bims-midmar 2021-10-10 papers

bims-midmar

Biomed News

on Mitochondrial DNA maintenance and replication

Issue of 2021‒10‒10
twenty-one papers selected by
Flavia Söllner
Ludwig-Maximilians University

TFAM knockdown-triggered mtDNA-nucleoid aggregation and a decrease in mtDNA copy number induce the reorganization of nucleoid populations and mitochondria-associated ER-membrane contacts.
Beyond Base Excision Repair: An evolving picture of mitochondrial DNA repair.
Using Atomic Force Microscopy to Study the Real Time Dynamics of DNA Unwinding by Mitochondrial Twinkle Helicase.
Quantification of mtDNA content in cultured cells by direct droplet digital PCR.
Remdesivir triphosphate blocks DNA synthesis and increases exonucleolysis by the replicative mitochondrial DNA polymerase, Pol γ.
Mitochondrial DNA copy number and heteroplasmy load correlate with skeletal muscle oxidative capacity by P31 MR spectroscopy.
A replication-linked mutational gradient drives somatic mutation accumulation and influences germline polymorphisms and genome composition in mitochondrial DNA.
Topoisomerase Assays.
Neonatal-onset mitochondrial disease: clinical features, molecular diagnosis and prognosis.
Oklahoma Nathan Shock Aging Center - assessing the basic biology of aging from genetics to protein and function.
Molecular Machinery and Pathophysiology of Mitochondrial Dynamics.
A Mild Phenotype of Mitochondrial DNA Depletion Syndrome Type 13 with a Novel FBXL4 Variant.
DeepLearnMOR: a deep-learning framework for fluorescence image-based classification of organelle morphology.
Humanin-induced autophagy plays important roles in skeletal muscle function and lifespan extension.
Mitonuclear Interactions Produce Diverging Responses to Mild Stress in Drosophila Larvae.
The Gro3p factor: Restoring NAD+/NADH homeostasis to ameliorate mitochondrial disease.
The multifaceted ATPase inhibitory factor 1 (IF1) in energy metabolism reprogramming and mitochondrial dysfunction: a new player in age-associated disorders?
The mitochondrial genome and Epigenome of the Golden lion Tamarin from fecal DNA using Nanopore adaptive sequencing.
Visualizing formation of the active site in the mitochondrial ribosome.
New quaternized pyridinium derivatives of betulin: Synthesis and evaluation of membranotropic properties on liposomes, pro- and eukaryotic cells, and isolated mitochondria.
Morphological principles of neuronal mitochondria.

Biochem Biophys Rep. 2021 Dec;28 101142

TFAM knockdown-triggered mtDNA-nucleoid aggregation and a decrease in mtDNA copy number induce the reorganization of nucleoid populations and mitochondria-associated ER-membrane contacts.

Koit Aasumets, Yuliya Basikhina, Jaakko L Pohjoismäki, Steffi Goffart, Joachim Gerhold.

  The correct organization of mitochondrial DNA (mtDNA) in nucleoids and the contacts of mitochondria with the ER play an important role in maintaining the mitochondrial genome distribution within the cell. Mitochondria-associated ER membranes (MAMs) consist of interacting proteins and lipids located in the outer mitochondrial membrane and ER membrane, forming a platform for the mitochondrial inner membrane-associated genome replication factory as well as connecting the nucleoids with the mitochondrial division machinery. We show here that knockdown of a core component of mitochondrial nucleoids, TFAM, causes changes in the mitochondrial nucleoid populations, which subsequently impact ER-mitochondria membrane contacts. Knockdown of TFAM causes a significant decrease in the copy number of mtDNA as well as aggregation of mtDNA nucleoids. At the same time, it causes significant upregulation of the replicative TWNK helicase in the membrane-associated nucleoid fraction. This is accompanied by a transient elevation of MAM proteins, indicating a rearrangement of the linkage between ER and mitochondria triggered by changes in mitochondrial nucleoids. Reciprocal knockdown of the mitochondrial replicative helicase TWNK causes a decrease in mtDNA copy number and modifies mtDNA membrane association, however, it does not cause nucleoid aggregation and considerable alterations of MAM proteins in the membrane-associated fraction. Our explanation is that the aggregation of mitochondrial nucleoids resulting from TFAM knockdown triggers a compensatory mechanism involving the reorganization of both mitochondrial nucleoids and MAM. These results could provide an important insight into pathological conditions associated with impaired nucleoid organization or defects of mtDNA distribution.

Keywords:  Mitochondrial DNA; Mitochondrial transcription factor A (TFAM); Nucleoids; Organellar membranes; TWNK helicase

DOI:  https://doi.org/10.1016/j.bbrep.2021.101142
Biosci Rep. 2021 Oct 05. pii: BSR20211320. [Epub ahead of print]

Beyond Base Excision Repair: An evolving picture of mitochondrial DNA repair.

Kathrin Allkanjari, Robert A Baldock.

  Mitochondria are highly specialised organelles required for cellular processes including ATP-production through cellular respiration and controlling apoptosis. Mitochondria contain their own DNA genome which encodes both protein and RNA required for cellular respiration. Each cell may contain hundreds to thousands of copies of the mitochondrial genome, which is essential for normal cellular function - deviation of mitochondrial DNA (mtDNA) copy number is associated with cellular aging and disease. Furthermore, mtDNA lesions can arise from both endogenous or exogenous sources and must either be tolerated or corrected to preserve mitochondrial function. Importantly, replication of damaged mtDNA can lead to stalling and introduction of mutations or genetic loss, mitochondria have adapted mechanisms to repair damaged DNA. These mechanisms rely on nuclear encoded DNA repair proteins that are translocated into the mitochondria. Despite the presence of many known nuclear DNA repair proteins being found in the mitochondrial proteome, it remains to be established which DNA repair mechanisms are functional in mammalian mitochondria. Here, we summarise the existing and emerging research, alongside examining proteomic evidence, demonstrating that mtDNA damage can be repaired using Base Excision Repair (BER), Homologous Recombination (HR) and Microhomology-mediated End Joining (MMEJ). Critically, these repair mechanisms do not operate in isolation and evidence for interplay between pathways and repair associated with replication is discussed. Importantly, characterising non-canonical functions of key proteins and understanding the bespoke pathways used to tolerate, repair or bypass DNA damage will be fundamental in fully understanding the causes of mitochondrial genome mutations and mitochondrial dysfunction.

Keywords:  DNA replication and recombination; DNA synthesis and repair; genome integrity; mtDNA

DOI:  https://doi.org/10.1042/BSR20211320
Bio Protoc. 2021 Sep 05. 11(17): e4139

Using Atomic Force Microscopy to Study the Real Time Dynamics of DNA Unwinding by Mitochondrial Twinkle Helicase.

Parminder Kaur, Hai Pan, Matthew J Longley, William C Copeland, Hong Wang.

  Understanding the structure and dynamics of DNA-protein interactions during DNA replication is crucial for elucidating the origins of disorders arising from its dysfunction. In this study, we employed Atomic Force Microscopy as a single-molecule imaging tool to examine the mitochondrial DNA helicase Twinkle and its interactions with DNA. We used imaging in air and time-lapse imaging in liquids to observe the DNA binding and unwinding activities of Twinkle hexamers at the single-molecule level. These procedures helped us visualize Twinkle loading onto and unloading from the DNA in the open-ring conformation. Using traditional methods, it has been shown that Twinkle is capable of unwinding dsDNA up to 20-55 bps. We found that the addition of mitochondrial single-stranded DNA binding protein (mtSSB) facilitates a 5-fold increase in the DNA unwinding rate for the Twinkle helicase. The protocols developed in this study provide new platforms to examine DNA replication and to explore the mechanism driving DNA deletion and human diseases. Graphic abstract: Mitochondrial Twinkle Helicase Dynamics.

Keywords:  Atomic Force Microscope; Liquid AFM imaging; Mitochondria; Mitochondrial replication; Single molecule imaging; Twinkle helicase

DOI:  https://doi.org/10.21769/BioProtoc.4139
Mitochondrion. 2021 Oct 01. pii: S1567-7249(21)00140-9. [Epub ahead of print]

Quantification of mtDNA content in cultured cells by direct droplet digital PCR.

Natalya Kozhukhar, Anthony Fant, Mikhail F Alexeyev.

  Although alterations in cellular mitochondrial DNA (mtDNA) content have been linked to various pathological conditions, the mechanisms that govern mtDNA copy number (mtCN) control remain poorly understood. Moreover, techniques for mtDNA quantification do not allow for direct comparisons of absolute mtCNs between labs. Here we report the development of a direct droplet digital PCR technique for the determination of mtCNs in whole-cell lysates. Using this technique, we demonstrate that cellular mtDNA content can fluctuate in culture by as much as 50% and provide evidence for both cell proliferation-coupled and uncoupled mtDNA replication.

Keywords:  Mitochondrial DNA; X-ray; cell cycle; cell proliferation; mtDNA copy number; mtDNA replication

DOI:  https://doi.org/10.1016/j.mito.2021.09.014
Mitochondrion. 2021 Oct 04. pii: S1567-7249(21)00136-7. [Epub ahead of print]

Remdesivir triphosphate blocks DNA synthesis and increases exonucleolysis by the replicative mitochondrial DNA polymerase, Pol γ.

Elena J Ciesielska, Shalom Kim, Hyacintha-Ghislaine M Bisimwa, Cody Grier, Md Mostafijur Rahman, Carolyn K J Young, Matthew J Young, Marcos T Oliveira, Grzegorz L Ciesielski.

  The COVID-19 pandemic prompted the FDA to authorize a new nucleoside analogue, remdesivir, for emergency use in affected individuals. We examined the effects of its active metabolite, remdesivir triphosphate (RTP), on the activity of the replicative mitochondrial DNA polymerase, Pol γ. We found that while RTP is not incorporated by Pol γ into a nascent DNA strand, it remains associated with the enzyme impeding its synthetic activity and stimulating exonucleolysis. In spite of that, we found no evidence for deleterious effects of remdesivir treatment on the integrity of the mitochondrial genome in human cells in culture.

Keywords:  Antiviral nucleoside analogues; COVID-19; DNA polymerase gamma; Mitochondrial DNA; Remdesivir

DOI:  https://doi.org/10.1016/j.mito.2021.09.010
Aging Cell. 2021 Oct 06. e13487

Mitochondrial DNA copy number and heteroplasmy load correlate with skeletal muscle oxidative capacity by P31 MR spectroscopy.

Qu Tian, Ann Zenobia Moore, Richard Oppong, Jun Ding, Marta Zampino, Kenneth W Fishbein, Richard G Spencer, Luigi Ferrucci.

  The association between blood-based estimates of mitochondrial DNA parameters, mitochondrial DNA copy number (mtDNA-CN) and heteroplasmy load, with skeletal muscle bioenergetic capacity was evaluated in 230 participants of the Baltimore Longitudinal Study of Aging (mean age:74.7 years, 53% women). Participants in the study sample had concurrent data on muscle oxidative capacity (τPCr ) assessed by 31 P magnetic resonance spectroscopy, and mitochondrial DNA parameters estimated from whole-genome sequencing data. In multivariable linear regression models, adjusted for age, sex, extent of phosphocreatine (PCr) depletion, autosomal sequencing coverage, white blood cell total, and differential count, as well as platelet count, mtDNA-CN and heteroplasmy load were not significantly associated with τPCr (both p > 0.05). However, in models evaluating whether the association between mtDNA-CN and τPCr varied by heteroplasmy load, there was a significant interaction between mtDNA-CN and heteroplasmy load (p = 0.037). In stratified analysis, higher mtDNA-CN was significantly associated with lower τPCr among participants with high heteroplasmy load (n = 84, β (SE) = -0.236 (0.115), p-value = 0.044), but not in those with low heteroplasmy load (n = 146, β (SE) = 0.046 (0.119), p-value = 0.702). Taken together, mtDNA-CN and heteroplasmy load provide information on muscle bioenergetics. Thus, mitochondrial DNA parameters may be considered proxy measures of mitochondrial function that can be used in large epidemiological studies, especially when comparing subgroups.

Keywords:  aging; mitochondrial DNA; skeletal muscle

DOI:  https://doi.org/10.1111/acel.13487
Nucleic Acids Res. 2021 Oct 06. pii: gkab901. [Epub ahead of print]

A replication-linked mutational gradient drives somatic mutation accumulation and influences germline polymorphisms and genome composition in mitochondrial DNA.

Monica Sanchez-Contreras, Mariya T Sweetwyne, Brendan F Kohrn, Kristine A Tsantilas, Michael J Hipp, Elizabeth K Schmidt, Jeanne Fredrickson, Jeremy A Whitson, Matthew D Campbell, Peter S Rabinovitch, David J Marcinek, Scott R Kennedy.

Mutations in mitochondrial DNA (mtDNA) cause maternally inherited diseases, while somatic mutations are linked to common diseases of aging. Although mtDNA mutations impact health, the processes that give rise to them are under considerable debate. To investigate the mechanism by which de novo mutations arise, we analyzed the distribution of naturally occurring somatic mutations across the mouse and human mtDNA obtained by Duplex Sequencing. We observe distinct mutational gradients in G→A and T→C transitions delimited by the light-strand origin and the mitochondrial Control Region (mCR). The gradient increases unequally across the mtDNA with age and is lost in the absence of DNA polymerase γ proofreading activity. In addition, high-resolution analysis of the mCR shows that important regulatory elements exhibit considerable variability in mutation frequency, consistent with them being mutational 'hot-spots' or 'cold-spots'. Collectively, these patterns support genome replication via a deamination prone asymmetric strand-displacement mechanism as the fundamental driver of mutagenesis in mammalian DNA. Moreover, the distribution of mtDNA single nucleotide polymorphisms in humans and the distribution of bases in the mtDNA across vertebrate species mirror this gradient, indicating that replication-linked mutations are likely the primary source of inherited polymorphisms that, over evolutionary timescales, influences genome composition during speciation.

DOI: https://doi.org/10.1093/nar/gkab901
Curr Protoc. 2021 Oct;1(10): e250

Topoisomerase Assays.

John L Nitiss, Kostantin Kiianitsa, Yilun Sun, Karin C Nitiss, Nancy Maizels.

  Topoisomerases are enzymes that play essential roles in DNA replication, transcription, chromosome segregation, and recombination. All cells have two major forms of DNA topoisomerases: type I enzymes, which make single-stranded cuts in DNA, and type II enzymes, which cut and decatenate double-stranded DNA. DNA topoisomerases are important targets of approved and experimental anti-cancer agents. Provided in this article are protocols to assess activities of topoisomerases and their inhibitors. Included are an assay for topoisomerase I activity based on relaxation of supercoiled DNA; an assay for topoisomerase II based on the decatenation of double-stranded DNA; and approaches for enriching and quantifying DNA-protein covalent complexes formed as obligatory intermediates in the reactions of type I and II topoisomerases with DNA; and assays for measuring DNA cleavage in vitro. Topoisomerases are not the only proteins that form covalent adducts with DNA in living cells, and the approaches described here are likely to find use in characterizing other protein-DNA adducts and exploring their utility as targets for therapy. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Assay of topoisomerase I activity Basic Protocol 2: Assay of topoisomerase II activity Basic Protocol 3: In vivo determination of topoisomerase covalent complexes using the in vivo complex of enzyme (ICE) assay Support Protocol 1: Preparation of mouse tissue for determination of topoisomerase covalent complexes using the ICE assay Support Protocol 2: Using recombinant topoisomerase standard for absolute quantification of cellular TOP2CC Basic Protocol 4: Quantification of topoisomerase-DNA covalent complexes by RADAR/ELISA: The rapid approach to DNA adduct recovery (RADAR) combined with the enzyme-linked immunosorbent assay (ELISA) Basic Protocol 5: Analysis of protein-DNA covalent complexes by RADAR/Western Support Protocol 3: Adduct-Seq to characterize adducted DNA Support Protocol 4: Nuclear fractionation and RNase treatment to reduce sample complexity Basic Protocol 6: Determination of DNA cleavage by purified topoisomerase I Basic Protocol 7: Determination of inhibitor effects on DNA cleavage by topoisomerase II using a plasmid linearization assay Alternate Protocol: Gel electrophoresis determination of topoisomerase II cleavage.

Keywords:  DNA cleavage; ICE assay; RADAR enrichment; camptothecin; etoposide; protein-DNA adduct; topoisomerase; topoisomerase I; topoisomerase II; topoisomerase poison

DOI:  https://doi.org/10.1002/cpz1.250
Arch Dis Child Fetal Neonatal Ed. 2021 Oct 07. pii: fetalneonatal-2021-321633. [Epub ahead of print]

Neonatal-onset mitochondrial disease: clinical features, molecular diagnosis and prognosis.

Tomohiro Ebihara, Taro Nagatomo, Yohei Sugiyama, Tomoko Tsuruoka, Yoshiteru Osone, Masaru Shimura, Makiko Tajika, Tetsuro Matsuhashi, Keiko Ichimoto, Ayako Matsunaga, Nana Akiyama, Minako Ogawa-Tominaga, Yukiko Yatsuka, Kazuhiro R Nitta, Yoshihito Kishita, Takuya Fushimi, Atsuko Imai-Okazaki, Akira Ohtake, Yasushi Okazaki, Kei Murayama.

  OBJECTIVE: Neonatal-onset mitochondrial disease has not been fully characterised owing to its heterogeneity. We analysed neonatal-onset mitochondrial disease in Japan to clarify its clinical features, molecular diagnosis and prognosis.DESIGN: Retrospective observational study from January 2004 to March 2020.
SETTING: Population based.
PATIENTS: Patients (281) with neonatal-onset mitochondrial disease diagnosed by biochemical and genetic approaches.
INTERVENTIONS: None.
MAIN OUTCOME MEASURES: Disease types, initial symptoms, biochemical findings, molecular diagnosis and prognosis.
RESULTS: Of the 281 patients, multisystem mitochondrial disease was found in 194, Leigh syndrome in 26, cardiomyopathy in 38 and hepatopathy in 23 patients. Of the 321 initial symptoms, 236 occurred within 2 days of birth. Using biochemical approaches, 182 patients were diagnosed by mitochondrial respiratory chain enzyme activity rate and 89 by oxygen consumption rate. The remaining 10 patients were diagnosed using a genetic approach. Genetic analysis revealed 69 patients had nuclear DNA variants in 36 genes, 11 of 15 patients had mitochondrial DNA variants in five genes and four patients had single large deletion. The Cox proportional hazards regression analysis showed the effects of Leigh syndrome (HR=0.15, 95% CI 0.04 to 0.63, p=0.010) and molecular diagnosis (HR=1.87, 95% CI 1.18 to 2.96, p=0.008) on survival.
CONCLUSIONS: Neonatal-onset mitochondrial disease has a heterogenous aetiology. The number of diagnoses can be increased, and clarity regarding prognosis can be achieved by comprehensive biochemical and molecular analyses using appropriate tissue samples.

Keywords:  data collection; mortality; neonatology; statistics

DOI:  https://doi.org/10.1136/archdischild-2021-321633
Geroscience. 2021 Oct 04.

Oklahoma Nathan Shock Aging Center - assessing the basic biology of aging from genetics to protein and function.

Holly Van Remmen, Willard M Freeman, Benjamin F Miller, Michael Kinter, Jonathan D Wren, Ann Chiao, Rheal A Towner, Timothy A Snider, William E Sonntag, Arlan Richardson.

  The Oklahoma Shock Nathan Shock Center is designed to deliver unique, innovative services that are not currently available at most institutions. The focus of the Center is on geroscience and the development of careers of young investigators. Pilot grants are provided through the Research Development Core to junior investigators studying aging/geroscience throughout the USA. However, the services of our Center are available to the entire research community studying aging and geroscience. The Oklahoma Nathan Shock Center provides researchers with unique services through four research cores. The Multiplexing Protein Analysis Core uses the latest mass spectrometry technology to simultaneously measure the levels, synthesis, and turnover of hundreds of proteins associated with pathways of importance to aging, e.g., metabolism, antioxidant defense system, proteostasis, and mitochondria function. The Genomic Sciences Core uses novel next-generation sequencing that allows investigators to study the effect of age, or anti-aging manipulations, on DNA methylation, mitochondrial genome heteroplasmy, and the transcriptome of single cells. The Geroscience Redox Biology Core provides investigators with a comprehensive state-of-the-art assessment of the oxidative stress status of a cell, e.g., measures of oxidative damage and redox couples, which are important in aging as well as many major age-related diseases as well as assays of mitochondrial function. The GeroInformatics Core provides investigators assistance with data analysis, which includes both statistical support as well as analysis of large datasets. The Core also has developed number of unique software packages to help with interpretation of results and discovery of new leads relevant to aging. In addition, the Geropathology Research Resource in the Program Enhancement Core provides investigators with pathological assessments of mice using the recently developed Geropathology Grading Platform.

Keywords:  Genomic Sciences Core; Geropathology Grading Platform; Geroscience

DOI:  https://doi.org/10.1007/s11357-021-00454-7
Front Cell Dev Biol. 2021 ;9 743892

Molecular Machinery and Pathophysiology of Mitochondrial Dynamics.

Yi-Han Chiu, Shu-Chuan Amy Lin, Chen-Hsin Kuo, Chia-Jung Li.

  Mitochondria are double-membraned organelles that exhibit fluidity. They are the main site of cellular aerobic respiration, providing energy for cell proliferation, migration, and survival; hence, they are called "powerhouses." Mitochondria play an important role in biological processes such as cell death, cell senescence, autophagy, lipid synthesis, calcium homeostasis, and iron balance. Fission and fusion are active processes that require many specialized proteins, including mechanical enzymes that physically alter mitochondrial membranes, and interface proteins that regulate the interaction of these mechanical proteins with organelles. This review discusses the molecular mechanisms of mitochondrial fusion, fission, and physiopathology, emphasizing the biological significance of mitochondrial morphology and dynamics. In particular, the regulatory mechanisms of mitochondria-related genes and proteins in animal cells are discussed, as well as research trends in mitochondrial dynamics, providing a theoretical reference for future mitochondrial research.

Keywords:  fission; fusion; machinery; mitochondrial dynamics; pathophysiology

DOI:  https://doi.org/10.3389/fcell.2021.743892
Mol Syndromol. 2021 Aug;12(5): 294-299

A Mild Phenotype of Mitochondrial DNA Depletion Syndrome Type 13 with a Novel FBXL4 Variant.

Ummuhan Oncul, Engin Kose, Fatma Tuba Eminoglu.

  Mitochondrial DNA depletion syndromes (MDDS) are a group of rare genetic disorders caused by defects in multiple genes involved in mitochondrial DNA maintenance. Among these, FBXL4 gene variants result in encephalomyopathic mtDNA depletion syndrome 13 (MTDPS13), which commonly presents as a combination of failure to thrive, neurodevelopmental delays, encephalopathy, hypotonia, a pattern of mild facial dysmorphisms, and persistent lactic acidosis. To date, 53 pathogenic FBXL4 variants and 100 cases have been described in the literature. In the present case report, we report on a 4.5-year-old boy with MTDPS13 and a novel variant. The patient had a history of antenatal hydrocephalus, severe developmental delay and mental motor retardation with psychomotor delay, severe hypotonia, mild left ventricular hypertrophic cardiomyopathy, mild facial dysmorphism, and elevated lactate levels. Symptoms suggested mitochondrial myopathy; subsequently, whole-exome sequencing was performed and a novel homozygous variant FBXL4 (NM_012160.4): c.486T>G (p.Tyr162Ter) was identified. While most of the patients with FBLX4 gene mutation have severe clinical manifestation and die at a very young age, clinical progress of our case was milder than previously reported. MDDS are very rare and can present with many different clinical signs and symptoms. In this report, we identified a novel pathogenic variant in the FBXL4 gene. This report shows that patients with FBLX4 gene mutations may present with a milder clinical phenotype than previously reported.

Keywords:  Encephalopathy; FBXL4; Mitochondrial DNA depletion; Myopathy, MTDPS13; mtDNA depletion syndrome

DOI:  https://doi.org/10.1159/000515928
Plant Physiol. 2021 Aug 03. 186(4): 1786-1799

DeepLearnMOR: a deep-learning framework for fluorescence image-based classification of organelle morphology.

Jiying Li, Jinghao Peng, Xiaotong Jiang, Anne C Rea, Jiajie Peng, Jianping Hu.

The proper biogenesis, morphogenesis, and dynamics of subcellular organelles are essential to their metabolic functions. Conventional techniques for identifying, classifying, and quantifying abnormalities in organelle morphology are largely manual and time-consuming, and require specific expertise. Deep learning has the potential to revolutionize image-based screens by greatly improving their scope, speed, and efficiency. Here, we used transfer learning and a convolutional neural network (CNN) to analyze over 47,000 confocal microscopy images from Arabidopsis wild-type and mutant plants with abnormal division of one of three essential energy organelles: chloroplasts, mitochondria, or peroxisomes. We have built a deep-learning framework, DeepLearnMOR (Deep Learning of the Morphology of Organelles), which can rapidly classify image categories and identify abnormalities in organelle morphology with over 97% accuracy. Feature visualization analysis identified important features used by the CNN to predict morphological abnormalities, and visual clues helped to better understand the decision-making process, thereby validating the reliability and interpretability of the neural network. This framework establishes a foundation for future larger-scale research with broader scopes and greater data set diversity and heterogeneity.

DOI: https://doi.org/10.1093/plphys/kiab223
Biochim Biophys Acta Gen Subj. 2021 Oct 05. pii: S0304-4165(21)00176-8. [Epub ahead of print] 130017

Humanin-induced autophagy plays important roles in skeletal muscle function and lifespan extension.

Su-Jeong Kim, Anjali Devgan, Brendan Miller, Sam Mool Lee, Hiroshi Kumagai, Kenneth A Wilson, Gabriella Wassef, Richard Wong, Hemal H Mehta, Pinchas Cohen, Kelvin Yen.

  BACKGROUND: Autophagy, a highly conserved homeostatic mechanism, is essential for cell survival. The decline of autophagy function has been implicated in various diseases as well as aging. Although mitochondria play a key role in the autophagy process, whether mitochondrial-derived peptides are involved in this process has not been explored.METHODS: We developed a high through put screening method to identify potential autophagy inducers among mitochondrial-derived peptides. We used three different cell lines, mice, c.elegans, and a human cohort to validate the observation.
RESULTS: Humanin, a mitochondrial-derived peptide, increases autophagy and maintains autophagy flux in several cell types. Humanin administration increases the expression of autophagy-related genes and lowers accumulation of harmful misfolded proteins in mice skeletal muscle, suggesting that humanin-induced autophagy potentially contributes to the improved skeletal function. Moreover, autophagy is a critical role in humanin-induced lifespan extension in C. elegans.
CONCLUSIONS: Humanin is an autophagy inducer.
GENERAL SIGNIFICANCE: This paper presents a significant, novel discovery regarding the role of the mitochondrial derived peptide humanin in autophagy regulation and as a possible therapeutic target for autophagy in various age-related diseases.

Keywords:  Autophagy; Humanin; Lifespan; Mitochondrial-derived peptides

DOI:  https://doi.org/10.1016/j.bbagen.2021.130017
Front Genet. 2021 ;12 734255

Mitonuclear Interactions Produce Diverging Responses to Mild Stress in Drosophila Larvae.

Enrique Rodríguez, Finley Grover Thomas, M Florencia Camus, Nick Lane.

  Mitochondrial function depends on direct interactions between respiratory proteins encoded by genes in two genomes, mitochondrial and nuclear, which evolve in very different ways. Serious incompatibilities between these genomes can have severe effects on development, fitness and viability. The effect of subtle mitonuclear mismatches has received less attention, especially when subject to mild physiological stress. Here, we investigate how two distinct physiological stresses, metabolic stress (high-protein diet) and redox stress [the glutathione precursor N-acetyl cysteine (NAC)], affect development time, egg-to-adult viability, and the mitochondrial physiology of Drosophila larvae with an isogenic nuclear background set against three mitochondrial DNA (mtDNA) haplotypes: one coevolved (WT) and two slightly mismatched (COX and BAR). Larvae fed the high-protein diet developed faster and had greater viability in all haplotypes. The opposite was true of NAC-fed flies, especially those with the COX haplotype. Unexpectedly, the slightly mismatched BAR larvae developed fastest and were the most viable on both treatments, as well as control diets. These changes in larval development were linked to a shift to complex I-driven mitochondrial respiration in all haplotypes on the high-protein diet. In contrast, NAC increased respiration in COX larvae but drove a shift toward oxidation of proline and succinate. The flux of reactive oxygen species was increased in COX larvae treated with NAC and was associated with an increase in mtDNA copy number. Our results support the notion that subtle mitonuclear mismatches can lead to diverging responses to mild physiological stress, undermining fitness in some cases, but surprisingly improving outcomes in other ostensibly mismatched fly lines.

Keywords:  Drosophila melanogaster; N-acetyl cysteine; diet; high-protein; larvae; mitochondria; mitonuclear interactions; oxidative stress

DOI:  https://doi.org/10.3389/fgene.2021.734255
Cell Metab. 2021 Oct 05. pii: S1550-4131(21)00425-3. [Epub ahead of print]33(10): 1905-1907

The Gro3p factor: Restoring NAD+/NADH homeostasis to ameliorate mitochondrial disease.

Divakar S Mithal, Navdeep S Chandel.

Leigh syndrome, a mitochondrial disease, can be modeled in mice with a deficiency in mitochondrial complex I that results in a decreased NAD+/NADH ratio. In this issue of Cell Metabolism, Liu et al. (2021) identify glycerol-3-phosphate (Gro3P) biosynthesis as a method for regenerating cytosolic NAD+ to ameliorate pathology in this mitochondrial disease model.

DOI: https://doi.org/10.1016/j.cmet.2021.09.006
Antioxid Redox Signal. 2021 Oct 04.

The multifaceted ATPase inhibitory factor 1 (IF1) in energy metabolism reprogramming and mitochondrial dysfunction: a new player in age-associated disorders?

Emilia Gore, Thibaut Duparc, Annelise Genoux, Bertrand Perret, Souad Najib, Laurent O Martinez.

Significance The mitochondrial oxidative phosphorylation (OXPHOS) system, comprising the electron transport chain and ATP synthase, generates membrane potential, drives ATP synthesis, governs energy metabolism, and maintains redox balance. OXPHOS dysfunction is associated with a plethora of diseases ranging from rare inherited disorders to common conditions including diabetes, cancer, neurodegenerative diseases, as well as aging. There has been great interest in studying regulators of OXPHOS. Among these, ATPase inhibitory factor 1 (IF1) is an endogenous inhibitor of ATP synthase that has long been thought to avoid the consumption of cellular ATP when ATP synthase acts as an ATP hydrolysis enzyme. Recent Advances Recent data indicates that IF1 inhibits ATP synthesis and is involved in a multitude of mitochondrial-related functions, such as mitochondrial quality control, energy metabolism, redox balance, and cell fate. IF1 also inhibits the ATPase activity of cell-surface ATP synthase, and it is used as a cardiovascular disease biomarker. Critical Issues Although recent data have led to a paradigm shift regarding IF1 functions, these have been poorly studied in entire organisms and in different organs. The understanding of the cellular biology of IF1 is, therefore, still limited. The aim of this review was to provide an overview of the current understanding of the role of IF1 in mitochondrial functions, health, and diseases. Future Directions Further investigations of IF1 functions at the cell, organ, and whole-organism levels and in different pathophysiological conditions will help decipher the controversies surrounding its involvement in mitochondrial function and could unveil therapeutic strategies in human pathology.

DOI: https://doi.org/10.1089/ars.2021.0137
BMC Genomics. 2021 Oct 07. 22(1): 726

The mitochondrial genome and Epigenome of the Golden lion Tamarin from fecal DNA using Nanopore adaptive sequencing.

Nicole Wanner, Peter A Larsen, Adam McLain, Christopher Faulk.

  BACKGROUND: The golden lion tamarin (Leontopithecus rosalia) is an endangered Platyrrhine primate endemic to the Atlantic coastal forests of Brazil. Despite ongoing conservation efforts, genetic data on this species remains scarce. Complicating factors include limitations on sample collection and a lack of high-quality reference sequences. Here, we used nanopore adaptive sampling to resequence the L. rosalia mitogenome from feces, a sample which can be collected non-invasively.RESULTS: Adaptive sampling doubled the fraction of both host-derived and mitochondrial sequences compared to sequencing without enrichment. 258x coverage of the L. rosalia mitogenome was achieved in a single flow cell by targeting the unfinished genome of the distantly related emperor tamarin (Saguinus imperator) and the mitogenome of the closely related black lion tamarin (Leontopithecus chrysopygus). The L. rosalia mitogenome has a length of 16,597 bp, sharing 99.68% sequence identity with the L. chrysopygus mitogenome. A total of 38 SNPs between them were identified, with the majority being found in the non-coding D-loop region. DNA methylation and hydroxymethylation were directly detected using a neural network model applied to the raw signal from the MinION sequencer. In contrast to prior reports, DNA methylation was negligible in mitochondria in both CpG and non-CpG contexts. Surprisingly, a quarter of the 642 CpG sites exhibited DNA hydroxymethylation greater than 1% and 44 sites were above 5%, with concentration in the 3' side of several coding regions.
CONCLUSIONS: Overall, we report a robust new mitogenome assembly for L. rosalia and direct detection of cytosine base modifications in all contexts.

Keywords:  DNA hydroxymethylation; DNA methylation; Lion Tamarin; Mitochondria; Poop; Primates

DOI:  https://doi.org/10.1186/s12864-021-08046-7
Elife. 2021 10 05. pii: e68806. [Epub ahead of print]10

Visualizing formation of the active site in the mitochondrial ribosome.

Viswanathan Chandrasekaran, Nirupa Desai, Nicholas O Burton, Hanting Yang, Jon Price, Eric A Miska, V Ramakrishnan.

  Ribosome assembly is an essential and conserved process that is regulated at each step by specific factors. Using cryo-electron microscopy (cryo-EM), we visualize the formation of the conserved peptidyl transferase center (PTC) of the human mitochondrial ribosome. The conserved GTPase GTPBP7 regulates the correct folding of 16S ribosomal RNA (rRNA) helices and ensures 2'-O-methylation of the PTC base U3039. GTPBP7 binds the RNA methyltransferase NSUN4 and MTERF4, which sequester H68-71 of the 16S rRNA and allow biogenesis factors to access the maturing PTC. Mutations that disrupt binding of their Caenorhabditis elegans orthologs to the large subunit potently activate mitochondrial stress and cause viability, development, and sterility defects. Next-generation RNA sequencing reveals widespread gene expression changes in these mutant animals that are indicative of mitochondrial stress response activation. We also answer the long-standing question of why NSUN4, but not its enzymatic activity, is indispensable for mitochondrial protein synthesis.

Keywords:  C. elegans; RNA modifications; biochemistry; chemical biology; cryo-EM; human; mitochondrial ribosome; molecular biophysics; peptidyl transferase center; structural biology

DOI:  https://doi.org/10.7554/eLife.68806
Chem Biol Interact. 2021 Oct 01. pii: S0009-2797(21)00316-1. [Epub ahead of print]349 109678

New quaternized pyridinium derivatives of betulin: Synthesis and evaluation of membranotropic properties on liposomes, pro- and eukaryotic cells, and isolated mitochondria.

Mikhail V Dubinin, Alena A Semenova, Anna I Ilzorkina, Natalia Y Markelova, Nikita V Penkov, Elvira R Shakurova, Konstantin N Belosludtsev, Lyudmila V Parfenova.

  The present study describes the synthesis of pyridinium derivatives of betulin, including new 4-methyl- and 3,5-methyl-pyridinium analogs, their effect on artificial membrane systems (liposomes), cytotoxicity in models of prokaryotic (E. coli K-12 MG1655) and eukaryotic cells (rat thymocytes), as well as their effect on the functioning of membrane systems of rat liver mitochondria. We have shown that the presence of methyl groups in the pyridine ring of compounds determines the ability of the derivatives to effectively permeabilize the artificial membrane of lecithin liposomes for the fluorescent probe sulforhodamine B. The 4-methyl- and 3,5-methyl-pyridinium analogs inhibit the growth of E. coli K-12 MG1655 and, at the same time, did not have a cytotoxic effect on rat thymocytes. However, in the latter case, we noted a decrease in the mitochondrial potential of cells. The studied compounds reduced the functional activity of mitochondria, suppressing the activity of complexes of the respiratory chain and reducing the membrane potential. In addition, compounds containing methyl groups in the p- and m-positions of the pyridine ring were also able to permeabilize the inner membrane of mitochondria, causing them to swell. In this case, the most lipophilic compound containing two methyl substituents at the m-position of the pyridine fragment was most effective and had a protonophore effect on mitochondria. The paper discusses the dependence of the membranotropic and biological actions of the quaternized pyridine derivatives of betulin on their structure and lipophilicity.

Keywords:  Betulin; Cytotoxicity; Liposomes; Membrane permeability; Mitochondria; Quaternized pyridinium salts

DOI:  https://doi.org/10.1016/j.cbi.2021.109678
J Comp Neurol. 2021 Oct 05.

Morphological principles of neuronal mitochondria.

Rachel Mendelsohn, Guadalupe C Garcia, Thomas M Bartol, Christopher T Lee, P Khandelwal, Emily Liu, Donald J Spencer, Adam Husar, Eric A Bushong, Sebastien Phan, Guy Perkins, Mark H Ellisman, Alexander Skupin, Terrence J Sejnowski, Padmini Rangamani.

  In the highly dynamic metabolic landscape of a neuron, mitochondrial membrane architectures can provide critical insight into the unique energy balance of the cell. Current theoretical calculations of functional outputs like ATP and heat often represent mitochondria as idealized geometries and therefore can miscalculate the metabolic uxes. To analyze mitochondrial morphology in neurons of mouse cerebellum neuropil, 3D tracings of complete synaptic and axonal mitochondria were constructed using a database of serial TEM tomography images and converted to watertight meshes with minimal distortion of the original microscopy volumes with a granularity of 1.64 nanometer isotropic voxels. The resulting in silico representations were subsequently quanti ed by di erential geometry methods in terms of the mean and Gaussian curvatures, surface areas, volumes, and membrane motifs, all of which can alter the metabolic output of the organelle. Finally, we identify structural motifs present across this population of mitochondria, which may contribute to future modeling studies of mitochondrial physiology and metabolism in neurons. This article is protected by copyright. All rights reserved.

Keywords:  EM tomography; energetics; mitochondria; morphology; neuronal

DOI:  https://doi.org/10.1002/cne.25254