bims-mitper Biomed News
on Mitochondrial Permeabilization
Issue of 2023‒02‒26
twenty-one papers selected by
Bradley Irizarry
Thomas Jefferson University

  1. Biochem Soc Trans. 2023 Feb 23. pii: BST20221525. [Epub ahead of print]
      Cytosolic DNA is recognized by the innate immune system as a potential threat. During apoptotic cell death, mitochondrial DNA (mtDNA) release activates the DNA sensor cyclic GMP-AMP synthase (cGAS) to promote a pro-inflammatory type I interferon response. Inflammation following mtDNA release during apoptotic cell death can be exploited to engage anti-tumor immunity and represents a potential avenue for cancer therapy. Additionally, various studies have described leakage of mtDNA, independent of cell death, with different underlying cues such as pathogenic infections, changes in mtDNA packaging, mtDNA stress or reduced mitochondrial clearance. The interferon response in these scenarios can be beneficial but also potentially disadvantageous, as suggested by a variety of disease phenotypes. In this review, we discuss mechanisms underlying mtDNA release governed by cell death pathways and summarize release mechanisms independent of cell death. We further highlight the similarities and differences in mtDNA release pathways, outlining gaps in our knowledge and questions for further research. Together, a deeper understanding of how and when mtDNA is released may enable the development of drugs to specifically target or inhibit mtDNA release in different disease settings.
    Keywords:  apoptosis; cell death; mitochondria; mtDNA; pyroptosis
  2. Handb Clin Neurol. 2023 ;pii: B978-0-12-821751-1.00001-4. [Epub ahead of print]194 3-6
      This chapter provides a overview of this volume of the Handbook of Clinical Neurology, placing recent advances in our understanding of mitochondrial disorders in a historical context, and speculates about the future.
    Keywords:  Clinical medicine; Diagnosis; Genomics; Mitochondria; Mitochondrial diseases; Neurology; Treatments; mtDNA
  3. Methods Mol Biol. 2023 ;2615 79-88
      Mitochondrial DNA (mtDNA) encodes a variety of rRNAs, tRNAs, and respiratory chain complex proteins. The integrity of mtDNA supports the mitochondrial functions and plays an essential role in numerous physiological and pathological processes. Mutations in mtDNA cause metabolic diseases and aging. The mtDNA within the human cells are packaged into hundreds of nucleoids within the mitochondrial matrix. Knowledge of how the nucleoids are dynamically distributed and organized within mitochondria is key to understanding mtDNA structure and functions. Therefore, visualizing the distribution and dynamics of mtDNA within mitochondria is a powerful approach to gain insights into the regulation of mtDNA replication and transcription. In this chapter, we describe the methods of observing mtDNA and its replication with fluorescence microscopy in both fixed and live cells using different labeling strategies.
    Keywords:  BrdU; EdU; Mitochondrial DNA (mtDNA); POLG2; PdG; TFAM
  4. Methods Mol Biol. 2023 ;2615 219-228
      Mitochondria are eukaryotic organelles of endosymbiotic origin that contain their own genetic material, mitochondrial DNA (mtDNA), and dedicated systems for mtDNA maintenance and expression. MtDNA molecules encode a limited number of proteins that are nevertheless all essential subunits of the mitochondrial oxidative phosphorylation system. Here, we describe protocols to monitor DNA and RNA synthesis in intact, isolated mitochondria. These in organello synthesis protocols are valuable techniques for studying the mechanisms and regulation of mtDNA maintenance and expression.
    Keywords:  Mitochondria; Radioactive labeling of nucleic acids; in organello replication and transcription; mtDNA; mtDNA maintenance and expression
  5. Methods Mol Biol. 2023 ;2615 427-441
      Mitochondrial DNA (mtDNA) encodes components essential for cellular respiration. Low levels of point mutations and deletions accumulate in mtDNA during normal aging. However, improper maintenance of mtDNA results in mitochondrial diseases, stemming from progressive loss of mitochondrial function through the accelerated formation of deletions and mutations in mtDNA. To better understand the molecular mechanisms underlying the creation and propagation of mtDNA deletions, we developed the LostArc next-generation DNA sequencing pipeline to detect and quantify rare mtDNA species in small tissue samples. LostArc procedures are designed to minimize PCR amplification of mtDNA and instead achieve enrichment of mtDNA by selective destruction of nuclear DNA. This approach leads to cost-effective, high-depth sequencing of mtDNA with a sensitivity sufficient to identify one mtDNA deletion per million mtDNA circles. Here, we describe detailed protocols for isolation of genomic DNA from mouse tissues, enrichment of mtDNA through enzymatic destruction of linear nuclear DNA, and preparation of libraries for unbiased next-generation sequencing of mtDNA.
    Keywords:  DNA deletions; Mitochondrial DNA; Mitochondrial DNA Replication; Mitochondrial disease; Next-Generation Sequencing; POLG
  6. Biochem Soc Trans. 2023 Feb 23. pii: BST20220014. [Epub ahead of print]
      Mitochondria have long been appreciated as the metabolic hub of cells. Emerging evidence also posits these organelles as hubs for innate immune signalling and activation, particularly in macrophages. Macrophages are front-line cellular defenders against endogenous and exogenous threats in mammals. These cells use an array of receptors and downstream signalling molecules to respond to a diverse range of stimuli, with mitochondrial biology implicated in many of these responses. Mitochondria have the capacity to both divide through mitochondrial fission and coalesce through mitochondrial fusion. Mitochondrial dynamics, the balance between fission and fusion, regulate many cellular functions, including innate immune pathways in macrophages. In these cells, mitochondrial fission has primarily been associated with pro-inflammatory responses and metabolic adaptation, so can be considered as a combative strategy utilised by immune cells. In contrast, mitochondrial fusion has a more protective role in limiting cell death under conditions of nutrient starvation. Hence, fusion can be viewed as a cellular survival strategy. Here we broadly review the role of mitochondria in macrophage functions, with a focus on how regulated mitochondrial dynamics control different functional responses in these cells.
    Keywords:  inflammation; macrophages; mitochondrial dynamics; mitochondrial fission; mitochondrial fusion; neuroinflammation
  7. Methods Mol Biol. 2023 ;2615 281-292
      Mitochondrial DNA (mtDNA) mutations are found in several human pathologies and are associated with aging. Deletion mutations in mtDNA result in the loss of essential genes for mitochondrial function. Over 250 deletion mutations have been reported and the common deletion is the most frequent mtDNA deletion linked to disease. This deletion removes 4977 base pairs of mtDNA. It has previously been shown that exposure to UVA radiation can promote the formation of the common deletion. Furthermore, aberrations in mtDNA replication and repair are associated with formation of the common deletion. However, molecular mechanisms describing the formation of this deletion are poorly characterized. This chapter describes a method to irradiate human skin fibroblasts with physiological doses of UVA and the subsequent detection of the common deletion by quantitative PCR analysis.
    Keywords:  Common Deletion; Deletion Mutations; Mitochondrial DNA; Replication and Repair; UVA radiation
  8. J Biomol Struct Dyn. 2023 Feb 23. 1-17
      The mitochondrial permeability transition pore (mtPTP) plays a vital role in altering the structure and function of mitochondria. Cyclophilin D (CypD) is a mitochondrial protein that regulates mtPTP function and a known drug target for therapeutic studies involving mitochondria. While the effect of aromatase inhibition on the mtPTP has been studied previously, the effect of anastrozole on the mtPTP has not been completely elucidated. The role of anastrozole in modulating the mtPTP was evaluated by docking, molecular dynamics and network-guided studies using human CypD data. The peripheral blood mononuclear cells (PBMCs) of patients with mitochondrial disorders and healthy controls were treated with anastrozole and evaluated for mitochondrial permeability transition pore (mtPTP) function and apoptosis using a flow cytometer. Spectrophotometry was employed for estimating total ATP levels. The anastrozole-CypD complex is more stable than cyclosporin A (CsA)-CypD. Anastrozole performed better than cyclosporine in inhibiting mtPTP. Additional effects included inducing mitochondrial membrane depolarization and a reduction in mitochondrial swelling and superoxide generation, intrinsic caspase-3 activity and cellular apoptosis, along with an increase in ATP levels. Anastrozole may serve as a potential therapeutic agent for mitochondrial disorders and ameliorate the clinical phenotype by regulating the activity of mtPTP. However, further studies are required to substantiate our preliminary findings.Communicated by Ramaswamy H. Sarma.
    Keywords:  Anastrozole; MD simulation; binding sites; docking complexes; mitochondrial disorders; mitochondrial permeability transition pore; molecular docking; protein targets; structural modelling
  9. Biomedicines. 2023 Feb 12. pii: 532. [Epub ahead of print]11(2):
      Mitochondrial diseases are a large class of human hereditary diseases, accompanied by the dysfunction of mitochondria and the disruption of cellular energy synthesis, that affect various tissues and organ systems. Mitochondrial DNA mutation-caused disorders are difficult to study because of the insufficient number of clinical cases and the challenges of creating appropriate models. There are many cellular models of mitochondrial diseases, but their application has a number of limitations. The most proper and promising models of mitochondrial diseases are animal models, which, unfortunately, are quite rare and more difficult to develop. The challenges mainly arise from the structural features of mitochondria, which complicate the genetic editing of mitochondrial DNA. This review is devoted to discussing animal models of human mitochondrial diseases and recently developed approaches used to create them. Furthermore, this review discusses mitochondrial diseases and studies of metabolic disorders caused by the mitochondrial DNA mutations underlying these diseases.
    Keywords:  animal model; cellular model; gene editing; mitochondrial diseases; mitochondrial mutations
  10. Handb Clin Neurol. 2023 ;pii: B978-0-12-821751-1.00014-2. [Epub ahead of print]194 99-116
      Mitochondria are essential for the health and viability of both motor and sensory neurons and their axons. Processes that disrupt their normal distribution and transport along axons will likely cause peripheral neuropathies. Similarly, mutations in mtDNA or nuclear encoded genes result in neuropathies that either stand alone or are part of multisystem disorders. This chapter focuses on the more common genetic forms and characteristic clinical phenotypes of "mitochondrial" peripheral neuropathies. We also explain how these various mitochondrial abnormalities cause peripheral neuropathy. In a patient with a neuropathy either due to a mutation in a nuclear or an mtDNA gene, clinical investigations aim to characterize the neuropathy and make an accurate diagnosis. In some patients, this may be relatively straightforward, where a clinical assessment and nerve conduction studies followed by genetic testing is all that is needed. In others, multiple investigations including a muscle biopsy, CNS imaging, CSF analysis, and a wide range of metabolic and genetic tests in blood and muscle may be needed to establish diagnosis.
    Keywords:  Axonal transport; Charcot–Marie–Tooth disease; Mitochondrial DNA; Mitochondrial fusion/fission; Nuclear mitochondrial genes; Respiratory chain enzymes
  11. Methods Mol Biol. 2023 ;2615 381-395
      Over the last 10 years, next generation sequencing (NGS) became the gold standard for both diagnosis and discovery of new disease genes responsible for heterogeneous disorders, such as mitochondrial encephalomyopathies. The application of this technology to mtDNA mutations poses extra challenges compared to other genetic conditions because of the peculiarities of mitochondrial genetics and the requirement for proper NGS data management and analysis. Here, we describe a detailed, clinically relevant protocol to sequence the whole mtDNA and quantify heteroplasmy levels of mtDNA variants, starting from total DNA through the generation of a single PCR amplicon.
    Keywords:  Heteroplasmy; Mitochondrial DNA; Mitochondrial disease; Mitochondrial haplogroups; Next generation sequencing; Single amplicon
  12. Autophagy. 2023 Feb 20.
      Mitochondrial DNA (mtDNA) is prone to the accumulation of mutations. To prevent harmful mtDNA mutations from being passed on to the next generation, the female germline, through which mtDNA is exclusively inherited, has evolved extensive mtDNA quality control. To dissect the molecular underpinnings of this process, we recently performed a large RNAi screen in Drosophila and uncovered a programmed germline mitophagy (PGM) that is essential for mtDNA quality control. We found that PGM begins as germ cells enter meiosis, induced, at least in part, by the inhibition of the mTor (mechanistic Target of rapamycin) complex 1 (mTorC1). Interestingly, PGM requires the general macroautophagy/autophagy machinery and the mitophagy adaptor BNIP3, but not the canonical mitophagy genes Pink1 and park (parkin), even though they are critical for germline mtDNA quality control. We also identified the RNA-binding protein Atx2 as a major regulator of PGM. This work is the first to identify and implicate a programmed mitophagy event in germline mtDNA quality control, and it highlights the utility of the Drosophila ovary for studying developmentally regulated mitophagy and autophagy in vivo.
    Keywords:  Drosophila; autophagy; germline; mitochondria; mitochondrial DNA; mitophagy; mtDNA; purifying selection
  13. Chemistry. 2023 Feb 20. e202204021
      Mitochondrial DNA (mtDNA) plays an essential role in maintaining normal cellular activities. Its heteroplasmic mutations are known to cause various genetic diseases. Current genetic engineering strategies, such as those based on RNA interference (RNAi) and antisense technology, are difficult to genetically alter mtDNA, however, due to the inability of highly negatively charged oligonucleotides to translocate across the double-membrane mitochondria. We report herein a universal mitochondria-targeted gene-delivery approach by using cell-penetrating poly(disulfide)s (CPDs). Novel CPD-based mitochondrial transporters, named Mito-CPDs, were synthesized by using triphenylphosphonium (TPP)-fused propagating monomers containing either disulfide or diselenide backbones. Upon spontaneous complex formation with an oligonucleotide (single- or double-stranded), the resulting nanoscale Mito-CPD@Oligo exhibited excellent properties in common biological media. While the intracellular gene-delivery efficiency of these Mito-CPDs was comparable to that of commercial transfection agents, their unique mitochondria-localized properties enabled effective release of the loaded cargo inside these organelles. Subsequent mitochondrial delivery of siRNA and antisense oligonucleotides against suitable mtDNA-encoded proteins showed successful down-regulation of target protein expression, leading to profound effects on mitochondrial functions. Mito-CPDs thus provide a useful tool for future investigations of mitochondrial biology and treatment of mitochondria-related diseases.
    Keywords:  Antisense oligonucleotides; Cell-penetrating poly(disulfide)s; Mitochondrial functions; Mitochondrial gene; RNA interference
  14. Methods Mol Biol. 2023 ;2611 269-282
      Mitochondria are unique organelles of eukaryotic cells that carry their own multicopy number and circular genome. In most mammals, including humans and mice, the size of the chromosome is ~16,000 base pairs and unlike nuclear DNA, mitochondrial DNA (mtDNA) is not densely compacted. This results in mtDNA to be highly accessible for enzymes such as the Tn5 transposase, commonly used for accessible chromatin profiling of nuclear chromatinized DNA. Here, we describe a method for the concomitant sequencing of mtDNA and accessible chromatin in thousands of individual cells via the mitochondrial single-cell assay for transposase accessible chromatin by sequencing (mtscATAC-seq). Our approach extends the utility of existing scATAC-seq products and protocols as we (Nam et al, Nat Rev Genet 22:3-18, 2021) fix cells using formaldehyde to retain mitochondria and its mtDNA within its originating cell, (Buenrostro et al, Nat Methods 10:1213-1218, 2013) modify lysis conditions to permeabilize cells and mitochondria, and (Corces et al, Nat Methods 14:959-962, 2017) optimize bioinformatic processing protocols to collectively increase mitochondrial genome coverage for downstream analysis. Here, we discuss the essentials for the experimental and computational methodologies to generate and analyze thousands of multiomic profiles of single cells over the course of a few days, enabling the profiling of accessible chromatin and mtDNA genotypes to reconstruct clonal relationships and studies of mitochondrial genetics and disease.
    Keywords:  Accessible chromatin profiling; Lineage tracing; Mitochondrial DNA; Mitochondrial disease; Pathogenic mutation; Single cell multiomics; Somatic mutation
  15. Handb Clin Neurol. 2023 ;pii: B978-0-12-821751-1.00011-7. [Epub ahead of print]194 141-165
      Mitochondrial diseases are a genetically and phenotypically variable set of monogenic disorders. The main characteristic of mitochondrial diseases is a defective oxidative phosphorylation. Both nuclear and mitochondrial DNA encode the approximately 1500 mitochondrial proteins. Since identification of the first mitochondrial disease gene in 1988 a total of 425 genes have been associated with mitochondrial diseases. Mitochondrial dysfunctions can be caused both by pathogenic variants in the mitochondrial DNA or the nuclear DNA. Hence, besides maternal inheritance, mitochondrial diseases can follow all modes of Mendelian inheritance. The maternal inheritance and tissue specificity distinguish molecular diagnostics of mitochondrial disorders from other rare disorders. With the advances made in the next-generation sequencing technology, whole exome sequencing and even whole-genome sequencing are now the established methods of choice for molecular diagnostics of mitochondrial diseases. They reach a diagnostic rate of more than 50% in clinically suspected mitochondrial disease patients. Moreover, next-generation sequencing is delivering a constantly growing number of novel mitochondrial disease genes. This chapter reviews mitochondrial and nuclear causes of mitochondrial diseases, molecular diagnostic methodologies, and their current challenges and perspectives.
    Keywords:  Diagnostic; Genetic; Heteroplasmy; Mitochondrial DNA; Mitochondrial disease; Multi-omic; Mutation
  16. Curr Protoc. 2023 Feb;3(2): e679
      To cope with DNA damage, mitochondria have developed a pathway whereby severely damaged or unrepairable mitochondrial DNA (mtDNA) molecules can be discarded and degraded, after which new molecules are synthesized using intact templates. In this unit, we describe a method that harnesses this pathway to eliminate mtDNA from mammalian cells by transiently overexpressing the Y147A mutant of human uracil-N-glycosylase (mUNG1) in mitochondria. We also provide alternate protocols for mtDNA elimination using either combined treatment with ethidium bromide (EtBr) and dideoxycytidine (ddC) or clustered regulatory interspersed short palindromic repeat (CRISPR)-Cas9-mediated knockout of TFAM or other genes essential for mtDNA replication. Support protocols detail approaches for several processes: (1) genotyping ρ0 cells of human, mouse, and rat origin by polymerase chain reaction (PCR); (2) quantification of mtDNA by quantitative PCR (qPCR); (3) preparation of calibrator plasmids for mtDNA quantification; and (4) quantification of mtDNA by direct droplet digital PCR (dddPCR). © 2023 Wiley Periodicals LLC. Basic Protocol: Inducing mtDNA loss with mUNG1 Alternate Protocol 1: Generation of ρ0 cells by mtDNA depletion with EtBr and ddC Alternate Protocol 2: Generation of ρ0 cells by knocking out genes critical for mtDNA replication Support Protocol 1: Genotyping ρ0 cells by DirectPCR Support Protocol 2: Determination of mtDNA copy number by qPCR Support Protocol 3: Preparation of calibrator plasmid for qPCR Support Protocol 4: Determination of mtCN by direct droplet digital PCR (dddPCR).
    Keywords:  cybrids; mtDNA; mtDNA copy number; mtDNA damage; ρ0 cells
  17. Int J Biol Macromol. 2023 Feb 20. pii: S0141-8130(23)00649-9. [Epub ahead of print] 123755
      Mitochondria are centers of energy metabolism. The mitochondrial network is shaped by mitochondrial dynamics, including the processes of mitochondrial fission and fusion and cristae remodeling. The cristae folded by the inner mitochondrial membrane are sites of the mitochondrial oxidative phosphorylation (OXPHOS) system. However, the factors and their coordinated interplay in cristae remodeling and linked human diseases have not been fully demonstrated. In this review, we focus on key regulators of cristae structure, including the mitochondrial contact site and cristae organizing system, optic atrophy-1, mitochondrial calcium uniporter, and ATP synthase, which function in the dynamic remodeling of cristae. We summarized their contribution to sustaining functional cristae structure and abnormal cristae morphology, including a decreased number of cristae, enlarged cristae junctions, and cristae as concentric ring structures. These abnormalities directly impact cellular respiration and are caused by dysfunction or deletion of these regulators in diseases such as Parkinson's disease, Leigh syndrome, and dominant optic atrophy. Identifying the important regulators of cristae morphology and understanding their role in sustaining mitochondrial morphology could be applied to explore the pathologies of diseases and to develop relevant therapeutic tools.
    Keywords:  ATP synthase; Cristae ultrastructure; MICOS; MICU1; Mitochondria; OPA1
  18. Methods Mol Biol. 2023 ;2615 139-151
      Mitochondrial transcription factor A (TFAM) is a mitochondrial DNA (mtDNA)-binding protein that plays a crucial dual role in the initiation of mitochondrial transcription initiation and mtDNA maintenance. Because TFAM directly interacts with mtDNA, assessing its DNA-binding property can provide useful information. This chapter describes two in vitro assay methods, an electrophoretic mobility shift assay (EMSA) and a DNA-unwinding assay with recombinant TFAM proteins, which both require simple agarose gel electrophoresis. These are used to investigate the effects of mutations, truncation, and posttranslational modifications on this key mtDNA regulatory protein.
    Keywords:  DNA-unwinding assay; EMSA; Electrophoretic mobility shift assay; Mitochondrial DNA; Mitochondrial nucleoids; Mitochondrial transcription factor A; TFAM; mtDNA
  19. Handb Clin Neurol. 2023 ;pii: B978-0-12-821751-1.00003-8. [Epub ahead of print]194 117-124
      Mitochondrial diseases typically involve organs highly dependent on aerobic metabolism and are often progressive with high morbidity and mortality. In the previous chapters of this book, classical mitochondrial phenotypes and syndromes are extensively described. However, these well-known clinical pictures are more the exception rather than the rule in mitochondrial medicine. In fact, more complex, unspecified, incomplete, and/or overlap clinical entities may be even more frequent, with multisystem appearance or progression. In this chapter, we describe some complex neurological presentations, as well as the multisystem manifestations of mitochondrial diseases, ranging from the brain to the other organs.
    Keywords:  Cardiomyopathies; Hearing loss; Leukoencephalopathies; Mitochondrial diseases; Multisystem involvement; Parkinsonism; mtDNA
  20. Cells. 2023 Feb 16. pii: 641. [Epub ahead of print]12(4):
      Dissecting and identifying the major actors and pathways in the genesis, progression and aggressive advancement of breast cancer is challenging, in part because neoplasms arising in this tissue represent distinct diseases and in part because the tumors themselves evolve. This review attempts to illustrate the complexity of this mutational landscape as it pertains to the RUNX genes and their transcription co-factor CBFβ. Large-scale genomic studies that characterize genetic alterations across a disease subtype are a useful starting point and as such have identified recurring alterations in CBFB and in the RUNX genes (particularly RUNX1). Intriguingly, the functional output of these mutations is often context dependent with regards to the estrogen receptor (ER) status of the breast cancer. Therefore, such studies need to be integrated with an in-depth understanding of both the normal and corrupted function in mammary cells to begin to tease out how loss or gain of function can alter the cell phenotype and contribute to disease progression. We review how alterations to RUNX/CBFβ function contextually ascribe to breast cancer subtypes and discuss how the in vitro analyses and mouse model systems have contributed to our current understanding of these proteins in the pathogenesis of this complex set of diseases.
    Keywords:  CBFβ; RUNX1; RUNX2; RUNX3; breast cancer; estrogen receptor (ER); mammary; metastasis
  21. Handb Clin Neurol. 2023 ;pii: B978-0-12-821751-1.00002-6. [Epub ahead of print]194 229-250
      Primary mitochondrial diseases are some of the most common and complex inherited inborn errors of metabolism. Their molecular and phenotypic diversity has led to difficulties in finding disease-modifying therapies and clinical trial efforts have been slow due to multiple significant challenges. Lack of robust natural history data, difficulties in finding specific biomarkers, absence of well-validated outcome measures, and small patient numbers have made clinical trial design and conduct difficult. Encouragingly, new interest in treating mitochondrial dysfunction in common diseases and regulatory incentives to develop therapies for rare conditions have led to significant interest and efforts to develop drugs for primary mitochondrial diseases. Here, we review past and present clinical trials and future strategies of drug development in primary mitochondrial diseases.
    Keywords:  Antioxidants; Clinical trials; Gene therapy; Mitochondria; Mitochondrial biogenesis; Mitophagy; Nucleosides; Primary mitochondrial disease; Treatment