bims-mitdis Biomed News
on Mitochondrial disorders
Issue of 2024–11–10
sixty papers selected by
Catalina Vasilescu, Helmholz Munich



  1. Methods Enzymol. 2024 ;pii: S0076-6879(24)00408-7. [Epub ahead of print]707 501-517
      The mitochondrial import machinery is regulated by several protein kinases that phosphorylate key components. This allows an adjustment of the protein flux to changing cellular demands and allow a dynamic organellar proteome. PhosTag electrophoresis has been proven as highly valuably tool to study these signalling machanisms at the import machinery.
    Keywords:  TOM complex; assembly; biogenesis; import machinery; precursor protein; protein kinase; reversible phosphorylation; signalling
    DOI:  https://doi.org/10.1016/bs.mie.2024.07.063
  2. Methods Enzymol. 2024 ;pii: S0076-6879(24)00365-3. [Epub ahead of print]707 543-564
      The mitochondrial unfolded protein response (UPRmt) is a mitochondria-to-nuclear signaling pathway that mediates the transcription of genes required to maintain mitochondrial function during development as well as during aging. In this chapter, we describe the approaches and techniques that we and others have used to elucidate the mechanism(s) by which cells detect mitochondrial stress or dysfunction and communicate with the nucleus to induce transcription of a protective stress response. We also describe approaches to evaluate the impact of UPRmt activation on mitochondrial function and mitochondrial biogenesis including imaging-based approaches as well as approaches to evaluate mitochondrial genome (mtDNA) copy number.
    Keywords:  Deleterious mtDNA heteroplasmy; Mito-nuclear communication; Mitochondrial biogenesis; Mitochondrial unfolded protein response; Molecular chaperones; MtDNA replication
    DOI:  https://doi.org/10.1016/bs.mie.2024.07.029
  3. Methods Enzymol. 2024 ;pii: S0076-6879(24)00395-1. [Epub ahead of print]707 565-584
      The functionality of mitochondria depends on the import of proteins synthesized on cytosolic ribosomes. Impaired import into mitochondria results in mitochondrial dysfunction and proteotoxic accumulation of precursor proteins in the cytosol. All proteins sorted to inner mitochondrial compartments are imported via the translocase of the outer membrane (TOM) complex. Premature protein folding, a reduction of the mitochondrial membrane potential or defects in translocases can result in precursor arrest during translocation, thereby clogging the TOM channel and blocking protein import. In recent years, different pathways have been identified, which employ the cytosolic ubiquitin-proteasome system in the extraction and turnover of precursor proteins from the TOM complex. Central events in this process are the modification of arrested precursor proteins with ubiquitin, their extraction by AAA-ATPases and subsequent degradation by the 26 S proteasome. Analysis of these processes is largely facilitated by the expression of model proteins that function as efficient "cloggers" of the import machinery. Here we describe the use of such clogger proteins and how their handling by the protein quality control machinery can be monitored. We provide protocols to study the extent of clogging, the ubiquitin-modification of arrested precursor proteins and their turnover by the 26 S proteasome.
    Keywords:  Mitochondria; Proteasome; Protein import; Protein quality control; Ubiquitin
    DOI:  https://doi.org/10.1016/bs.mie.2024.07.050
  4. Proc Natl Acad Sci U S A. 2024 Nov 19. 121(47): e2414187121
      Mitochondrial biogenesis relies on both the nuclear and mitochondrial genomes, and imbalance in their expression can lead to inborn errors of metabolism, inflammation, and aging. Here, we investigate N6AMT1, a nucleo-cytosolic methyltransferase that exhibits genetic codependency with mitochondria. We determine transcriptional and translational profiles of N6AMT1 and report that it is required for the cytosolic translation of TRMT10C (MRPP1) and PRORP (MRPP3), two subunits of the mitochondrial RNAse P enzyme. In the absence of N6AMT1, or when its catalytic activity is abolished, RNA processing within mitochondria is impaired, leading to the accumulation of unprocessed and double-stranded RNA, thus preventing mitochondrial protein synthesis and oxidative phosphorylation, and leading to an immune response. Our work sheds light on the function of N6AMT1 in protein synthesis and highlights a cytosolic program required for proper mitochondrial biogenesis.
    Keywords:  OXPHOS; RNA processing; mitochondria; mitochondrial RNA granules; translation
    DOI:  https://doi.org/10.1073/pnas.2414187121
  5. Methods Enzymol. 2024 ;pii: S0076-6879(24)00387-2. [Epub ahead of print]707 173-208
      Mitochondria play multiple essential roles in eukaryotic cells. To perform their functions, mitochondria require an adequate supply of externally produced proteins and an intact two-membrane structure. The structure of mitochondrial membranes separates these organelles from their cytosolic environment, with proteins that make up the mitochondrial proteome either being embedded into or enveloped by these membranes. From the experimental point of view, the structural properties of mitochondria contribute to the relative ease of isolating these organelles from other cellular components. The ability to isolate intact mitochondria and analyze them in a well-controlled environment opens up the possibility of tracking any proteins that enter or escape the membrane-formed enclosure. This chapter discusses assays that monitor the movement of proteins out of mitochondria through intact membranes. These protocols provide insight into the mechanisms behind mitochondrial protein quality control. It was discovered that the retro-translocation of IMS proteins regulates the protein content of this specific sub-compartment of the organelle. Additionally, proteins can move out of the mitochondria to resolve failed import events. Assays based on isolated mitochondria precisely tackle such intricate 'dance' of proteins crossing mitochondrial membranes during import and export, maintaining the dynamics of the organellar proteome.
    Keywords:  Intermembrane space; MIA pathway; Mitochondria; Protease; Protein import; Protein quality control; Protein retro-translocation; Protein transport; Proteostasis
    DOI:  https://doi.org/10.1016/bs.mie.2024.07.047
  6. Methods Enzymol. 2024 ;pii: S0076-6879(24)00401-4. [Epub ahead of print]707 441-473
      The development of easy-to-use gene-editing approaches has revolutionized the study of mitochondrial protein complex assembly in mammalian cells. Once the domain of classical cell biology models such Saccharomyces cerevisiae, human knockout cell lines lacking expression of a specific protein can be made at low cost and in as little as two to three weeks. In this chapter we outline our approach to generation of knockouts in commonly used transformed laboratory cell lines, with a view toward their use in the study of mitochondrial respiratory chain complex assembly and mitochondrial biology. Common pitfalls and caveats are discussed along with recommendations on how to validate a knockout cell line through sequencing of genomic edits and stable complementation to exclude the influence of off-target effects and enable further studies of protein function.
    Keywords:  CRISPR; Cas9; complementation; gene-editing; knockout; mammalian cell line; rescue
    DOI:  https://doi.org/10.1016/bs.mie.2024.07.056
  7. Methods Enzymol. 2024 ;pii: S0076-6879(24)00400-2. [Epub ahead of print]707 39-62
      Mitochondria are surrounded by two membranes, the outer and inner membrane. The outer membrane contains a few dozen integral membrane proteins that mediate transport, fusion and fission processes, form contact sites and are involved in signaling pathways. There are two different types of outer membrane proteins. A few proteins are membrane-integrated by a transmembrane β-barrel, while other proteins are embedded by single or multiple α-helical membrane segments. All outer membrane proteins are produced on cytosolic ribosomes, but their import mechanisms differ. The translocase of the outer mitochondrial membrane (TOM complex) and the sorting and assembly machinery (SAM complex) import β-barrel proteins. Different import pathways have been reported for proteins with α-helical membrane anchors. The mitochondrial import (MIM) complex is the major insertase for this type of proteins. The in vitro import of radiolabeled precursor proteins into isolated mitochondria is a versatile technique to study protein import into the outer mitochondrial membrane. The import of these proteins does not involve proteolytic processing and is not dependent on the membrane potential. Therefore, the import assay has to be combined with blue native electrophoresis, carbonate extraction or protease accessibility assays to determine the import efficiency. These techniques allow to define import steps, assembly intermediates and study membrane integration. The in vitro import assay has been a central tool to uncover specific import routes and mechanisms.
    Keywords:  MIM complex; Mitochondria; Protein sorting; SAM complex; TOM complex
    DOI:  https://doi.org/10.1016/bs.mie.2024.07.055
  8. Methods Enzymol. 2024 ;pii: S0076-6879(24)00406-3. [Epub ahead of print]707 673-683
      Eukaryotic cells require energy to perform diverse cellular functions critical for survival. Mitochondria are multifunctional organelles that generate energy in the form of Adenosine triphosphate by oxidative phosphorylation, emphasizing their importance to eukaryotic cell viability. The ability of mitochondria to consume oxygen for respiration is a key parameter in assessing mitochondrial health. Therefore, developing new techniques to monitor mitochondrial respiration are crucial for advancing our understanding of organelle functioning. Recently, Seahorse technology has emerged as a valuable tool to analyze various aspects of mitochondrial bioenergetics. Although the Seahorse assay is well established in adherent cell lines and other model organisms, it remains challenging to employ it efficiently in yeast, a powerful genetic system for studying mitochondrial biology. In this chapter, we provide a comprehensive methodology for assessing oxygen consumption rate in baker's yeast using Seahorse.
    Keywords:  Mitochondrial respiration; Saccharomyces cerevisiae; Seahorse; electron transport chain; oxygen consumption rate
    DOI:  https://doi.org/10.1016/bs.mie.2024.07.061
  9. Sci Rep. 2024 11 05. 14(1): 26778
      This study evaluates long-read and short-read sequencing for mitochondrial DNA (mtDNA) heteroplasmy detection. 592,315 bootstrapped datasets generated from two single-nucleotide mismatched ultra-deep sequenced mtDNA samples were used to assess basecalling error and accuracy, limit of heteroplasmy detection, and heteroplasmy detection across various coverage depths. Results showed high Phred scores of data with GC-rich sequence bias for long reads. Limit of detection of 12% heteroplasmy was identified, showing strong correlation (R2 ≥ 0.955) with expected heteroplasmy but underreporting tendency of high-level variants. Nanopore sequencing shows potential for direct applicability in mitochondrial diseases diagnostics, but stringent validation processes to ensure diagnostic result quality are required.
    Keywords:  Heteroplasmy; Long-read sequencing; Mitochondrial disease; mtDNA
    DOI:  https://doi.org/10.1038/s41598-024-78270-0
  10. Methods Enzymol. 2024 ;pii: S0076-6879(24)00382-3. [Epub ahead of print]707 3-22
      Mitochondria import the vast majority of proteins from the cytosol. Protein translocation machineries in outer and inner membranes facilitate precursor recognition and transport. Most mitochondrial proteins utilize N-terminal presequences as targeting signals that eventually direct them across the inner mitochondrial membrane. These precursors are transported by the TOM complex across the outer-, and subsequently by the TIM23 complex across the inner membrane. During this process the translocases align and the polypeptide chain is translocated across both membranes in a coupled manner. A transient precursor-containing TOM-TIM23 supercomplex is formed. This TOM-TIM23 supercomplex provides a fascinating import intermediate which can be stabilized if the precursor contains a tightly folded moiety at the C-terminus that is not able to pass through the TOM complex. Such a supercomplex can be generated during in vitro import, and in vivo. The stabilized TOM-TIM23 supercomplex can be purified for downstream analysis. The possibility of pausing translocation at this step provides a means to understand the mechanisms underlying precursor translocation.
    Keywords:  Import; Mitochondria; Supercomplex; TIM23; TOM
    DOI:  https://doi.org/10.1016/bs.mie.2024.07.042
  11. Methods Enzymol. 2024 ;pii: S0076-6879(24)00398-7. [Epub ahead of print]707 329-366
      Mitochondrial protein import and sorting relies on sophisticated molecular machineries or translocases, of which channels are integral. Channels are built upon membrane proteins whose functions are driven by conformational changes. This implies that structural and functional information need to be integrated to gain a deep understanding of their dynamic behavior. Patch-clamp approaches are well suited for this purpose. This chapter provides a detailed description and practical guidance for applying the patch-clamp methodology to the electrophysiological characterization of mitochondrial protein import. Implementing the technique to intact mitochondria, mitoplasts, and reconstituted proteoliposomes, combined with genetically modified yeast strains, expands the scope of these studies. Focused on the TOM, TIM23, and TIM22 translocases, an analysis of the patch-clamp contribution to the field is outlined.
    Keywords:  Mitochondria; Mitochondrial protein import; Patch-clamp; Protein import channels; TIM22 translocase; TIM23 translocase; TOM translocase
    DOI:  https://doi.org/10.1016/bs.mie.2024.07.053
  12. Curr Biol. 2024 Nov 04. pii: S0960-9822(24)01346-0. [Epub ahead of print]34(21): R1067-R1068
      Eve Kakudji and Samantha Lewis discuss the structure and function of mitochondrial nucleoids - large nucleoprotein complexes containing mitochondrial DNA and the regulatory factors necessary for its packaging, replication, transcription, and repair.
    DOI:  https://doi.org/10.1016/j.cub.2024.09.078
  13. Methods Enzymol. 2024 ;pii: S0076-6879(24)00388-4. [Epub ahead of print]707 475-498
      Deficits of mitochondrial functions have been identified in many human pathologies, in particular in age-related human neurodegenerative diseases. Hence, the molecular causes for mitochondrial dysfunction and potential protection mechanisms have become a major topic in modern cell biology. Apart from defects in their structural integrity, problems in mitochondrial protein biogenesis, including polypeptide transport, folding and assembly to active enzymes, all may result in some degree of functional defects of the organelle. An accumulation of misfolded polypeptides inside mitochondria, confounded by the dual source of mitochondrial polypeptides, will result in the formation of protein aggregates. Such aggregate accumulation bears a cell-toxic potential, resulting in mitochondrial and correlated cellular damages, summarized in the term "aggregate proteotoxicity". Here, we discuss methods to analyze protein aggregation in the mitochondrial matrix compartment. We also address techniques to characterize the biochemical mechanisms that reduce aggregate proteotoxicity, the disaggregation or resolubilization of aggregated polypeptides and the sequestration and neutralization of mitochondrial aggregates at specific sites inside a cell.
    Keywords:  ATP-dependent proteases; Human cells; Mitochondria; Molecular chaperones; Protein aggregation; Proteotoxicity; Yeast
    DOI:  https://doi.org/10.1016/bs.mie.2024.07.048
  14. Methods Enzymol. 2024 ;pii: S0076-6879(24)00404-X. [Epub ahead of print]707 101-152
      The multiple functions of mitochondria are governed by their proteome comprising 1000-1500 proteins depending on the organism. However, only few proteins are synthesized inside mitochondria, whereas most are "born" outside mitochondria. To reach their destined location, these mitochondrial proteins follow specific import routes established by a mitochondrial translocase network. A detailed understanding of the role and interplay of the different translocases is imperative to understand mitochondrial biology and how mitochondria are integrated into the cellular network. Mass spectrometry (MS) proved to be effective to study the translocase network regarding composition, functions, interplay, and cellular responses evoked by dysfunction. In this chapter, we provide protocols tailored to MS-enabled functional analysis of mutants and interactomes of mitochondrial translocation proteins. In the first part, we exemplify the MS-based proteomics analysis of translocation mutants for delineating the human mitochondrial importome following depletion of the central translocation protein TOMM40. The protocol comprises metabolic stable isotope labeling, TOMM40 knockdown, preparation of mitochondrial fractions, and sample preparation for liquid chromatography (LC)-MS. For deep MS analysis, prefractionation of peptide mixtures by high pH reversed-phase LC is described. In the second part, we outline an affinity purification MS approach to reveal the association of an orphaned protein with the translocase TIM23. The protocol covers FLAG-tag affinity purification of protein complexes from mitochondrial fractions and downstream sample preparation for interactome analysis. In the last unifying part, we describe methods for LC-MS, data processing, statistical analysis and visualization of quantitative MS data, and provide a Python code for effective, customizable analysis.
    Keywords:  Affinity-purification mass spectrometry; Data analysis; Importome; Interactome; Mass spectrometry; Mitochondria; Protein quantification; Proteomics; SILAC; Translocation mutants
    DOI:  https://doi.org/10.1016/bs.mie.2024.07.059
  15. Cell Calcium. 2024 Oct 23. pii: S0143-4160(24)00120-9. [Epub ahead of print]124 102962
      In a recent publication, Hernansanz-Agusti̒n et al. propose that a sodium gradient across the inner mitochondrial membrane, generated by a Na+/H+ activity integral to Complex I can account for half of the mitochondrial membrane potential. This conflicts with conventional electrophysiological and chemiosmotic understanding.
    Keywords:  Calcium signaling; Goldman equation; Membrane potential; Mitochondria; Sodium proton exchange
    DOI:  https://doi.org/10.1016/j.ceca.2024.102962
  16. Methods Enzymol. 2024 ;pii: S0076-6879(24)00397-5. [Epub ahead of print]707 299-327
      The mitochondrial translocases of the outer membrane (TOM) and of the inner membrane (TIM) act together to facilitate the import of nuclear-encoded proteins across the mitochondrial membranes. Stimulated Emission Depletion (STED) super-resolution microscopy enables the in situ imaging of such complexes in single cells at sub-diffraction resolution. STED microscopy requires only conventional sample preparation techniques and provides super-resolved raw data without the need for further image processing. In this chapter, we provide a detailed example protocol for STED microscopy of TOM20 and mitochondrial DNA in fixed mammalian cells. The protocol includes instructions on sample preparation for immunolabeling, including cell line selection, fixation, permeabilization, blocking, labeling and mounting, but also recommendations for sample and microscope performance evaluation. The protocol is supplemented by considerations on key factors that influence the quality of the final image and also includes some considerations for the analysis of the acquired images. While the protocol described here is aimed at imaging TOM20 and DNA, it contains all the information for an immediate adaptation to other cellular targets.
    DOI:  https://doi.org/10.1016/bs.mie.2024.07.052
  17. Nat Commun. 2024 Nov 01. 15(1): 9438
      Mitochondria serve as the cellular powerhouse, and their distinct DNA makes them a prospective target for gene editing to treat genetic disorders. However, the impact of genome editing on mitochondrial DNA (mtDNA) stability remains a mystery. Our study reveals previously unknown risks of genome editing that both nuclear and mitochondrial editing cause discernible transfer of mitochondrial DNA segments into the nuclear genome in various cell types including human cell lines, primary T cells, and mouse embryos. Furthermore, drug-induced mitochondrial stresses and mtDNA breaks exacerbate this transfer of mtDNA into the nuclear genome. Notably, we observe that mitochondrial editors, including mitoTALEN and recently developed base editor DdCBE, can also enhance crosstalk between mtDNA and the nuclear genome. Moreover, we provide a practical solution by co-expressing TREX1 or TREX2 exonucleases during DdCBE editing. These findings imply genome instability of mitochondria during induced DNA breaks and explain the origins of mitochondrial-nuclear DNA segments.
    DOI:  https://doi.org/10.1038/s41467-024-53806-0
  18. Methods Enzymol. 2024 ;pii: S0076-6879(24)00399-9. [Epub ahead of print]707 63-99
      Mitochondrial function is dependent on the correct localization and insertion of membrane proteins into the outer mitochondrial membrane (OM). In mammals, the OM contains ∼150 proteins, the majority of which contain α-helical transmembrane domains. This family of α-helical proteins has significantly expanded in metazoans and has evolved to mediate critical signaling and regulatory processes including mitochondrial fusion and fission, mitophagy, apoptosis and aspects of the innate immune response. Recently, the conserved OM protein MTCH2 has been identified as an insertase for α-helical proteins in human mitochondria. However, our understanding of the targeting, insertion, folding and quality control of α-helical OM proteins remains incomplete. Here we highlight three methods to monitor α-helical protein insertion both in human cells and in vitro. First, we describe a versatile split fluorescent reporter system that can be used to monitor the insertion of α-helical proteins into the OM in human cells. Second, we delineate a streamlined approach to isolating functional, insertion competent mitochondria from human cells that are compatible with in vitro import assays. Finally, we explain in detail how to reconstitute the insertion of α-helical proteins in a minimal system, by creating functional proteoliposomes containing purified MTCH2. Together these tools represent an integrated platform to enable the detailed mechanistic analysis of biogenesis of the diverse and physiologically essential α-helical OM proteome.
    Keywords:  Insertion assay; MTCH2; Mitochondria; Outer mitochondrial membrane; Proteoliposomes; Split fluorescent reporter; α-helical membrane protein
    DOI:  https://doi.org/10.1016/bs.mie.2024.07.054
  19. Methods Enzymol. 2024 ;pii: S0076-6879(24)00402-6. [Epub ahead of print]707 257-298
      Protein translocation is a highly dynamic process and, in addition, mitochondrial protein import is especially complicated as the majority of nuclear encoded precursor proteins must engage with multiple translocases before they are assembled in the correct mitochondrial subcompartment. In this chapter, we describe assays for engineered disulfide bond formation and cysteine specific crosslinking to analyze the rearrangement of translocase subunits or to probe protein-protein interactions between precursor proteins and translocase subunits. Such assays were used to characterize the translocase of the outer membrane, the presequence translocase of the inner membrane and the sorting and assembly machinery for the biogenesis of β-Barrel proteins. Moreover, these approaches were also employed to determine the translocation path of precursor proteins (identification of import receptors and specific domains required for translocation) as well as the analysis, location and translocase subunit dependence for the formation of β-Barrel proteins. Here we describe how engineered disulfide bond formation and cysteine specific crosslinking assays are planned and performed and discuss important aspects for its application to study mitochondrial protein translocation.
    Keywords:  Mitochondria; Precursor protein; Protein biogenesis; Protein import; Protein-protein interaction; SAM complex; TIM complex; TOM complex; Translocase
    DOI:  https://doi.org/10.1016/bs.mie.2024.07.057
  20. Mol Genet Metab. 2023 Oct 20. pii: S1096-7192(23)00341-4. [Epub ahead of print] 107711
      Fatty acid oxidation disorders (FAOD) are inborn errors of metabolism that occur due to deficiency of specific enzyme activities and transporter proteins involved in the mitochondrial metabolism of fatty acids, causing a deficiency in ATP production. The identification of suitable biomarkers plays a crucial role in predicting the future risk of disease and monitoring responses to therapies. Acyl-CoAs are directly involved in the steps of fatty acid oxidation and are the primary biomarkers associated with FAOD. However, acyl-CoAs are not used as diagnostic biomarkers in hospitals and clinics as they are present intracellularly with low endogenous levels. Additionally, the analytical method development of acyl-CoAs is quite challenging due to diverse physicochemical properties and instability. Hence, secondary biomarkers such as acylcarnitines are used for the identification of FAOD. In this review, the focus is on the analytical techniques that have evolved over the years for the identification and quantitation of acyl-CoAs. Among these techniques, liquid chromatography-mass spectrometry clearly has an advantage in terms of sensitivity and selectivity. Stable isotope labeling by essential nutrients in cell culture (SILEC) enables the generation of labeled internal standards. Each acyl-CoA species has a distinct pattern of instability and degradation, and the use of appropriately matched internal standards can compensate for such issues. Although significant progress has been made in measuring acyl-CoAs, more efforts are needed for bringing these technical advancements to hospitals and clinics. This review also highlights the difficulties involved in the routine use of acyl-CoAs as a diagnostic biomarker and some of the measures that can be adopted by clinics and hospitals for overcoming these limitations.
    Keywords:  Acyl-CoA; Biomarkers; Fatty acid oxidation disorders; LC-MS; Newborn screening; SILEC
    DOI:  https://doi.org/10.1016/j.ymgme.2023.107711
  21. J Pediatr Genet. 2024 Dec;13(4): 253-257
      The mitochondrial DNA (mtDNA) variant m.13513G > A is increasingly recognized as a cause of syndromic and nonsyndromic mitochondrial disorders (MIDs). This minireview aims a summarizing and discussing recent and previous findings about the phenotypic heterogeneity of this variant. A systematic literature review using the databases PubMed and Google Scholar by application of specific search terms was performed. As per the end of July 2021, at least 50 patients carrying the mtDNA variant m.13513G > A have been reported. Age ranged between 0 and 63 years, and of these patients, 28 were male and 22 were female. The phenotype was highly variable. The most common phenotypes were Leigh syndrome (LS; n  = 25), mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS; n  = 11), Leigh-like syndrome ( n  = 4), MELAS/LS ( n  = 3), progressive external ophthalmoplegia ( n  = 3), MELAS/Leber's hereditary optic neuropathy (LHON; n  = 1), and LHON ( n  = 1). More rarely are nonsyndromic phenotypes. Heteroplasmy rates were highly variable ranging from 0 to 86% depending on the investigated tissue. The outcome was reported in only a few cases but was worse in patients with LS compared with those with MELAS. The variant m.13513G > A is responsible for syndromic or nonsyndromic MIDs. Syndromic MIDs in which this variant should be particularly considered include LS, MELAS, and LHON and their overlaps. Patients with suspected MID and maternal inheritance should undergo sequencing of the entire mtDNA not to miss rare mtDNA variants.
    Keywords:  Leigh's syndrome; MELAS; heteroplasmy; m.13513G > A; mtDNA
    DOI:  https://doi.org/10.1055/s-0043-1768474
  22. Sci Rep. 2024 11 01. 14(1): 26324
      Glaucoma is a sight threatening neurodegenerative condition of the optic nerve head associated with ageing and marked by the loss of retinal ganglion cells. Mitochondrial dysfunction plays a crucial role in the pathogenesis of neurodegeneration in the most prevalent type of glaucoma: primary open angle glaucoma (POAG). All previous mitochondrial genome sequencing studies in POAG analyzed mitochondrial DNA (mtDNA) isolated from peripheral blood leukocytes and have not evaluated cells derived from ocular tissue, which better represent the glaucomatous disease context. In this study, we evaluated mitochondrial genome variation and heteroplasmy using massively parallel sequencing of mtDNA in a cohort of patients with POAG, and in a subset assess the role of somatic mitochondrial genome mutations in disease pathogenesis using paired samples of peripheral blood leukocytes and ocular tissue (Tenon's ocular fibroblasts). An enrichment of potentially pathogenic nonsynonymous mtDNA variants was identified in Tenon's ocular fibroblasts from participants with POAG. The absence of oxidative DNA damage and predominance of transition variants support the concept that errors in mtDNA replication represent the predominant mutation mechanism in Tenon's ocular fibroblasts from patients with POAG. Pathogenic somatic mitochondrial genome mutations were observed in people with POAG. This supports the role of somatic mitochondrial genome variants in the etiology of glaucoma.
    Keywords:  Glaucoma; Massively parallel sequencing; Mitochondrial DNA; Mitochondrial genome; Somatic mutations; Tenon’s fibroblasts
    DOI:  https://doi.org/10.1038/s41598-024-72684-6
  23. Brain Res Bull. 2024 Nov 01. pii: S0361-9230(24)00248-X. [Epub ahead of print]218 111114
      Nicotinamide phosphoribosyltransferase (NAMPT) is the key enzyme in the salvaging synthesize pathway of nicotinamide adenine dinucleotide (NAD). The neuroprotective roles of NAMPT on neurodegeneration have been explored in aging brain and Alzheimer's Disease. However, its roles in Parkinson's Disease (PD) remain to be elucidated. We found that the dopaminergic neurons in substantia nigra expressed higher levels of NAMPT than the other types of neurons. Using conditional knockout of the Nampt gene in dopaminergic neurons and utilizing a NAMPT inhibitor in the substantia nigra of mice, we found that the NAMPT deficiency triggered the time-dependent loss of dopaminergic neurons, the impairment of the dopamine nigrostriatal pathway, and the development of PD-like motor dysfunction. In the rotenone-induced PD mouse model, nicotinamide ribose (NR), a precursor of NAD, rescued the loss of dopaminergic neurons, the impairment of dopamine nigrostriatal pathway, and mitigated PD-like motor dysfunction. In SH-SY5Y cells, NAD suppression induced the accumulation of reactive oxygen species (ROS), mitochondrial impairment, and cell death, which was reversed by N-acetyl cysteine, an antioxidant and ROS scavenger. Rotenone decreased NAD level, induced the accumulation of ROS and the impairment of mitochondria, which was reversed by NR. In summary, our findings show that the ablation of NAMPT in dopaminergic neurons leads to neurodegeneration and contributes to the development of PD. The NAD precursors have the potential to protect the degeneration of dopaminergic neurons, and offering a therapeutic approach for the treatment of PD.
    Keywords:  Mitochondria; Nicotinamide adenine dinucleotide (NAD); Nicotinamide phosphoribosyltransferase (NAMPT); Nicotinamide ribose (NR); Oxidative stress; Parkinson’s disease (PD)
    DOI:  https://doi.org/10.1016/j.brainresbull.2024.111114
  24. Nature. 2024 Nov 06.
      Mitochondria serve a crucial role in cell growth and proliferation by supporting both ATP synthesis and the production of macromolecular precursors. Whereas oxidative phosphorylation (OXPHOS) depends mainly on the oxidation of intermediates from the tricarboxylic acid cycle, the mitochondrial production of proline and ornithine relies on reductive synthesis1. How these competing metabolic pathways take place in the same organelle is not clear. Here we show that when cellular dependence on OXPHOS increases, pyrroline-5-carboxylate synthase (P5CS)-the rate-limiting enzyme in the reductive synthesis of proline and ornithine-becomes sequestered in a subset of mitochondria that lack cristae and ATP synthase. This sequestration is driven by both the intrinsic ability of P5CS to form filaments and the mitochondrial fusion and fission cycle. Disruption of mitochondrial dynamics, by impeding mitofusin-mediated fusion or dynamin-like-protein-1-mediated fission, impairs the separation of P5CS-containing mitochondria from mitochondria that are enriched in cristae and ATP synthase. Failure to segregate these metabolic pathways through mitochondrial fusion and fission results in cells either sacrificing the capacity for OXPHOS while sustaining the reductive synthesis of proline, or foregoing proline synthesis while preserving adaptive OXPHOS. These findings provide evidence of the key role of mitochondrial fission and fusion in maintaining both oxidative and reductive biosyntheses in response to changing nutrient availability and bioenergetic demand.
    DOI:  https://doi.org/10.1038/s41586-024-08146-w
  25. Methods Enzymol. 2024 ;pii: S0076-6879(24)00405-1. [Epub ahead of print]707 519-539
      Of all the causes of metabolic and neurological disorders, oxidative stress distinguishes itself by its sweeping effect on the dynamic cellular redox homeostasis and, in its wake, exposing the vulnerabilities of the protein machinery of the cell. High levels of Reactive Oxygen Species (ROS) that mitochondria produce during ATP synthesis can damage mtDNA, lipids, and essential mitochondrial proteins. ROS majorly oxidizes cysteine and methionine amino acids in peptides, which can lead to protein unfolding or misfolding of proteins, which ultimately can have a toll on their function. As mitochondrial biogenesis relies on the continuous import of nuclear-encoded proteins into mitochondria mediated by mitochondrial protein import complexes, oxidative stress triggered by mitochondria can rapidly and detrimentally affect mitochondrial biogenesis and homeostasis. Functional Mge1 is a homodimer and acts as a cochaperone and a nucleotide exchange factor of mitochondrial heat shock protein 70 (mHsp70), crucial for mitochondrial protein import. Oxidative stress like ROS, oxidizes Met 155 in Mge1, compromising its ability to dimerize and interact with mHsp70. The cell employs Methionine sulphoxide reductase 2 (Mxr2), a member of the methionine sulphoxide reductase family, to reduce oxidized Met 155 and thereby restore the essential function of Mge1. Oxidation of methionine as a regulated post-translational modification has been gaining traction. Future high throughput studies that can scan the entire mitochondrial proteome to interrogate methionine oxidation and reversal may increase the repertoire of mitochondrial proteins undergoing regulated oxidation and reduction. In this chapter, we describe the methods followed in our laboratory to study the oxidation of Mge1 and its reduction by Mxr2 in vitro.
    Keywords:  Cross linking; Methionine oxidation; Methionine sulfoixde reductase 2; Mge1; Mitochondria; Reactive Oxygen Species
    DOI:  https://doi.org/10.1016/bs.mie.2024.07.060
  26. Methods Enzymol. 2024 ;pii: S0076-6879(24)00381-1. [Epub ahead of print]707 153-171
      Precise protein localization is essential for normal cellular functions. However, recent studies have revealed that protein targeting is error-prone, and tail-anchored proteins mistargeted to mitochondria are transferred to the endoplasmic reticulum (ER) by an ATPase Msp1 (yeast)/ATAD1 (human) in the mitochondrial outer membrane for further quality examination in the ER to determine their fate, degradation or re-targeting. Analysis of the inter-organelle transfer of proteins requires a combination of time-lapse fluorescence microscopy and a system to achieve regulation of the protein levels of both transfer substrates and factors regulating the transfer in a coordinated manner at precise timing. This can be achieved by using a promoter switch for expression and acute depletion of involved factors through the degron-based proteasome system. In this chapter, we will describe methods to analyze inter-organelle protein transfer by fluorescence microscope within living yeast cells, by using the example of Msp1-mediated transfer of mistargeted proteins from mitochondria to the ER.
    Keywords:  AID degradation system; Endoplasmic reticulum; Fluorescence microscope; GET system; Inter-organelle protein transfer; Mitochondria; Msp1; Tail-anchored protein
    DOI:  https://doi.org/10.1016/bs.mie.2024.07.041
  27. Methods Enzymol. 2024 ;pii: S0076-6879(24)00403-8. [Epub ahead of print]707 23-38
      The translocase of the mitochondrial inner membrane (TIM23) complex mediates the import and membrane insertion of presequence-carrying mitochondrial proteins. It is experimentally challenging to determine whether the segment of the polypeptide is imported to the matrix or inserted into the inner membrane. Utilizing the unique topogenesis of Mgm1p, a versatile experimental approach to study the TIM23-mediated membrane insertion is developed and described in this chapter. This method combines a simple SDS-gel based assay with the quantification of the relative fractions of membrane inserted and non-inserted products, enabling the quantitative measurement of the membrane insertion efficiencies of a transmembrane segment into the mitochondrial inner membrane.
    Keywords:  Mgm1; TIM17; TIM23; inner membrane protein; membrane insertion
    DOI:  https://doi.org/10.1016/bs.mie.2024.07.058
  28. Adv Sci (Weinh). 2024 Nov 03. e2405147
      Oxidative phosphorylation (OXPHOS) and fatty acid oxidation (FAO) are key bioenergetics pathways. The machineries for both processes are localized in mitochondria. Secondary OXPHOS defects have been documented in patients with primary FAO deficiencies, and vice versa. However, the underlying mechanisms remain unclear. Intrigued by the observations that regulation of supercomplexes (SCs) assembly in a mouse OXPHOS deficient cell line and its derivatives is associated with the changes in lipid metabolism, a proteomics analysis is carried out and identified mitochondrial trifunctional protein (MTP) subunit alpha (hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit alpha, HADHA) as a potential regulatory factor for SCs assembly. HADHA-Knockdown cells and mouse embryonic fibroblasts (MEFs) derived from HADHA-Knockout mice displayed both reduced SCs assembly and defective OXPHOS. Stimulation of OXPHOS induced in cell culture by replacing glucose with galactose and of lipid metabolism in mice with a high-fat diet (HFD) both exhibited increased HADHA expression. HADHA Heterozygous mice fed with HFD showed enhanced steatosis associated with a reduction of SCs assembly and OXPHOS function. The results indicate that HADHA participates in SCs assembly and couples FAO and OXPHOS.
    Keywords:  HADHA; fatty acid oxidation (FAO); mitochondrial respiratory chain; mitochondrial trifunctional protein (MTP); respiratory complex I
    DOI:  https://doi.org/10.1002/advs.202405147
  29. Methods Enzymol. 2024 ;pii: S0076-6879(24)00425-7. [Epub ahead of print]707 585-610
      Mitochondria are critical for cellular function in health, disease and aging. Mitochondria-associated degradation (MAD), a pathway for quality control of the organelle, recognizes and ubiquitinates unfolded mitochondrial proteins, removes them from the organelle using a conserved segregase complex, which contains an AAA-ATPase Cdc48 and its cofactors, and degrades them using the ubiquitin-proteasome system (UPS). Here, we describe an approach to (1) study the turnover and ubiquitination of candidate MAD substrates, (2) assay retrotranslocation and export of MAD substrates from the mitochondrial matrix in vitro, and (3) study interactions between MAD substrates and Cdc48 using the budding yeast, Saccharomyces cerevisiae, as a model organism.
    Keywords:  Affinity purification; Budding yeast; Co-immunoprecipitation; Mitochondrial isolation; Mitochondrial quality control
    DOI:  https://doi.org/10.1016/bs.mie.2024.09.001
  30. Methods Enzymol. 2024 ;pii: S0076-6879(24)00380-X. [Epub ahead of print]707 611-634
      Yeast deletion mutants of crucial genes are often associated with a number of secondary defects, which hamper the analysis of primary protein function. Therefore, temperature-sensitive mutants are valuable tools to evaluate protein function in a focused and often reversible manner. However, temperature-sensitive mutants are uncommon for non-essential genes that nevertheless may have strong defects. Here we describe a screening method for generating temperature-sensitive mutants of non-essential genes in synthetic lethal backgrounds of Saccharomyces cerevisiae. As proof of principle, we describe a successful screen for the yeast mitochondrial inner membrane protease iAAA subunit Yme1 utilizing two screening approaches: a random mutagenesis and rational design approach. We then describe how candidate temperature-sensitive mutants are validated.
    Keywords:  Mitochondria; Protease; Proteolysis; Saccharomyces cerevisiae; Temperature-sensitive screen; Yeast; Yme1; i-AAA
    DOI:  https://doi.org/10.1016/bs.mie.2024.07.040
  31. Methods Enzymol. 2024 ;pii: S0076-6879(24)00407-5. [Epub ahead of print]707 237-256
      The BPA photo-crosslinking method exploits the property of p-benzoyl-L-phenylalanine (pBpa), an amino acid containing a photoreactive side chain, and allows for the crosslinking with nearby proteins upon Ultraviolet irradiation. This feature enables the capture of two proteins within a close proximity with high spatial resolution at the level of amino acid residues. In this chapter, we introduce an example of the employment of the BPA photo-crosslinking method to the Translocase of the Outer Mitochondrial membrane complex of mitochondria in Saccharomyces cerevisiae as a model protein translocase. Here in, we provide three procedures (i) the introduction of pBpa into proteins of interest in living yeast cells by in vivo suppressor tRNA system; (ii) analysis of in vivo subunit-subunit interactions intra-complex; and (iii) analysis of translocase channel-substrate interactions in organello. The use of in vivo and in organello crosslinking tools enable the robust analysis of translocases in a near-to physiological condition.
    Keywords:  Mitochondrial protein import; Photo-crosslink; Protein-protein interaction; TOM complex; Tom40; Unnatural amino acid
    DOI:  https://doi.org/10.1016/bs.mie.2024.07.062
  32. Brain Commun. 2024 ;6(6): fcae361
      Hearing impairment is a frequent clinical feature in patients with mitochondrial disease harbouring the pathogenic variant, m.3243A>G. However, auditory neural dysfunction, its perceptual consequences and implications for patient management are not established. Similarly, the association with vestibular impairment has not yet been explored. This case-control study investigated in 12 adults with genetically confirmed m.3243A>G adults [9 females; 45.5 ± 16.3 years (range 18-66); 47.1 ± 21.5 hearing level, dB] compared with 12 age, sex and hearing level-matched controls with sensory (cochlear level) hearing loss [9 females; 46.6 ± 11.8 years (range 23-59); 47.7 ± 25.4 hearing level, dB]. Participants underwent a battery of electroacoustic, electrophysiologic and perceptual tests, which included pure tone audiometry, otoacoustic emissions, auditory brainstem responses, auditory temporal processing measures, monaural/binaural speech perception, balance and vestibular testing and self-reported questionnaires (dizziness and hearing disability). Our findings showed evidence of auditory neural abnormality and perceptual deficits greater than expected for cochlear pathology. Compared with matched controls with sensory hearing loss, adults with mitochondrial disease harbouring m.3243A>G had abnormal electrophysiologic responses from the VIII nerve and auditory brainstem (P = 0.005), an impaired capacity to encode rapidly occurring acoustic signal changes (P = 0.005), a reduced ability to localize sound sources (P = 0.028) and impaired speech perception in background noise (P = 0.008). Additionally, vestibular dysfunction (P = 0.011), greater perceived dizziness (P = 0.001) and reduced stance time (balance, P = 0.009) were also seen in participants with m.3243A>G mitochondrial disease when compared with matched counterparts. This pilot study revealed that auditory evaluation including evoked potential responses from the auditory nerve/brainstem and speech perception in noise tests should form an important part of the management for individuals with m.3243A>G-related mitochondrial disease. Those presenting with hearing impairment and symptoms concerning balance and dizziness should undergo vestibular testing and appropriate management.
    Keywords:  auditory neuropathy; hearing impairment; m.3243A>G mitochondrial disease; speech perception; vestibular dysfunction
    DOI:  https://doi.org/10.1093/braincomms/fcae361
  33. Biomed Pharmacother. 2024 Nov 04. pii: S0753-3322(24)01391-X. [Epub ahead of print]181 117505
      Skeletal system-related diseases, such as osteoporosis, arthritis, osteosarcoma and sarcopenia, are becoming major public health concerns. These diseases are characterized by insidious progression, which seriously threatens patients' health and quality of life. Early diagnosis and prevention in high-risk populations can effectively prevent the deterioration of these patients. Mitochondria are essential organelles for maintaining the physiological activity of the skeletal system. Mitochondrial functions include contributing to the energy supply, modulating the Ca2+ concentration, maintaining redox balance and resisting the inflammatory response. They participate in the regulation of cellular behaviors and the responses of osteoblasts, osteoclasts, chondrocytes and myocytes to external stimuli. In this review, we describe the pathogenesis of skeletal system diseases, focusing on mitochondrial function. In addition to osteosarcoma, a characteristic of which is active mitochondrial metabolism, mitochondrial damage occurs during the development of other diseases. Impairment of mitochondria leads to an imbalance in osteogenesis and osteoclastogenesis in osteoporosis, cartilage degeneration and inflammatory infiltration in arthritis, and muscle atrophy and excitationcontraction coupling blockade in sarcopenia. Overactive mitochondrial metabolism promotes the proliferation and migration of osteosarcoma cells. The copy number of mitochondrial DNA and mitochondria-derived peptides can be potential biomarkers for the diagnosis of these disorders. High-risk factor detection combined with mitochondrial component detection contributes to the early detection of these diseases. Targeted mitochondrial intervention is an effective method for treating these patients. We analyzed skeletal system-related diseases from the perspective of mitochondria and provided new insights for their diagnosis, prevention and treatment by demonstrating the relationship between mitochondria and the skeletal system.
    Keywords:  Mitochondria; Mitophagy; ROS; Skeletal system; TCA
    DOI:  https://doi.org/10.1016/j.biopha.2024.117505
  34. Pharmacol Res. 2024 Oct 30. pii: S1043-6618(24)00429-8. [Epub ahead of print] 107484
      Loss of proteostasis is well documented during physiological aging and depends on the progressive decline in the activity of two major degradative mechanisms: the ubiquitin-proteasome system (UPS) and the autophagy-lysosomal pathway. This decline in proteostasis is exacerbated in age-associated neurodegenerative diseases, such as Parkinson's Disease (PD). In PD, patients develop an accumulation of aggregated proteins and dysfunctional mitochondria, which leads to ROS production, neuroinflammation and neurodegeneration. We recently reported that inhibition of the deubiquitinating enzyme USP14, which is known to enhance both the UPS and autophagy, increases lifespan and rescues the pathological phenotype of two Drosophila models of PD. Studies on the effects of USP14 inhibition in mammalian neurons have not yet been conducted. To close this gap, we exploited iNeurons differentiated from human embryonic stem cells (hESCs), and investigated the effect of inhibiting USP14 in these cultured neurons. Quantitative global proteomics analysis performed following genetic ablation or pharmacological inhibition of USP14 demonstrated that USP14 loss of function specifically promotes mitochondrial autophagy in iNeurons. Biochemical and imaging data also showed that USP14 inhibition enhances mitophagy. The mitophagic effect of USP14 inhibition proved to be PINK1/Parkin- independent, instead relying on expression of the mitochondrial E3 Ubiquitin Ligase MITOL/MARCH5. Notably, USP14 inhibition normalized the mitochondrial defects of Parkin KO human neurons.
    Keywords:  Autophagy; MARCH5/MITOL; Mitophagy; PINK1; Parkin; UPS; USP14
    DOI:  https://doi.org/10.1016/j.phrs.2024.107484
  35. Hum Genomics. 2024 11 05. 18(1): 120
    Omics Sciences - Bioinformatics and Epigenetics Working Groups of the Italian Society of Human Genetics (SIGU)
      This comprehensive review provides insights and suggested strategies for the analysis of germline variants using second- and third-generation sequencing technologies (SGS and TGS). It addresses the critical stages of data processing, starting from alignment and preprocessing to quality control, variant calling, and the removal of artifacts. The document emphasized the importance of meticulous data handling, highlighting advanced methodologies for annotating variants and identifying structural variations and methylated DNA sites. Special attention is given to the inspection of problematic variants, a step that is crucial for ensuring the accuracy of the analysis, particularly in clinical settings where genetic diagnostics can inform patient care. Additionally, the document covers the use of various bioinformatics tools and software that enhance the precision and reliability of these analyses. It outlines best practices for the annotation of variants, including considerations for problematic genetic alterations such as those in the human leukocyte antigen region, runs of homozygosity, and mitochondrial DNA alterations. The document also explores the complexities associated with identifying structural variants and copy number variations, underscoring the challenges posed by these large-scale genomic alterations. The objective is to offer a comprehensive framework for researchers and clinicians, ensuring that genetic analyses conducted with SGS and TGS are both accurate and reproducible. By following these best practices, the document aims to increase the diagnostic accuracy for hereditary diseases, facilitating early diagnosis, prevention, and personalized treatment strategies. This review serves as a valuable resource for both novices and experts in the field, providing insights into the latest advancements and methodologies in genetic analysis. It also aims to encourage the adoption of these practices in diverse research and clinical contexts, promoting consistency and reliability across studies.
    Keywords:  Bioinformatics; DNA methylation; Genetic diagnostics; Germline variants; Hereditary diseases; NGS
    DOI:  https://doi.org/10.1186/s40246-024-00684-8
  36. Methods Enzymol. 2024 ;pii: S0076-6879(24)00383-5. [Epub ahead of print]707 367-388
      Recent developments in cryo-electron microscopy techniques have facilitated intensive research into determining protein structures. Nevertheless, the structures of some mitochondrial membrane protein complexes remain undetermined. One possible reason for this research gap is that mitochondrial membrane protein complexes are difficult to overexpress and purify. Even using high-resolution cryo-electron microscopy, structural determination is not possible without first obtaining purified homogeneous proteins. As determining novel structures of protein complexes would provide opportunities to answer many unresolved biological questions, it is important to generalize purification methods, which often become bottlenecks in protein research. In this chapter, we introduce purification methods for mitochondrial membrane protein complexes and mitochondria-localized soluble protein complexes using a yeast expression system. We also describe the recent development of a mitochondrial membrane isolation method that enables the extraction of large amounts of protein complexes for structural analyses.
    Keywords:  Mitochondria preparation; Mitochondrial proteins; Multi-gene expression; Protein complex purification; Structural analysis; Yeast expression system
    DOI:  https://doi.org/10.1016/bs.mie.2024.07.043
  37. Methods Enzymol. 2024 ;pii: S0076-6879(24)00396-3. [Epub ahead of print]707 391-422
      Holdase chaperones are essential in the mitochondrial membrane-protein biogenesis as they stabilize preproteins and keep them in an import-competent state as they travel through the aqueous cytosol and intermembrane space. The small TIM chaperones of the mitochondrial intermembrane space function within a fine balance of client promiscuity and high affinity binding, while being also able to release their client proteins without significant energy barrier to the downstream insertases/translocases. The tendency of the preproteins to aggregate and the dynamic nature of the preprotein-chaperone complexes makes the preparation of these complexes challenging. Here we present two optimized methods for complex formation of highly hydrophobic precursor proteins and chaperones: a pull-down approach and an in-vitro translation strategy. In the former, attaching the client protein to an affinity resin keeps the individual client protein copies apart from each other and decreases the client self-aggregation probability, thereby favouring complex formation. In the latter approach, a purified chaperone, added to the cell-free protein synthesis, captures the nascent precursor protein. The choice of method will depend on the desired client-chaperone complex amount, or the need for specific labeling scheme.
    Keywords:  Cell-free protein production; Chaperone; Mitochondrial protein import; Promiscuous binding; Pull-down assay
    DOI:  https://doi.org/10.1016/bs.mie.2024.07.051
  38. Methods Enzymol. 2024 ;pii: S0076-6879(24)00367-7. [Epub ahead of print]707 637-671
      Oxidation of cysteine residues in proteins can take place as part of an enzymatic reaction cycle, during oxidative protein folding or as a consequence of redox signalling or oxidative stress. Following changes in protein thiol redox states allows to investigate the mechanisms underlying thiol-disulphide redox processes. In this book chapter, we provide information and protocols on different methods for redox state determination with a focus on these processes in the context of oxidation-dependent protein import into the mitochondrial intermembrane space. These methods include assessing the cysteine redox state of mature proteins, methods to investigate oxidative protein folding in radioactive pulse chase assays and methods to follow specifically the formation of oxidative folding intermediates between oxidoreductases and substrates.
    Keywords:  ALR; MIA40; disulphide bond formation; import; mitochondria; oxidative protein folding; redox
    DOI:  https://doi.org/10.1016/bs.mie.2024.07.031
  39. Bratisl Lek Listy. 2024 ;125(11): 693-700
      Important metabolic variables that lead to the development of many diseases, including "mitochondrial diseases," include increased oxidative stress and mitochondrial malfunction. Given that the clinical picture and metabolic alterations in individuals suspected of having mitochondrial illnesses lack distinct characteristics, the development of sensitive and specific diagnostic techniques to detect alterations in mitochondrial bioenergetics is imperative. High-resolution respirometry (HRR), is a minimally invasive technique that enables the analysis of mitochondrial function in platelets taken from peripheral blood. This method allows for the detection of even the most subtle changes prior to disease development. HRR can identify minute variations in mitochondrial bioenergetics. Determining mitochondrial function and endogenous levels of CoQ10 in platelets can aid in the early detection of pathobiochemical changes in mitochondria and assessment of treatment efficacy. When combined with the measurement of endogenous coenzyme Q10 levels, HRR may be an effective approach for early identification of compromised mitochondrial function along with monitoring the therapeutic outcomes. Supplementing with coenzyme Q10, applying molecular hydrogen, transplanting mitochondria, and applying platelet-rich plasma (PRP) are some of the therapeutic strategies utilized to enhance mitochondrial function and reduce oxidative stress (Tab. 1, Fig. 1, Ref. 62). Text in PDF www.elis.sk Keywords: mitochondrial dysfunction, oxidative stress, mitochondrial bioenergetics, high-resolution respirometry, therapeutic approaches.
    DOI:  https://doi.org/10.4149/BLL_2024_105
  40. Front Neurol. 2024 ;15 1435749
      Cochlear outer hair cells (OHCs) play a fundamental role in the hearing sensitivity and frequency selectivity of mammalian hearing and are especially vulnerable to noise-induced damage. The OHCs depend on Ca2+ homeostasis, which is a balance between Ca2+ influx and extrusion, as well as Ca2+ buffering by proteins and organelles. Alterations in OHC Ca2+ homeostasis is not only an immediate response to noise, but also associated with impaired auditory function. However, there is little known about the contribution of Ca2+ buffering proteins and organelles to the vulnerability of OHCs to noise. In this study, we used a knockout (KO) mouse model where oncomodulin (Ocm), the major Ca2+ binding protein preferentially expressed in OHCs, is deleted. We show that Ocm KO mice were more susceptible to noise induced hearing loss compared to wildtype (WT) mice. Following noise exposure (106 dB SPL, 2 h), Ocm KO mice had higher threshold shifts and increased OHC loss and TUNEL staining, compared to age-matched WT mice. Mitochondrial morphology was significantly altered in Ocm KO OHCs compared to WT OHCs. Before noise exposure, Ocm KO OHCs showed decreased mitochondrial abundance, volume, and branching compared to WT OHCs, as measured by immunocytochemical staining of outer mitochondrial membrane protein, TOM20. Following noise exposure, mitochondrial proteins were barely visible in Ocm KO OHCs. Using a mammalian cell culture model of prolonged cytosolic Ca2+ overload, we show that OCM has protective effects against changes in mitochondrial morphology and apoptosis. These experiments suggest that disruption of Ca2+ buffering leads to an increase in noise vulnerability and mitochondrial-associated changes in OHCs.
    Keywords:  Ca2+; Ca2+ buffer; cell death; cochlea; mitochondria; noise-induced hearing loss; oncomodulin; outer hair cells
    DOI:  https://doi.org/10.3389/fneur.2024.1435749
  41. Brain. 2024 Nov 06. pii: awae277. [Epub ahead of print]
      Mitochondrial disease is a group of rare conditions, with no approved treatment to date, except for Leber hereditary optic neuropathy. Therapeutic options to alleviate the symptoms of mitochondrial disease are urgently needed. Sonlicromanol is a promising candidate, as it positively alters the key metabolic and inflammatory pathways associated with mitochondrial disease. Sonlicromanol is a reductive and oxidative distress modulator, selectively inhibiting microsomal prostaglandin E1 synthase activity. This Phase 2b program, aiming at evaluating sonlicromanol in adults with m.3243A>G mutation and primary mitochondrial disease, consisted of a randomized controlled (RCT) study (dose-selection) followed by a 52-week open-label extension study (EXT, long-term tolerability, safety, and efficacy of sonlicromanol). Patients were randomized (1:1:1) to receive 100- or 50-mg sonlicromanol, or placebo twice daily (bid) for 28 days with ≥2-week wash-out period between treatments. Patients who completed the RCT study entered the EXT study wherein they received 100-mg sonlicromanol bid. Overall, 27 patients were randomized (24 RCT patients completed all periods). 15 patients entered the EXT, and 12 patients were included in the EXT analysis set. All patients reported good tolerability and favourable safety, with pharmacokinetic results comparable to the earlier Phase 2a study. The RCT primary endpoint (change from placebo in the attentional domain of cognition score [IDN: visual identification, Cogstate]) did not reach statistical significance. Using a categorisation of the subject's period baseline a treatment effect over placebo was observed if their baseline was more affected (p=0.0338). Using this approach, there were signals of improvements over placebo in at least one dose in the Beck Depression Inventory (BDI, p=0.0143), Cognitive Failure Questionnaire (CFQ, p=0.0113), and the Depression subscale of the Hospital Anxiety and Depression Scale (p=0.0256). Statistically and/or clinically meaningful improvements were observed in the patient- and clinician-reported outcome measures at the end of the EXT study (Test of attentional performance [TAP] with alarm, p=0.0102; TAP without alarm, p=0.0047; BDI somatic, p=0.0261; BDI Total, p=0.0563; SF12 physical component score, p=0.0008). Seven of nine domains of RAND-Short form-36 like SF-36 pain improved (p=0.0105). Other promising results were observed in Neuro QoL-Fatigue-SF (p=0.0036), MiniBESTest (p=0.0009), McGill Pain Questionnaire (p=0.0105), EQ-5D-5L-VAS (p=0.0213) and EQ-5D-5L-index (p=0.0173). Most patients showed improvement in the 5× sit-to-stand test. Sonlicromanol was well-tolerated and demonstrated a favourable benefit/risk ratio for up to one year. Sonlicromanol was efficacious in patients when affected at baseline, as seen across a variety of clinically relevant domains. Long-term treatment showed more pronounced changes from baseline.
    Keywords:  MELAS; MIDD; m.3243A>G; primary mitochondrial disease; sonlicromanol
    DOI:  https://doi.org/10.1093/brain/awae277
  42. Mech Ageing Dev. 2024 Oct 25. pii: S0047-6374(24)00101-5. [Epub ahead of print] 112001
      Alzheimer's disease (AD) accounts for the majority of dementia cases, with aging being the primary risk factor for developing this neurodegenerative condition. Aging and AD share several characteristics, including the formation of amyloid plaques and neurofibrillary tangles, synaptic loss, and neuroinflammation. This overlap suggests that mechanisms driving the aging process might also promote AD; however, the underlying processes are not yet fully understood. In this narrative review, we will focus on the role of mitochondria, not only as the "powerhouse of the cell", but also in programmed cell death, immune response, macromolecular synthesis, and calcium regulation. We will explore both the common changes between aging and AD and the differences between them. Additionally, we will provide an overview of interventions aimed at maintaining mitochondrial function in an attempt to slow the progression of AD. This will include a discussion of antioxidant molecules, factors that trigger mitochondrial biogenesis, compounds capable of restoring the fission/fusion balance, and a particular focus on recent techniques for mitochondrial DNA gene therapy.
    Keywords:  Alzheimer’s disease; aging; calcium homeostasis; mitochondrial DNA; mitochondrial membrane dynamics; oxidative phosphorylation
    DOI:  https://doi.org/10.1016/j.mad.2024.112001
  43. Cell Metab. 2024 Oct 29. pii: S1550-4131(24)00401-7. [Epub ahead of print]
      Aging is a complex process manifesting at molecular, cellular, organ, and organismal levels. It leads to functional decline, disease, and ultimately death, but the relationship between these fundamental biomedical features remains elusive. By applying elastic net regularization to plasma proteome data of over 50,000 human subjects in the UK Biobank and other cohorts, we report interpretable organ-specific and conventional aging models trained on chronological age, mortality, and longitudinal proteome data. These models predict organ/system-specific disease and indicate that men age faster than women in most organs. Accelerated organ aging leads to diseases in these organs, and specific diets, lifestyles, professions, and medications influence organ aging rates. We then identify proteins driving these associations with organ-specific aging. Our analyses reveal that age-related chronic diseases epitomize accelerated organ- and system-specific aging, modifiable through environmental factors, advocating for both universal whole-organism and personalized organ/system-specific anti-aging interventions.
    Keywords:  aging models; blood plasma; diet; disease; elastic net; lifestyle; longevity interventions; mortality; organ-specific aging; proteomic clocks
    DOI:  https://doi.org/10.1016/j.cmet.2024.10.005
  44. Methods Enzymol. 2024 ;pii: S0076-6879(24)00394-X. [Epub ahead of print]707 423-440
      The mitochondrial 60 kDa heat shock protein (mHsp60) is an oligomeric, barrel-like structure that mediates protein folding in cooperation with its cochaperonin Hsp10, in an ATP-dependent manner. In contrast to the extremely stable oligomeric structure of the bacterial chaperonin, GroEL, the human mHsp60 exists in equilibrium between single and double heptameric units, which dissociate easily to inactive monomers under laboratory conditions. Consequently, purification and manipulation of active mHsp60 oligomers is not straightforward. In this manuscript, we present an improved protocol for the purification of functional mHsp60, following its expression in bacteria. This method is based upon a previously published strategy that exploits the notorious instability of mHsp60 to purify the monomeric form, which is subsequently reconstituted to functional oligomers under controlled conditions. In our protocol, we use affinity chromatography on a Ni NTA-agarose resin as the initial step, facilitating purification of substantial amounts of highly pure active protein. The resulting Hsp60 is suitable for both functional and structural analyses, including crystallography and electron cryo-microscopy (cryo-EM) studies, to obtain high resolution structures of the mHsp60 oligomers alone and in various complexes.
    Keywords:  Chaperone; Chaperonin; Cryo-EM; HSPD1; Hsp60; Mitochondria; Protein folding; Protein purification
    DOI:  https://doi.org/10.1016/bs.mie.2024.07.049
  45. Mitochondrion. 2024 Oct 30. pii: S1567-7249(24)00134-X. [Epub ahead of print] 101976
      The impact of mutations on the mitochondria deserves specific interest due to the crucial role played by these organelles on numerous cellular functions. This study examines the effects of repeated bottlenecks on mitochondrial function and fitness. Daphnia pulex mutation accumulation lines (MA) lines were maintained for over 120 generations under copper and no copper conditions. Following the MA propagation, Daphnia from MA lines were raised under optimal and high temperatures for two generations before assessing mitochondrial and phenotypic traits. Spontaneous mutation accumulation under copper led to a later age at maturity and lowered fecundity in the MA lines. Mitochondrial respiration was found to be 10% lower in all mutation accumulation (MA) lines as compared to the non-MA control. MtDNA copy number was elevated in MA lines compared to the control under optimal temperature suggesting a compensatory mechanism. Three MA lines propagated under low copper had very low mtDNA copy number and fitness, suggesting mutations might have affected genes involved in mtDNA replication or mitochondrial biogenesis. Overall, our study suggests that mutation accumulation had an impact on life history traits, mtDNA copy number, and mitochondrial respiration. Some phenotypic effects were magnified under high temperatures. MtDNA copy number appears to be an important mitigation factor to allow mitochondria to cope with mutation accumulation up to a certain level beyond which it can no longer compensate.
    Keywords:  Daphnia; Fitness; Mitochondria; Mitochondria copy number; Mutation accumulation
    DOI:  https://doi.org/10.1016/j.mito.2024.101976
  46. J Lipid Res. 2024 Oct 26. pii: S0022-2275(24)00194-9. [Epub ahead of print] 100689
      
    DOI:  https://doi.org/10.1016/j.jlr.2024.100689
  47. Lancet. 2024 Nov 02. pii: S0140-6736(24)02414-0. [Epub ahead of print]404(10464): 1701
      
    DOI:  https://doi.org/10.1016/S0140-6736(24)02414-0
  48. MicroPubl Biol. 2024 ;2024
      Mitochondria are essential for supporting the high metabolic demands that are required for brain function. Impairments in mitochondria have been linked to age-related decline in brain functions. Here, we investigate whether the mitochondrial respiratory capacity of brain cells is changed in cognitive aging. We used a rat model of normal cognitive aging and analyzed mitochondrial oxidative phosphorylation in frozen brain samples. Mitochondrial oxygen consumption rate analysis of the frontal cortex did not show any differences between young rats and aged rats with either intact memory or impaired spatial memory. Mitochondrial ATP synthase activity and quantity also did not differ between young and aged rats. These results suggest that the total level of mitochondrial respiratory capacity is preserved in the frontal cortex of aged rats and may not explain aging-associated cognitive impairment.
    DOI:  https://doi.org/10.17912/micropub.biology.001359
  49. J Med Genet. 2024 Nov 05. pii: jmg-2024-109862. [Epub ahead of print]
       INTRODUCTION: Hypomyelinating leukodystrophies are a group of genetic disorders, characterised by severe permanent myelin deficiency. Their clinical features include developmental delay with or without neuroregression, nystagmus, central hypotonia, progressing to spasticity and ataxia. HSPD1 encodes the HSP60 chaperonin protein, mediating ATP-dependent folding of imported proteins in the mitochondrial matrix. Pathogenic variants in HSPD1 have been related to a number of neurological phenotypes, including the dominantly inherited pure hereditary spastic paraplegia (MIM 605280) and the recessively inherited hypomyelinating leukodystrophy 4 (MIM 612233). Subsequently, an additional phenotype of hypomyelinating leukodystrophy has been reported due to de novo heterozygous HSPD1 variants.In the current work, we expand the clinical and genetic spectrum of this hypomyelinating disorder by describing a cohort of three patients, being heterozygous for HSPD1 variants involving residue Ala536 of HSP60 (the novel p.Ala536Pro variant and the previously reported p.Ala536Val).
    METHODS: Clinical and radiological evaluation; whole exome sequencing, in vitro reconstitution assay and patient fibroblast cell lysate analysis.
    RESULTS: Clinical manifestation was of early-onset nystagmus, tremor and hypotonia evolving into spasticity and ataxia and childhood-onset neuroregression in one case. Brain MRI studies revealed diffuse hypomyelination.The 3D protein structure showed these variants to lie in spatial proximity to the previously reported Leu47Val variant, associated with a similar clinical phenotype. In vitro reconstitution assay and patient fibroblast cell lysate analysis demonstrated that these mutants display aberrant chaperonin protein complex assembly.
    DISCUSSION: We provide evidence that impaired oligomerisation of the chaperonin complex might underlie this HSPD1-related phenotype, possibly through exerting a dominant negative effect.
    Keywords:  Genetic Diseases, Inborn
    DOI:  https://doi.org/10.1136/jmg-2024-109862
  50. Nature. 2024 Nov 06.
      
    Keywords:  Chemistry; Microbiology; Plant sciences; Psychology
    DOI:  https://doi.org/10.1038/d41586-024-03646-1
  51. Mitochondrion. 2024 Nov 04. pii: S1567-7249(24)00137-5. [Epub ahead of print] 101979
      Genetic control is vital for the growth of cells and tissues, and it also helps living things, from single-celled organisms to complex creatures, maintain a stable internal environment. Within cells, structures called mitochondria act like tiny power plants, producing energy and keeping the cell balanced. The two primary categories of RNA are messenger RNA (mRNA) and non-coding RNA (ncRNA). mRNA carries the instructions for building proteins, while ncRNA does various jobs at the RNA level. There are different kinds of ncRNA, each with a specific role. Some help put RNA molecules together correctly, while others modify other RNAs or cut them into smaller pieces. Still others control how much protein is made from a gene. Scientists have recently discovered many more ncRNAs than previously known, and their functions are still being explored. This article analyzes the RNA molecules present within mitochondria, which have a crucial purpose in the operation of mitochondria. We'll also discuss how genes can be turned on and off without changing their DNA code, and how this process might be linked to mitochondrial RNA. Finally, we'll explore how scientists are using engineered particles to silence genes and develop new treatments based on manipulating ncRNA.
    Keywords:  Mitochondrial epigenetics; Neurodegeneration; Non-coding RNA; Transcriptomics; lnc-RNA; miRNA
    DOI:  https://doi.org/10.1016/j.mito.2024.101979
  52. Sci Adv. 2024 Nov 08. 10(45): eadm8212
      Mitochondrial dynamics orchestrate many essential cellular functions, including metabolism, which is instrumental in promoting cancer growth and metastatic progression. However, how mitochondrial dynamics influences metastatic progression remains poorly understood. Here, we show that breast cancer cells with low metastatic potential exhibit a more fused mitochondrial network compared to highly metastatic cells. To study the impact of mitochondrial dynamics on metastasis, we promoted mitochondrial elongation in metastatic breast cancer cells by individual genetic deletion of three key regulators of mitochondrial fission (Drp1, Fis1, Mff) or by pharmacological intervention with leflunomide. Omics analyses revealed that mitochondrial elongation causes substantial alterations in metabolic pathways and processes related to cell adhesion. In vivo, enhanced mitochondrial elongation by loss of mitochondrial fission mediators or treatment with leflunomide notably reduced metastasis formation. Furthermore, the transcriptomic signature associated with elongated mitochondria correlated with improved clinical outcome in patients with breast cancer. Overall, our findings highlight mitochondrial dynamics as a potential therapeutic target in breast cancer.
    DOI:  https://doi.org/10.1126/sciadv.adm8212
  53. Mol Metab. 2024 Nov 01. pii: S2212-8778(24)00187-X. [Epub ahead of print] 102056
      The gene encoding TOMM40 (Transporter of Outer Mitochondrial Membrane 40) is adjacent to that encoding APOE, which has a central role in lipid and lipoprotein metabolism. Human genetic variants near APOE and TOMM40 are strongly associated with plasma lipid levels, but a specific role for TOMM40 in lipid metabolism has not been established. We show here that suppression of TOMM40 in human hepatoma cells upregulates expression of APOE and LDLR in part via activation of LXRB (NR1H2) by oxysterols, with consequent increased uptake of VLDL and LDL. This is in part due to disruption of mitochondria-endoplasmic reticulum contact sites, with resulting accrual of reactive oxygen species and non-enzymatically derived oxysterols. With TOMM40 knockdown, cellular triglyceride and lipid droplet content are increased, effects attributable in part to receptor-mediated VLDL uptake, since lipid staining is significantly reduced by concomitant suppression of either LDLR or APOE. In contrast, cellular cholesterol content is reduced due to LXRB-mediated upregulation of the ABCA1 transporter as well as increased production and secretion of oxysterol-derived cholic acid. Consistent with the findings in hepatoma cells, in vivo knockdown of TOMM40 in mice results in significant reductions of plasma triglyceride and cholesterol concentrations, reduced hepatic cholesterol and increased triglyceride content, and accumulation of lipid droplets leading to development of steatosis. These findings demonstrate a role for TOMM40 in regulating hepatic lipid and plasma lipoprotein levels and identify mechanisms linking mitochondrial function with lipid metabolism.
    DOI:  https://doi.org/10.1016/j.molmet.2024.102056
  54. Mitochondrion. 2024 Nov 03. pii: S1567-7249(24)00136-3. [Epub ahead of print] 101978
      The encephalomyopathic mtDNA depletion syndrome with methylmalonic aciduria is associated with succinyl-CoA synthetase (SCS) deficiency caused by pathogenic variants in genes encoding its two subunits. SCS is a mitochondrial enzyme involved in several metabolic pathways and acts as a heterodimer composed of α and β subunits encoded by SUCLG1 and SUCLA2 genes, respectively. The purpose of this study was to analyze the effects of the most pathogenic non-synonymous single nucleotide polymorphisms (nsSNPs) by applying, using different prediction tools, a filtering strategy, on the 343 and 365 nsSNPs found in SUCLG1 and SUCLA2 genes, respectively, retrieved from the databases, then to evaluate their structural and functional effects using homology modeling and molecular docking. Results showed that most deleterious mutations selected for structural analysis were located in loop regions critical for protein stability and function, especially, variants altering glycine and proline residues in these regions supporting their importance. We also showed that variants leading to hydrophobic and hydrophilic residues can destabilize the folding and binding of the protein. Molecular docking has also been used to identify the most important regions of ligand binding site (CoA binding site, ADP-Mg2+ binding site and phosphate ion binding site) and between the two subunits themselves, which mainly involving the ligase CoA domain. Our structural analysis, performed on selected nsSNP, are in accordance with experimental studies reported in the literature and predicted that they would responsible to either nonfunctional protein, subunit instability resulting in reduced amounts of misassembled protein, or in a protein unable to phosphorylate ADP.
    Keywords:  Docking; Homology modeling; Missense SNPs; Structure-function relationship; Succinyl-CoA synthetase ADP- dependent
    DOI:  https://doi.org/10.1016/j.mito.2024.101978
  55. Nat Immunol. 2024 Nov 04.
      Acute lower respiratory tract infection (ALRI) remains a major worldwide cause of childhood mortality, compelling innovation in prevention and treatment. Children in Papua New Guinea (PNG) experience profound morbidity from ALRI caused by Streptococcus pneumoniae. As a result of evolutionary divergence, the human PNG population exhibits profound genetic variation and diversity. To address unmet health needs of children in PNG, we tested whether genetic variants increased ALRI morbidity. Whole-exome sequencing of a pilot child cohort identified homozygosity for a novel single-nucleotide variant (SNV) in coenzyme Q6 (COQ6) in cases with ALRI. COQ6 encodes a mitochondrial enzyme essential for biosynthesis of ubiquinone, an electron acceptor in the electron transport chain. A significant association of SNV homozygosity with ALRI was replicated in an independent ALRI cohort (P = 0.036). Mice homozygous for homologous mouse variant Coq6 exhibited increased mortality after pneumococcal lung infection, confirming causality. Bone marrow chimeric mice further revealed that expression of variant Coq6 in recipient (that is, nonhematopoietic) tissues conferred increased mortality. Variant Coq6 maintained ubiquinone biosynthesis, while accelerating metabolic remodeling after pneumococcal challenge. Identification of this COQ6 variant provides a genetic basis for increased pneumonia susceptibility in PNG and establishes a previously unrecognized role for the enzyme COQ6 in regulating inflammatory-mediated metabolic remodeling.
    DOI:  https://doi.org/10.1038/s41590-024-01998-4
  56. MicroPubl Biol. 2024 ;2024
      Two genomic genes, which rescue ammonium assimilation defect in the glutamate-requiring Schizosaccharomyces pombe glu1 mutant, were identified. The maa1 , encoding a mitochondrial aspartate aminotransferase, is the causative gene of glu1 mutation because an inseparable linkage between maa1 and glu1 on the chromosome, and also the glu1 mutant strain has a nonsense mutation within the maa1 coding region, which is responsible for its defective phenotype. The yhm2 , a mitochondrial 2-oxoglutarate carrier, was also isolated as a weak multicopy suppressor gene. These findings reiterate the importance of the mitochondria in utilizing the amino acids for cellular nitrogen metabolism.
    DOI:  https://doi.org/10.17912/micropub.biology.001338
  57. Nature. 2024 Nov 01.
      
    Keywords:  Chemistry; Drug discovery; Organic chemistry
    DOI:  https://doi.org/10.1038/d41586-024-03538-4
  58. STAR Protoc. 2024 Nov 01. pii: S2666-1667(24)00593-8. [Epub ahead of print]5(4): 103428
      Here, we present a NAD+/NADH detection assay for evaluating NAD+, NADH, and NAD+/NADH ratio across diverse biological models, including Caenorhabditis elegans, mouse muscle tissue, mouse whole blood, and human whole blood. We describe steps for sample collection and preparation from different models as well as detection and calculation of NAD+ and NADH levels. This protocol is applicable for quantifying cellular/tissue NAD+ and NADH levels across different biological models.
    Keywords:  Cell Biology; Metabolism; Molecular Biology; Neuroscience
    DOI:  https://doi.org/10.1016/j.xpro.2024.103428
  59. Nat Rev Drug Discov. 2024 Nov 04.
    N-of-1 Task Force of the International Rare Diseases Research Consortium (IRDiRC)
      In recent years, a small number of people with rare diseases caused by unique genetic variants have been treated with therapies developed specifically for them. This pioneering field of genetic N-of-1 therapies is evolving rapidly, giving hope for the individualized treatment of people living with very rare diseases. In this Review, we outline the concept of N-of-1 individualized therapies, focusing on genetic therapies, and illustrate advances and challenges in the field using cases for which therapies have been successfully developed. We discuss why the traditional drug development and reimbursement pathway is not fit for purpose in this field, and outline the pragmatic, regulatory and ethical challenges this poses for future access to N-of-1 therapies. Finally, we provide a roadmap for N-of-1 individualized therapy development.
    DOI:  https://doi.org/10.1038/s41573-024-01059-3