress. Surprisingly, LDs accumulate under certain conditions such as amino acid starvation. This review explores the biochemical aspects of FA utilization in both physiological contexts and within cancer cells, focusing on the metabolism of TGs, cholesteryl esters (CEs), and mitochondrial FA oxidation. Emphasis is placed on the potential toxicity associated with non-esterified FAs in cytosolic and mitochondrial compartments. Additionally, we discuss mechanisms that lead to increased LD biogenesis due to an inhibited mitochondrial import of FAs.
    Keywords:  CPT1; ferroptosis; lipid droplets; lipotoxicity; mitochondria; triglycerides
    DOI:  https://doi.org/10.7150/ijbs.105361
  38. J Immunol. 2025 Feb 01. 214(2): 238-252
    Aggregatin is a mitochondrial regulator of MAVS activation to drive innate immunity.
    Ju Gao, Mao Ding, Yanbin Xiyang, Siyue Qin, Devanshi Shukla, Jiawei Xu, Masaru Miyagi, Hisashi Fujioka, Jingjing Liang, Xinglong Wang.
      Mitochondrial antiviral-signaling protein (MAVS) is a key adapter protein required for inducing type I interferons (IFN-Is) and other antiviral effector molecules. The formation of MAVS aggregates on mitochondria is essential for its activation; however, the regulatory mitochondrial factor that mediates the aggregation process is unknown. Our recent work has identified the protein Aggregatin as a critical seeding factor for β-amyloid peptide aggregation. Here we show that Aggregatin serves as a cross-seed for MAVS aggregates on mitochondria to orchestrate innate immune signaling. Aggregatin is primarily localized to mitochondria in the cytosol and has the ability to induce MAVS aggregation and MAVS-dependent IFN-I responses alone in both HEK293 cells and human leukemia monocytic THP-1 cells. Mitochondrial Aggregatin level increases upon viral infection. Also, Aggregatin knockout suppresses viral infection-induced MAVS aggregation and IFN-I signal cascade activation. Nemo-like kinase is further identified as a kinase phosphorylating Aggregatin at Ser59 to regulate its stability and cross-seeding activity. Collectively, our finding reveals an important physiological function of Aggregatin in innate immunity by cross-seeding MAVS aggregation.
    Keywords:  Aggregatin; MAVS; innate immunity; interferon; mitochondria; viral infection
    DOI:  https://doi.org/10.1093/jimmun/vkae019
  39. Mol Med Rep. 2025 May;pii: 127. [Epub ahead of print]31(5):
    Mitochondria‑derived peptides: Promising microproteins in cardiovascular diseases (Review).
    Yutong Ran, Zhiliang Guo, Lijuan Zhang, Hong Li, Xiaoyun Zhang, Xiumei Guan, Xiaodong Cui, Hao Chen, Min Cheng.
      Mitochondria‑derived peptides (MDPs) are a unique class of peptides encoded by short open reading frames in mitochondrial DNA, including the mitochondrial open reading frame of the 12S ribosomal RNA type‑c (MOTS‑c). Recent studies suggest that MDPs offer therapeutic benefits in various diseases, including neurodegenerative disorders and types of cancer, due to their ability to increase cellular resilience. Mitochondrial dysfunction is a key factor in the onset and progression of cardiovascular diseases (CVDs), such as atherosclerosis and heart failure, as it disrupts energy metabolism, increases oxidative stress and promotes inflammation. MDPs such as humanin and MOTS‑c have emerged as important regulators of mitochondrial health, as they show protective effects against these processes. Recent studies have shown that MDPs can restore mitochondrial function, reduce oxidative damage and alleviate inflammation, thus counteracting the pathological mechanisms that drive CVDs. Therefore, MDPs hold promise as therapeutic agents that are capable of slowing, stopping, or even reversing CVD progression and their use presents a promising strategy for future treatments. However, the clinical application of MDPs remains challenging due to their low bioavailability, poor stability and high synthesis costs. Thus, it is necessary to improve drug delivery systems to enhance the bioavailability of MDPs. Moreover, integrating basic research with clinical trials is essential to bridge the gap between experimental findings and clinical applications.
    Keywords:  cardiovascular diseases; inflammation; mechanism; mitochondria; mitochondria‑derived peptides
    DOI:  https://doi.org/10.3892/mmr.2025.13492
  40. Sci Rep. 2025 Mar 08. 15(1): 8101
    Skeletal muscle mitochondrial dysfunction is associated with increased Gdf15 expression and circulating GDF15 levels in aged mice.
    J Chen, J Kastroll, F M Bello, M M Pangburn, A Murali, P M Smith, K Rychcik, K E Loughridge, A M Vandevender, N Dedousis, I J Sipula, J K Alder, M J Jurczak.
      Growth differentiation factor-15 (GDF15) is a biomarker of multiple disease states and circulating GDF15 levels are increased during aging in both pre-clinical animal models and human studies. Accordingly, multiple stressors have been identified, including mitochondrial dysfunction, that lead to induction of Gdf15 expression downstream of the integrated stress response (ISR). For some disease states, the source of increased circulating GDF15 is evident based on the specific pathology. Aging, however, presents a less tractable system for understanding the source of increased plasma GDF15 levels in that cellular dysfunction with aging can be pleiotropic and heterogeneous. To better understand which organ or organs contribute to increased circulating GDF15 levels with age, and whether changes in metabolic and mitochondrial dysfunction were associated with these potential changes, we compared young 12-week-old and middle-aged 52-week-old C57BL/6 J mice using a series of metabolic phenotyping studies and by comparing circulating levels of GDF15 and tissue-specific patterns of Gdf15 expression. Overall, we found that Gdf15 expression was increased in skeletal muscle but not liver, white or brown adipose tissue, kidney or heart of middle-aged mice, and that insulin sensitivity and mitochondrial respiratory capacity were impaired in middle-aged mice. These data suggest that early changes in skeletal muscle mitochondrial function and metabolism contribute to increased circulating GDF15 levels observed during aging.
    Keywords:  Aging; Energy expenditure; Insulin resistance; Integrated stress response; Respirometry
    DOI:  https://doi.org/10.1038/s41598-025-92572-x
  41. Nat Rev Genet. 2025 Mar 10.
    Adapting systems biology to address the complexity of human disease in the single-cell era.
    David S Fischer, Martin A Villanueva, Peter S Winter, Alex K Shalek.
      Systems biology aims to achieve holistic insights into the molecular workings of cellular systems through iterative loops of measurement, analysis and perturbation. This framework has had remarkable success in unicellular model organisms, and recent experimental and computational advances - from single-cell and spatial profiling to CRISPR genome editing and machine learning - have raised the exciting possibility of leveraging such strategies to prevent, diagnose and treat human diseases. However, adapting systems-inspired approaches to dissect human disease complexity is challenging, given that discrepancies between the biological features of human tissues and the experimental models typically used to probe function (which we term 'translational distance') can confound insight. Here we review how samples, measurements and analyses can be contextualized within overall multiscale human disease processes to mitigate data and representation gaps. We then examine ways to bridge the translational distance between systems-inspired human discovery loops and model system validation loops to empower precision interventions in the era of single-cell genomics.
    DOI:  https://doi.org/10.1038/s41576-025-00821-6
  42. Am J Hum Genet. 2025 Mar 05. pii: S0002-9297(25)00059-X. [Epub ahead of print]
    Data-driven insights to inform splice-altering variant assessment.
    Patricia J Sullivan, Julian M W Quinn, Pamela Ajuyah, Mark Pinese, Ryan L Davis, Mark J Cowley.
      Disease-causing genetic variants often disrupt mRNA splicing, an intricate process that is incompletely understood. Thus, accurate inference of which genetic variants will affect splicing and what their functional consequences will be is challenging, particularly for variants outside of the essential splice sites. Here, we describe a set of data-driven heuristics that inform the interpretation of human splice-altering variants (SAVs) based on the analysis of annotated exons, experimentally validated SAVs, and the currently understood principles of splicing biology. We defined requisite splicing criteria by examining around 202,000 canonical protein-coding exons and 19,000 experimentally validated splicing branchpoints. This analysis defined the sequence, spacing, and motif strength required for splicing, with 95.9% of the exons examined meeting these criteria. By considering over 12,000 experimentally validated variants from the SpliceVarDB, we defined a set of heuristics that inform the evaluation of putative SAVs. To ensure the applicability of each heuristic, only those supported by at least 10 experimentally validated variants were considered. This allowed us to establish a measure of spliceogenicity: the proportion of variants at a location (or motif site) that affected splicing in a given context. This study makes considerable advances toward bridging the gap between computational predictions and the biological process of splicing, offering an evidence-based approach to identifying SAVs and evaluating their impact. Our splicing heuristics enhance the current framework for genetic variant evaluation with a robust, detailed, and comprehensible analysis by adding valuable context over traditional binary prediction tools.
    Keywords:  RNA splicing; cancer genomics; clinical genomics; genomics; medical genomics; next-generation sequencing; personalized medicine; splice-altering variants; variant classification; whole-genome sequencing
    DOI:  https://doi.org/10.1016/j.ajhg.2025.02.012
  43. ACS Nano. 2025 Mar 13.
    Amplification-Free Quantification of Endogenous Mitochondrial DNA Copy Number Using Solid-State Nanopores.
    Sohini Pal, Diana Huttner, Navneet C Verma, Talya Nemirovsky, Oren Ziv, Noa Sher, Natalie Yivgi-Ohana, Amit Meller.
      Mitochondrial DNA (mtDNA) quantification is crucial in understanding mitochondrial dysfunction, which is linked to a variety of diseases, including cancer and neurodegenerative disorders. Traditional methods often rely on amplification-based techniques, which can introduce bias and lack the precision needed for clinical diagnostics. Solid-state nanopores, an emerging biosensing platform, have the advantage of offering single-molecule and label-free approaches by enabling the direct counting of DNA molecules without amplification. The ion-current signatures obtained from each DNA molecule contain rich information on the molecules' lengths and origin. In this study, we present an amplification-free method for mtDNA quantification using solid-state nanopores and machine learning. Intriguingly, we find that native (unamplified) mtDNA translocations harbor structurally distinctive features that can be exploited to specifically detect and quantify mtDNA copies over the background of genomic DNA fragments. By combining selective degradation of linear genomic DNA (gDNA) via exonuclease V with a support vector machine (SVM)-based model, we isolate and quantify mtDNA directly from biological samples. We validate our method using plasmids or isolated mtDNAs by spiking in predetermined quantities. We then quantify endogenous mtDNAs in a cancer cell line and in blood cells and compare our results with qPCR-based quantification of the mtDNA/nuclear DNA ratios. To elucidate the source of the ion-current signatures from the native mtDNA molecules, we perform synchronous electro-optical sensing of mtDNAs during passage through the nanopore after NHS ester reaction with fluorophore compounds. Our results show correlated electro-optical events, indicating that the mtDNA is complexed with packaging proteins. Our assay is robust, with a high classification accuracy and is capable of detecting mtDNA at picomolar levels, making it suitable for low-abundance samples. This technique requires minimal sample preparation and eliminates the need for amplification or purification steps. The developed approach has significant potential for point-of-care applications, offering a low-cost and scalable solution for accurate mtDNA quantification in clinical settings.
    Keywords:  TFAM; amplification-free quantification; electro-optical nanopore sensing; mitochondrial DNA; purification-free assay; single-molecule analysis; solid-state nanopores
    DOI:  https://doi.org/10.1021/acsnano.5c00732
  44. FEBS J. 2025 Mar 12.
    A steric gate prevents mutagenic dATP incorporation opposite 8-oxo-deoxyguanosine in mitochondrial DNA polymerases.
    Noe Baruch-Torres, Carlos H Trasviña-Arenas, Alexandru Ionut Gilea, Upeksha C Dissanayake, Missael Molina-Jiménez, Paola L García-Medel, Corina Díaz-Quezada, Tiziana Lodi, G Andrés Cisneros, Enrico Baruffini, Luis G Brieba.
      Reactive oxygen species (ROS) generate DNA lesions that alter genome integrity. Among those DNA lesions, 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG) is particularly mutagenic. 8-oxodG efficiently incorporates deoxycytidine monophosphate (dCMP) and deoxyadenosine monophosphate (dAMP) via base pairing mediated by its anti and syn conformations, respectively. In family-A DNA polymerases (DNAPs), the amino acids responsible for modulating dCMP or dAMP incorporation across 8-oxodG are located in a determined structural position. Those residues are a conserved tyrosine located at the N terminus of the α-helix O and a nonconserved residue located six amino acids after this conserved tyrosine. In yeast mitochondrial DNAP (DNA-directed DNA polymerase gamma MIP1 [Mip1]), those residues correspond to amino acids Y757 and F763. We hypothesized that the phenyl group of the F763 residue impinges on the syn conformation of 8-oxodG, therefore reducing dAMP misincorporation. Here, we measured dCMP and dAMP incorporation across 8-oxodG using wild-type and F763 Mip1 mutants. Our data suggest that both residue F763 and the universally conserved Y757 assemble a steric gate that obtrudes the 8-oxodG(syn) conformation. As the human orthologue of Mip1, DNA polymerase gamma (HsPolγ) or DNAP γ, also harbors phenylalanine at the corresponding position to Mip1-F763, the steric gate mechanism might similarly be responsible for controlling HsPolγ's fidelity when tolerating 8-oxodG lesions.
    Keywords:  8‐oxo‐deoxyguanosine; DNA polymerase; ROS; kinetic assay; mitochondrial DNA; translesion DNA synthesis
    DOI:  https://doi.org/10.1111/febs.70064
  45. Molecules. 2025 Feb 24. pii: 1025. [Epub ahead of print]30(5):
    Functional Nucleic Acid Nanostructures for Mitochondrial Targeting: The Basis of Customized Treatment Strategies.
    Wanchong He, Siyu Dong, Qinghua Zeng.
      Mitochondria, as vital organelles, play a central role in subcellular research and biomedical innovation. Although functional nucleic acid (FNA) nanostructures have witnessed remarkable progress across numerous biological applications, strategies specifically tailored to target mitochondria for molecular imaging and therapeutic interventions remain scarce. This review delves into the latest advancements in leveraging FNA nanostructures for mitochondria-specific imaging and cancer therapy. Initially, we explore the creation of FNA-based biosensors localized to mitochondria, enabling the real-time detection and visualization of critical molecules essential for mitochondrial function. Subsequently, we examine developments in FNA nanostructures aimed at mitochondrial-targeted cancer treatments, including modular FNA nanodevices for the precise delivery of therapeutic agents and programmable FNA nanostructures for disrupting mitochondrial processes. Emphasis is placed on elucidating the chemical principles underlying the design of mitochondrial-specific FNA nanotechnology for diverse biomedical uses. Lastly, we address the unresolved challenges and outline prospective directions, with the goal of advancing the field and encouraging the creation of sophisticated FNA tools for both academic inquiry and clinical applications centered on mitochondria.
    Keywords:  FNA nanostructures; customized treatment strategy; mitochondria
    DOI:  https://doi.org/10.3390/molecules30051025
  46. J Biol Chem. 2025 Mar 12. pii: S0021-9258(25)00252-2. [Epub ahead of print] 108403
    The E3 ubiquitin ligase RNF126 facilitates quality control of unimported mitochondrial membrane proteins.
    Di Liu, Xin-Yu Huo, Xiaoli Zhang, Zai-Rong Zhang.
      Pathological stress can lead to failure in the translocation of mitochondrial proteins, resulting in accumulation of unimported proteins within the cytosol and upregulation of proteasome for their quality control. Malfunction or delay in protein clearance causes dysregulation of mitochondrial protein homeostasis, cellular toxicity, and diseases. Ubiquilins (UBQLNs) are known to serve as chaperone which associates with unimported mitochondrial membrane protein precursors, and facilitates their proteasomal degradation. However, how UBQLN-engaged proteins are ubiquitinated and efficiently targeted to the proteasome are poorly understood. Here, using mitochondrial membrane protein ATP5G1 as a model substrate, we report that E3 ubiquitin ligase RNF126 interacts with substrate-engaged UBQLN1, thereby promoting ubiquitination and degradation of unimported proteins during mitochondrial stress. We find that UBQLN1's ubiquitin-associated domain (UBA) recruits RNF126 when its middle domain binds to unimported protein substrate. Recombinant RNF126 forms ternary complex with UBQLN1 and pATP5G1 in vitro and catalyzes ubiquitination of UBQLN1-bound ATP5G1. Without RNF126, proteasomal degradation of ATP5G1 was compromised. These results explain how RNF126 and ubiquilins interplay to ensure specific quality control of unimported mitochondrial membrane proteins under pathophysiological conditions.
    Keywords:  ATP synthase F(0) complex subunit C1; RNF126; Ubiquilin; cytosolic quality control; mitochondrial membrane protein degradation
    DOI:  https://doi.org/10.1016/j.jbc.2025.108403
  47. Cell Discov. 2025 Mar 11. 11(1): 22
    A positive feedback loop between SMAD3 and PINK1 in regulation of mitophagy.
    Mingzhu Tang, Dade Rong, Xiangzheng Gao, Guang Lu, Haimei Tang, Peng Wang, Ning-Yi Shao, Dajing Xia, Xin-Hua Feng, Wei-Feng He, Weilin Chen, Jia-Hong Lu, Wei Liu, Han-Ming Shen.
      PTEN-induced kinase-1 (PINK1) is a crucial player in selective clearance of damaged mitochondria via the autophagy-lysosome pathway, a process termed mitophagy. Previous studies on PINK1 mainly focused on its post-translational modifications, while the transcriptional regulation of PINK1 is much less understood. Herein, we reported a novel mechanism in control of PINK1 transcription by SMAD Family Member 3 (SMAD3), an essential component of the transforming growth factor beta (TGFβ)-SMAD signaling pathway. First, we observed that mitochondrial depolarization promotes PINK1 transcription, and SMAD3 is likely to be the nuclear transcription factor mediating PINK1 transcription. Intriguingly, SMAD3 positively transactivates PINK1 transcription independent of the canonical TGFβ signaling components, such as TGFβ-R1, SMAD2 or SMAD4. Second, we found that mitochondrial depolarization activates SMAD3 via PINK1-mediated phosphorylation of SMAD3 at serine 423/425. Therefore, PINK1 and SMAD3 constitute a positive feedforward loop in control of mitophagy. Finally, activation of PINK1 transcription by SMAD3 provides an important pro-survival signal, as depletion of SMAD3 sensitizes cells to cell death caused by mitochondrial stress. In summary, our findings identify a non-canonical function of SMAD3 as a nuclear transcriptional factor in regulation of PINK1 transcription and mitophagy and a positive feedback loop via PINK1-mediated SMAD3 phosphorylation and activation. Understanding this novel regulatory mechanism provides a deeper insight into the pathological function of PINK1 in the pathogenesis of neurodegenerative diseases such as Parkinson's disease.
    DOI:  https://doi.org/10.1038/s41421-025-00774-4
  48. Arch Soc Esp Oftalmol (Engl Ed). 2025 Mar 07. pii: S2173-5794(25)00027-1. [Epub ahead of print]
    Retinopathy associated with MELAS syndrome. A case report.
    A B González Escobar, E Barco Moreno, M A López-Egea Bueno, J M Galván Cano, R Luque Aranda, A González Gómez.
      MELAS syndrome (mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes) is an inherited disease frequently caused by a mutation in the mitochondrial DNA variant m.3243A>G in the MT-TL1 gene. The most frequent ophthalmologic finding present in 86-87% of patients with this mutation is mitochondrial retinopathy, where the clinical picture may vary from a macular and peripapillary salt-and-pepper granular pattern to chorioretinal atrophy. We present the case of a 47-year-old woman with type 1 diabetes mellitus, epilepsy, leukoencephalopathy, and deafness who was suspected of having mitochondrial disease after fundus examination. We would like to emphasize the importance of suspecting a mitochondrial disease in progressive multisystem disorders associated with neuro-ophthalmological manifestations, since early diagnosis allows for better monitoring of systemic manifestations, reducing morbidity and mortality.
    Keywords:  Atrofia coriorretiniana; Chorio-retinal atrophy; Encefalopatía; Encephalopathy; M.3243A>G; MELAS; MTTL1 gen; MTTL1 gene; Mitochondrial retinopathy; Retinopatía mitocondrial
    DOI:  https://doi.org/10.1016/j.oftale.2025.03.001
  49. JCI Insight. 2025 Mar 10. pii: e177999. [Epub ahead of print]10(5):
    Neurofilament accumulation disrupts autophagy in giant axonal neuropathy.
    Jean-Michel Paumier, James Zewe, Chiranjit Panja, Melissa R Pergande, Meghana Venkatesan, Eitan Israeli, Shikha Prasad, Natasha Snider, Jeffrey N Savas, Puneet Opal.
      Neurofilament accumulation is associated with many neurodegenerative diseases, but it is the primary pathology in giant axonal neuropathy (GAN). This childhood-onset autosomal recessive disease is caused by loss-of-function mutations in gigaxonin, the E3 adaptor protein that enables neurofilament degradation. Using a combination of genetic and RNA interference approaches, we found that dorsal root ganglia from mice lacking gigaxonin have impaired autophagy and lysosomal degradation through 2 mechanisms. First, neurofilament accumulations interfere with the distribution of autophagic organelles, impairing their maturation and fusion with lysosomes. Second, the accumulations attract the chaperone 14-3-3, which is responsible for the proper localization of the key autophagy regulator transcription factor EB (TFEB). We propose that this dual disruption of autophagy contributes to the pathogenesis of other neurodegenerative diseases involving neurofilament accumulations.
    Keywords:  Autophagy; Cell biology; Neurological disorders; Neuroscience; Ubiquitin-proteosome system
    DOI:  https://doi.org/10.1172/jci.insight.177999
  50. Stem Cell Reports. 2025 Feb 28. pii: S2213-6711(25)00052-9. [Epub ahead of print] 102448
    Metabolic remodeling in hiPSC-derived myofibers carrying the m.3243A>G mutation.
    Gabriel E Valdebenito, Anitta R Chacko, Chih-Yao Chung, Preethi Sheshadri, Haoyu Chi, Benjamin O'Callaghan, Monika J Madej, Henry Houlden, Hannah Rouse, Valle Morales, Katiuscia Bianchi, Francesco Saverio Tedesco, Robert D S Pitceathly, Michael R Duchen.
      Mutations in mitochondrial DNA cause severe multisystem disease frequently associated with muscle weakness. The m.3243A>G mutation is the major cause of mitochondrial encephalomyopathy lactic acidosis and stroke-like episodes (MELAS). Experimental models that recapitulate the disease phenotype in vitro for disease modeling or drug screening are very limited. We have therefore generated hiPSC-derived muscle fibers with variable heteroplasmic mtDNA mutation load without significantly affecting muscle differentiation potential. The cells exhibit physiological characteristics of muscle fibers and show a well-organized myofibrillar structure. In cells carrying the m.3243A>G mutation, the mitochondrial membrane potential and oxygen consumption were reduced in relation to the mutant load. We have shown through proteomic, phosphoproteomic, and metabolomic analyses that the m.3243A>G mutation variably affects the cell phenotype in relation to the mutant load. This variation is reflected by an increase in the NADH/NAD+ ratio, which in turn influences key nutrient-sensing pathways in the myofibers. This model enables a detailed study of the impact of the mutation on cellular bioenergetics and on muscle physiology with the potential to provide a platform for drug screening.
    Keywords:  iPSC-derived myofibers; mitochondria; mtDNA; mtDNA mutations
    DOI:  https://doi.org/10.1016/j.stemcr.2025.102448
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