bims-midmar Biomed News
on Mitochondrial DNA maintenance and replication
Issue of 2021‒08‒29
fifteen papers selected by
Flavia Söllner
Ludwig-Maximilians University
  1. Interactions of Mitochondrial Transcription Factor A with DNA Damage: Mechanistic Insights and Functional Implications.
  2. Targeted elimination of mutated mitochondrial DNA by a multi-functional conjugate capable of sequence-specific adenine alkylation.
  3. Mitochondria-Induced Immune Response as a Trigger for Neurodegeneration: A Pathogen from Within.
  4. Requirements for successful mitochondrial transplantation.
  5. Mechanism of transcription initiation and primer generation at the mitochondrial replication origin OriL.
  6. Substrates and interactors of the ClpP protease in the mitochondria.
  7. Approaches to monitor ATP levels in living cells: where do we stand?
  8. MitoPhen database: a human phenotype ontology-based approach to identify mitochondrial DNA diseases.
  9. Microvesicles transfer mitochondria and increase mitochondrial function in brain endothelial cells.
  10. In-depth understanding of Pearson syndrome arising from a novel large mitochondrial DNA deletion in an infant case.
  11. Voluntary exercise prevents abnormal muscle mitochondrial morphology in cancer cachexia mice.
  12. Structural and Functional Alterations in Mitochondria-Associated Membranes (MAMs) and in Mitochondria Activate Stress Response Mechanisms in an In Vitro Model of Alzheimer's Disease.
  13. Loosening ER-Mitochondria Coupling by the Expression of the Presenilin 2 Loop Domain.
  14. Dual-color dSTORM imaging and ThunderSTORM image reconstruction and analysis to study the spatial organization of the nuclear phosphatidylinositol phosphates.
  15. miQC: An adaptive probabilistic framework for quality control of single-cell RNA-sequencing data.
  1. Genes (Basel). 2021 Aug 15. pii: 1246. [Epub ahead of print]12(8):
    Interactions of Mitochondrial Transcription Factor A with DNA Damage: Mechanistic Insights and Functional Implications.
    Krystie Chew, Linlin Zhao.
      Mitochondria have a plethora of functions in eukaryotic cells, including cell signaling, programmed cell death, protein cofactor synthesis, and various aspects of metabolism. The organelles carry their own genomic DNA, which encodes transfer and ribosomal RNAs and crucial protein subunits in the oxidative phosphorylation system. Mitochondria are vital for cellular and organismal functions, and alterations of mitochondrial DNA (mtDNA) have been linked to mitochondrial disorders and common human diseases. As such, how the cell maintains the integrity of the mitochondrial genome is an important area of study. Interactions of mitochondrial proteins with mtDNA damage are critically important for repairing, regulating, and signaling mtDNA damage. Mitochondrial transcription factor A (TFAM) is a key player in mtDNA transcription, packaging, and maintenance. Due to the extensive contact of TFAM with mtDNA, it is likely to encounter many types of mtDNA damage and secondary structures. This review summarizes recent research on the interaction of human TFAM with different forms of non-canonical DNA structures and discusses the implications on mtDNA repair and packaging.
    Keywords:  DNA modification; DNA packing; DNA-protein interaction; G-quadruplex; epigenetics; nucleoid; post-translational modification
    DOI:  https://doi.org/10.3390/genes12081246
  2. Cell Chem Biol. 2021 Aug 24. pii: S2451-9456(21)00365-2. [Epub ahead of print]
    Targeted elimination of mutated mitochondrial DNA by a multi-functional conjugate capable of sequence-specific adenine alkylation.
    Takuya Hidaka, Kaori Hashiya, Toshikazu Bando, Ganesh N Pandian, Hiroshi Sugiyama.
      Mutations in mitochondrial DNA (mtDNA) cause mitochondrial diseases, characterized by abnormal mitochondrial function. Although eliminating mutated mtDNA has potential to cure mitochondrial diseases, no chemical-based drugs in clinical trials are capable of selective modulation of mtDNA mutations. Here, we construct a class of compounds encompassing pyrrole-imidazole polyamides (PIPs), mitochondria-penetrating peptide, and chlorambucil, an adenine-specific DNA-alkylating reagent. The sequence-selective DNA binding of PIPs allows chlorambucil to alkylate mutant adenine more efficiently than other sites in mtDNA. In vitro DNA alkylation assay shows that our compound 8950A-Chb(Cl/OH) targeting a nonpathogenic point mutation in HeLa S3 cells (m.8950G>A) can specifically alkylate the mutant adenine. Furthermore, the compound reduces the mtDNA possessing the target mutation in cultured HeLa S3 cells. The programmability of PIPs to target different sequences could allow this class of compounds to be developed as designer drugs targeting pathogenic mutations associated with mitochondrial diseases in future studies.
    Keywords:  DNA alkylation; DNA mutation; designer small molecule; heteroplasmy; mitochondria; mitochondrial DNA; mitochondrial disease; pyrrole-imidazole polyamide
    DOI:  https://doi.org/10.1016/j.chembiol.2021.08.003
  3. Int J Mol Sci. 2021 Aug 07. pii: 8523. [Epub ahead of print]22(16):
    Mitochondria-Induced Immune Response as a Trigger for Neurodegeneration: A Pathogen from Within.
    Marta Luna-Sánchez, Patrizia Bianchi, Albert Quintana.
      Symbiosis between the mitochondrion and the ancestor of the eukaryotic cell allowed cellular complexity and supported life. Mitochondria have specialized in many key functions ensuring cell homeostasis and survival. Thus, proper communication between mitochondria and cell nucleus is paramount for cellular health. However, due to their archaebacterial origin, mitochondria possess a high immunogenic potential. Indeed, mitochondria have been identified as an intracellular source of molecules that can elicit cellular responses to pathogens. Compromised mitochondrial integrity leads to release of mitochondrial content into the cytosol, which triggers an unwanted cellular immune response. Mitochondrial nucleic acids (mtDNA and mtRNA) can interact with the same cytoplasmic sensors that are specialized in recognizing genetic material from pathogens. High-energy demanding cells, such as neurons, are highly affected by deficits in mitochondrial function. Notably, mitochondrial dysfunction, neurodegeneration, and chronic inflammation are concurrent events in many severe debilitating disorders. Interestingly in this context of pathology, increasing number of studies have detected immune-activating mtDNA and mtRNA that induce an aberrant production of pro-inflammatory cytokines and interferon effectors. Thus, this review provides new insights on mitochondria-driven inflammation as a potential therapeutic target for neurodegenerative and primary mitochondrial diseases.
    Keywords:  antiviral response; inflammation; innate immunity; interferon; mitochondrial disorders; mitochondrial dysfunction; mtDNA; mtRNA; neurodegeneration
    DOI:  https://doi.org/10.3390/ijms22168523
  4. J Biochem Mol Toxicol. 2021 Aug 25. e22898
    Requirements for successful mitochondrial transplantation.
    Gokhan Burcin Kubat, Oner Ulger, Senay Akin.
      Maintenance of mitochondrial oxidative phosphorylation capacity and other mitochondrial functions are essential for the prevention of mitochondrial dysfunction-related diseases such as neurodegenerative, cardiovascular, and liver diseases. To date, no well-known treatment modality has been developed to prevent or reduce mitochondrial dysfunction. However, a novel approach that transplants fully functional mitochondria directly into defective cells has recently caught the attention of scientists. In this review, we provide an overview of the cell/tissue source of the mitochondria to prompt cell regeneration or tissue repair in vitro and in vivo applications. The animal and human models entail that effective procedures should be used in the isolation and confirmation of mitochondrial membrane potential and function. We believe that these procedures for mitochondrial transplantation for tissue or cell culture will confirm intact, viable, and free from contamination isolated mitochondria from the appropriate sources.
    Keywords:  mitochondria dysfunction; mitochondrial isolation; mitochondrial transplantation
    DOI:  https://doi.org/10.1002/jbt.22898
  5. EMBO J. 2021 Aug 23. e107988
    Mechanism of transcription initiation and primer generation at the mitochondrial replication origin OriL.
    Azadeh Sarfallah, Angelica Zamudio-Ochoa, Michael Anikin, Dmitry Temiakov.
      The intricate process of human mtDNA replication requires the coordinated action of both transcription and replication machineries. Transcription and replication events at the lagging strand of mtDNA prompt the formation of a stem-loop structure (OriL) and the synthesis of a ∼25 nt RNA primer by mitochondrial RNA polymerase (mtRNAP). The mechanisms by which mtRNAP recognizes OriL, initiates transcription, and transfers the primer to the replisome are poorly understood. We found that transcription initiation at OriL involves slippage of the nascent transcript. The transcript slippage is essential for initiation complex stability and its ability to translocate the mitochondrial DNA polymerase gamma, PolG, which pre-binds to OriL, downstream of the replication origin thus allowing for the primer synthesis. Our data suggest the primosome assembly at OriL-a complex of mtRNAP and PolG-can efficiently generate the primer, transfer it to the replisome, and protect it from degradation by mitochondrial endonucleases.
    Keywords:  POLRMT; PolG; mitochondrial replication; mitochondrial transcription; primosome
    DOI:  https://doi.org/10.15252/embj.2021107988
  6. Curr Opin Chem Biol. 2021 Aug 23. pii: S1367-5931(21)00100-9. [Epub ahead of print]
    Substrates and interactors of the ClpP protease in the mitochondria.
    Mark F Mabanglo, Vaibhav Bhandari, Walid A Houry.
      The ClpP protease is found across eukaryotic and prokaryotic organisms. It is well-characterized in bacteria where its function is important in maintaining protein homeostasis. Along with its ATPase partners, it has been shown to play critical roles in the regulation of enzymes involved in important cellular pathways. In eukaryotes, ClpP is found within cellular organelles. Proteomic studies have begun to characterize the role of this protease in the mitochondria through its interactions. Here, we discuss the proteomic techniques used to identify its interactors and present an atlas of mitochondrial ClpP substrates. The ClpP substrate pool is extensive and consists of proteins involved in essential mitochondrial processes such as the Krebs cycle, oxidative phosphorylation, translation, fatty acid metabolism, and amino acid metabolism. Discoveries of these associations have begun to illustrate the functional significance of ClpP in human health and disease.
    Keywords:  Cancer; ClpP protease; Mitochondria; Mitochondrial diseases; Parkinson's disease; Protein quality control; Proteolysis; Proteomics; Proteostasis
    DOI:  https://doi.org/10.1016/j.cbpa.2021.07.003
  7. FEBS J. 2021 Aug 26.
    Approaches to monitor ATP levels in living cells: where do we stand?
    Seyta Ley-Ngardigal, Giulia Bertolin.
      ATP is the most universal and essential energy molecule in cells. This is due to its ability to store cellular energy in form of high energy phosphate bonds, which are extremely stable and readily usable by the cell. This energy is key for a variety of biological functions such as cell growth and division, metabolism, signaling, and for the turnover of biomolecules. Understanding how ATP is produced and hydrolyzed with a spatiotemporal resolution is necessary to understand its functions both in physiological and pathological contexts. In this review, we will first describe the organization of the electron transport chain and ATP synthase, the main molecular motor for ATP production in mitochondria. Second, we will review the biochemical assays currently available to estimate ATP quantities in cells, and we will compare their readouts, strengths and weaknesses. Finally, we will explore the palette of genetically-encoded biosensors designed for microscopy-based approaches, and show how their spatiotemporal resolution opened up the possibility to follow ATP levels in living cells.
    Keywords:  ATP; ATP synthase; biochemical assays; fluorescence-based tools; mitochondria
    DOI:  https://doi.org/10.1111/febs.16169
  8. Nucleic Acids Res. 2021 Aug 24. pii: gkab726. [Epub ahead of print]
    MitoPhen database: a human phenotype ontology-based approach to identify mitochondrial DNA diseases.
    Thiloka E Ratnaike, Daniel Greene, Wei Wei, Alba Sanchis-Juan, Katherine R Schon, Jelle van den Ameele, Lucy Raymond, Rita Horvath, Ernest Turro, Patrick F Chinnery.
      Diagnosing mitochondrial disorders remains challenging. This is partly because the clinical phenotypes of patients overlap with those of other sporadic and inherited disorders. Although the widespread availability of genetic testing has increased the rate of diagnosis, the combination of phenotypic and genetic heterogeneity still makes it difficult to reach a timely molecular diagnosis with confidence. An objective, systematic method for describing the phenotypic spectra for each variant provides a potential solution to this problem. We curated the clinical phenotypes of 6688 published individuals with 89 pathogenic mitochondrial DNA (mtDNA) mutations, collating 26 348 human phenotype ontology (HPO) terms to establish the MitoPhen database. This enabled a hypothesis-free definition of mtDNA clinical syndromes, an overview of heteroplasmy-phenotype relationships, the identification of under-recognized phenotypes, and provides a publicly available reference dataset for objective clinical comparison with new patients using the HPO. Studying 77 patients with independently confirmed positive mtDNA diagnoses and 1083 confirmed rare disease cases with a non-mitochondrial nuclear genetic diagnosis, we show that HPO-based phenotype similarity scores can distinguish these two classes of rare disease patients with a false discovery rate <10% at a sensitivity of 80%. Enriching the MitoPhen database with more patients will improve predictions for increasingly rare variants.
    DOI:  https://doi.org/10.1093/nar/gkab726
  9. J Control Release. 2021 Aug 24. pii: S0168-3659(21)00444-2. [Epub ahead of print]
    Microvesicles transfer mitochondria and increase mitochondrial function in brain endothelial cells.
    Anisha D'Souza, Amelia Burch, Kandarp M Dave, Aravind Sreeram, Michael J Reynolds, Duncan X Dobbins, Yashika S Kamte, Wanzhu Zhao, Courtney Sabatelle, Gina M Joy, Vishal Soman, Uma R Chandran, Sruti S Shiva, Nidia Quillinan, Paco S Herson, Devika S Manickam.
      We have demonstrated, for the first time that microvesicles, a sub-type of extracellular vesicles (EVs) derived from hCMEC/D3: a human brain endothelial cell (BEC) line transfer polarized mitochondria to recipient BECs in culture and to neurons in mice acute brain cortical and hippocampal slices. This mitochondrial transfer increased ATP levels by 100 to 200-fold (relative to untreated cells) in the recipient BECs exposed to oxygen-glucose deprivation, an in vitro model of cerebral ischemia. We have also demonstrated that transfer of microvesicles, the larger EV fraction, but not exosomes resulted in increased mitochondrial function in hypoxic endothelial cultures. Gene ontology and pathway enrichment analysis of EVs revealed a very high association to glycolysis-related processes. In comparison to heterotypic macrophage-derived EVs, BEC-derived EVs demonstrated a greater selectivity to transfer mitochondria and increase endothelial cell survival under ischemic conditions.
    Keywords:  BBB protection; Exosomes; Extracellular vesicles; Ischemic stroke; Microvesicles; Mitochondrial function; Mitochondrial transfer
    DOI:  https://doi.org/10.1016/j.jconrel.2021.08.038
  10. Transl Pediatr. 2021 Jul;10(7): 1972-1973
    In-depth understanding of Pearson syndrome arising from a novel large mitochondrial DNA deletion in an infant case.
    Rui Liu, Gui-Ling Mo, Yuan-Zong Song.
      
    DOI:  https://doi.org/10.21037/tp-21-199
  11. Physiol Rep. 2021 Aug;9(16): e15016
    Voluntary exercise prevents abnormal muscle mitochondrial morphology in cancer cachexia mice.
    Yu Kitaoka, Mitsunori Miyazaki, Shin Kikuchi.
      This study aimed to examine the effects of voluntary wheel running on cancer cachexia-induced mitochondrial alterations in mouse skeletal muscle. Mice bearing colon 26 adenocarcinoma (C26) were used as a model of cancer cachexia. C26 mice showed a lower gastrocnemius and plantaris muscle weight, but 4 weeks of voluntary exercise rescued these changes. Further, voluntary exercise attenuated observed declines in the levels of oxidative phosphorylation proteins and activities of citrate synthase and cytochrome c oxidase in the skeletal muscle of C26 mice. Among mitochondrial morphology regulatory proteins, mitofusin 2 (Mfn2) and dynamin-related protein 1 (Drp1) were decreased in the skeletal muscle of C26 mice, but exercise resulted in similar improvements as seen in markers of mitochondrial content. In isolated mitochondria, 4-hydroxynonenal and protein carbonyls were elevated in C26 mice, but exercise blunted the increases in these markers of oxidative stress. In addition, electron microscopy revealed that exercise alleviated the observed increase in the percentage of damaged mitochondria in C26 mice. These results suggest that voluntary exercise effectively counteracts mitochondrial dysfunction to mitigate muscle loss in cachexia.
    Keywords:  cancer cachexia; mitochondria; oxidative stress; skeletal muscle; voluntary exercise
    DOI:  https://doi.org/10.14814/phy2.15016
  12. Biomedicines. 2021 Jul 24. pii: 881. [Epub ahead of print]9(8):
    Structural and Functional Alterations in Mitochondria-Associated Membranes (MAMs) and in Mitochondria Activate Stress Response Mechanisms in an In Vitro Model of Alzheimer's Disease.
    Tânia Fernandes, Rosa Resende, Diana F Silva, Ana P Marques, Armanda E Santos, Sandra M Cardoso, M Rosário Domingues, Paula I Moreira, Cláudia F Pereira.
      Alzheimer's disease (AD) is characterized by the accumulation of extracellular plaques composed by amyloid-β (Aβ) and intracellular neurofibrillary tangles of hyperphosphorylated tau. AD-related neurodegenerative mechanisms involve early changes of mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) and impairment of cellular events modulated by these subcellular domains. In this study, we characterized the structural and functional alterations at MAM, mitochondria, and ER/microsomes in a mouse neuroblastoma cell line (N2A) overexpressing the human amyloid precursor protein (APP) with the familial Swedish mutation (APPswe). Proteins levels were determined by Western blot, ER-mitochondria contacts were quantified by transmission electron microscopy, and Ca2+ homeostasis and mitochondria function were analyzed using fluorescent probes and Seahorse assays. In this in vitro AD model, we found APP accumulated in MAM and mitochondria, and altered levels of proteins implicated in ER-mitochondria tethering, Ca2+ signaling, mitochondrial dynamics, biogenesis and protein import, as well as in the stress response. Moreover, we observed a decreased number of close ER-mitochondria contacts, activation of the ER unfolded protein response, reduced Ca2+ transfer from ER to mitochondria, and impaired mitochondrial function. Together, these results demonstrate that several subcellular alterations occur in AD-like neuronal cells, which supports that the defective ER-mitochondria crosstalk is an important player in AD physiopathology.
    Keywords:  Alzheimer’s disease; Ca2+ signaling; ER-mitochondria contacts; mitochondrial dysfunction; subcellular fractions
    DOI:  https://doi.org/10.3390/biomedicines9080881
  13. Cells. 2021 Aug 03. pii: 1968. [Epub ahead of print]10(8):
    Loosening ER-Mitochondria Coupling by the Expression of the Presenilin 2 Loop Domain.
    Michela Rossini, Paloma García-Casas, Riccardo Filadi, Paola Pizzo.
      Presenilin 2 (PS2), one of the three proteins in which mutations are linked to familial Alzheimer's disease (FAD), exerts different functions within the cell independently of being part of the γ-secretase complex, thus unrelated to toxic amyloid peptide formation. In particular, its enrichment in endoplasmic reticulum (ER) membrane domains close to mitochondria (i.e., mitochondria-associated membranes, MAM) enables PS2 to modulate multiple processes taking place on these signaling hubs, such as Ca2+ handling and lipid synthesis. Importantly, upregulated MAM function appears to be critical in AD pathogenesis. We previously showed that FAD-PS2 mutants reinforce ER-mitochondria tethering, by interfering with the activity of mitofusin 2, favoring their Ca2+ crosstalk. Here, we deepened the molecular mechanism underlying PS2 activity on ER-mitochondria tethering, identifying its protein loop as an essential domain to mediate the reinforced ER-mitochondria connection in FAD-PS2 models. Moreover, we introduced a novel tool, the PS2 loop domain targeted to the outer mitochondrial membrane, Mit-PS2-LOOP, that is able to counteract the activity of FAD-PS2 on organelle tethering, which possibly helps in recovering the FAD-PS2-associated cellular alterations linked to an increased organelle coupling.
    Keywords:  Alzheimer’s disease; Ca2+ signaling; ER; MAM; Presenilin 2; mitochondria; organelle contacts
    DOI:  https://doi.org/10.3390/cells10081968
  14. MethodsX. 2021 ;8 101372
    Dual-color dSTORM imaging and ThunderSTORM image reconstruction and analysis to study the spatial organization of the nuclear phosphatidylinositol phosphates.
    Peter Hoboth, Ondřej Šebesta, Martin Sztacho, Enrique Castano, Pavel Hozák.
      Single molecule localization microscopy (SMLM) provided an unprecedented insight into the sub-nuclear organization of proteins and nucleic acids but apart from the nuclear envelope the role of the nuclear lipids in the functional organization of the cell nucleus was less studied. Nevertheless, nuclear lipids and specifically phosphatidylinositol phosphates (PIPs) play increasingly evident roles in gene expression. Therefore, here we provide the SMLM-based approach for the quantitative evaluation of the nuclear PIPs distribution while preserving the context of nuclear architecture. Specifically, on the example of phosphatidylinositol 4,5-bisphosphate (PIP2) we have:•Implemented and optimized the dual-color dSTORM imaging of nuclear PIP2.•Customized the Nearest Neighbor Distance analysis using ImageJ2 plug-in ThunderSTORM to quantitatively evaluate the spatial distribution of nuclear PIP2.•Developed an ImageJ2 tool for the visualization of the Nearest Neighbor Distance analysis results in cellulo.Our customization of the dual-color dSTORM imaging and quantitative analysis provide a tool that is independent of but complementary to the biochemical and lipidomic analyses of the nuclear PIPs. Contrary to the biochemical and lipidomic analyses, the advantage of our analysis is that it preserves the spatial context of the nuclear PIP distribution.
    Keywords:  Cell nucleus; Fibrillarin; ImageJ; Immunofluorescence; Nearest neighbor distance; Nuclear architecture; Nuclear speckles; RNA polymerase II; SON; Super-resolution microscopy; Wide-field microscopy
    DOI:  https://doi.org/10.1016/j.mex.2021.101372
  15. PLoS Comput Biol. 2021 Aug 24. 17(8): e1009290
    miQC: An adaptive probabilistic framework for quality control of single-cell RNA-sequencing data.
    Ariel A Hippen, Matias M Falco, Lukas M Weber, Erdogan Pekcan Erkan, Kaiyang Zhang, Jennifer Anne Doherty, Anna Vähärautio, Casey S Greene, Stephanie C Hicks.
      Single-cell RNA-sequencing (scRNA-seq) has made it possible to profile gene expression in tissues at high resolution. An important preprocessing step prior to performing downstream analyses is to identify and remove cells with poor or degraded sample quality using quality control (QC) metrics. Two widely used QC metrics to identify a 'low-quality' cell are (i) if the cell includes a high proportion of reads that map to mitochondrial DNA (mtDNA) encoded genes and (ii) if a small number of genes are detected. Current best practices use these QC metrics independently with either arbitrary, uniform thresholds (e.g. 5%) or biological context-dependent (e.g. species) thresholds, and fail to jointly model these metrics in a data-driven manner. Current practices are often overly stringent and especially untenable on certain types of tissues, such as archived tumor tissues, or tissues associated with mitochondrial function, such as kidney tissue [1]. We propose a data-driven QC metric (miQC) that jointly models both the proportion of reads mapping to mtDNA genes and the number of detected genes with mixture models in a probabilistic framework to predict the low-quality cells in a given dataset. We demonstrate how our QC metric easily adapts to different types of single-cell datasets to remove low-quality cells while preserving high-quality cells that can be used for downstream analyses. Our software package is available at https://bioconductor.org/packages/miQC.
    DOI:  https://doi.org/10.1371/journal.pcbi.1009290
This page is being maintained by Thomas Krichel. It was last updated on 2021‒09‒19 at 08:30:35.