bims-nenemi 2023-02-26 papers

bims-nenemi

Biomed News

on Neuroinflammation, neurodegeneration and mitochondria

Issue of 2023–02–26
seventeen papers selected by
Marco Tigano, Thomas Jefferson University

Visualize the Distribution and Dynamics of Mitochondrial DNA (mtDNA) Nucleoids with Multiple Labeling Strategies.
The role of programmed mitophagy in germline mitochondrial DNA quality control.
Mitochondrial DNA Enrichment for Sensitive Next-Generation Sequencing.
Generation of Mammalian Cells Devoid of Mitochondrial DNA (ρ0 cells).
Measurement of Nucleoid Size Using STED Microscopy.
Cell lineage-specific mitochondrial resilience during mammalian organogenesis.
Localization of Mitochondrial Nucleoids by Transmission Electron Microscopy Using the Transgenic Expression of the Mitochondrial Helicase Twinkle and APEX2.
Regulatory principles of human mitochondrial gene expression revealed by kinetic analysis of the RNA life cycle.
Mitochondrial DNA Sequencing and Heteroplasmy Quantification by Next Generation Sequencing.
High-throughput Assessment of Mitochondrial Protein Synthesis in Mammalian Cells Using Mito-FUNCAT FACS.
TEFM variants impair mitochondrial transcription causing childhood-onset neurological disease.
Mitochondria-Targeted Gene Silencing Facilitated by Mito-CPDs.
Coupling Differential Centrifugation with Exonuclease Treatment and Size Exclusion Chromatography (DIFSEC) for Purification of mtDNA from Mammalian Cells.
Detection of UV-Induced Deletions in Mitochondrial DNA.
Concomitant Sequencing of Accessible Chromatin and Mitochondrial Genomes in Single Cells Using mtscATAC-Seq.
Mitochondrial structure and function adaptation in residual triple negative breast cancer cells surviving chemotherapy treatment.
Transcriptomic Profiling and Pathway Analysis of Mesenchymal Stem Cells Following Low Dose-Rate Radiation Exposure.

Methods Mol Biol. 2023 ;2615 79-88

Visualize the Distribution and Dynamics of Mitochondrial DNA (mtDNA) Nucleoids with Multiple Labeling Strategies.

Xiangjun Di, Jinshan Qin, Yujie Sun, Qian Peter Su.

  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

DOI:  https://doi.org/10.1007/978-1-0716-2922-2_6
Autophagy. 2023 Feb 20.

The role of programmed mitophagy in germline mitochondrial DNA quality control.

Jonathan M Palozzi, Thomas R Hurd.

  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

DOI:  https://doi.org/10.1080/15548627.2023.2182595
Methods Mol Biol. 2023 ;2615 427-441

Mitochondrial DNA Enrichment for Sensitive Next-Generation Sequencing.

Shilan Wu, Matthew J Longley, Scott A Lujan, Thomas A Kunkel, William C Copeland.

  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

DOI:  https://doi.org/10.1007/978-1-0716-2922-2_28
Curr Protoc. 2023 Feb;3(2): e679

Generation of Mammalian Cells Devoid of Mitochondrial DNA (ρ0 cells).

Natalya Khozhukhar, Domenico Spadafora, Yelitza A Rodriguez Rodriguez, Rafik Fayzulin, Mikhail Alexeyev.

  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

DOI:  https://doi.org/10.1002/cpz1.679
Methods Mol Biol. 2023 ;2615 107-117

Measurement of Nucleoid Size Using STED Microscopy.

Elisa Motori.

  Mitochondria are equipped with their own DNA (mtDNA), which is packed into structures termed nucleoids . While nucleoids can be visualized in situ by fluorescence microscopy , the advent of super-resolution microscopy , and in particular of stimulated emission depletion (STED), has recently enabled the visualization of nucleoids at sub-diffraction resolution. Super-resolution microscopy has proved an invaluable tool for addressing fundamental questions in mitochondrial biology. In this chapter I describe how to achieve efficient labeling of mtDNA and how to quantify nucleoid diameter using an automated approach in fixed cultured cells by STED microscopy .

Keywords:  Fluorescence microscopy; ImageJ; Immunocytochemistry; Mitochondrial DNA; Nucleoid; STED; Stimulated emission depletion microscopy

DOI:  https://doi.org/10.1007/978-1-0716-2922-2_9
Cell. 2023 Feb 17. pii: S0092-8674(23)00093-4. [Epub ahead of print]

Cell lineage-specific mitochondrial resilience during mammalian organogenesis.

Stephen P Burr, Florian Klimm, Angelos Glynos, Malwina Prater, Pamella Sendon, Pavel Nash, Christopher A Powell, Marie-Lune Simard, Nina A Bonekamp, Julia Charl, Hector Diaz, Lyuba V Bozhilova, Yu Nie, Haixin Zhang, Michele Frison, Maria Falkenberg, Nick Jones, Michal Minczuk, James B Stewart, Patrick F Chinnery.

  Mitochondrial activity differs markedly between organs, but it is not known how and when this arises. Here we show that cell lineage-specific expression profiles involving essential mitochondrial genes emerge at an early stage in mouse development, including tissue-specific isoforms present before organ formation. However, the nuclear transcriptional signatures were not independent of organelle function. Genetically disrupting intra-mitochondrial protein synthesis with two different mtDNA mutations induced cell lineage-specific compensatory responses, including molecular pathways not previously implicated in organellar maintenance. We saw downregulation of genes whose expression is known to exacerbate the effects of exogenous mitochondrial toxins, indicating a transcriptional adaptation to mitochondrial dysfunction during embryonic development. The compensatory pathways were both tissue and mutation specific and under the control of transcription factors which promote organelle resilience. These are likely to contribute to the tissue specificity which characterizes human mitochondrial diseases and are potential targets for organ-directed treatments.

Keywords:  OXPHOS; RNA-seq; SCENIC; mitochondria; mt-Ta; mtDNA; organogenesis; single-cell

DOI:  https://doi.org/10.1016/j.cell.2023.01.034
Methods Mol Biol. 2023 ;2615 173-188

Localization of Mitochondrial Nucleoids by Transmission Electron Microscopy Using the Transgenic Expression of the Mitochondrial Helicase Twinkle and APEX2.

David Pla-Martín, Felix Babatz, Astrid C Schauss.

  Reminiscent of their evolutionary origin, mitochondria contain their own genome (mtDNA) compacted into the mitochondrial chromosome or nucleoid (mt-nucleoid). Many mitochondrial disorders are characterized by disruption of mt-nucleoids, either by direct mutation of genes involved in mtDNA organization or by interfering with other vital proteins for mitochondrial function. Thus, changes in mt-nucleoid morphology, distribution, and structure are a common feature in many human diseases and can be exploited as an indicator of cellular fitness. Electron microscopy provides the highest possible resolution that can be achieved, delivering spatial and structural information about all cellular structures. Recently, the ascorbate peroxidase APEX2 has been used to increase transmission electron microscopy (TEM) contrast by inducing diaminobenzidine (DAB) precipitation. DAB has the ability to accumulate osmium during classical EM sample preparation and, due to its high electron density, provides strong contrast for TEM. Among the nucleoid proteins, the mitochondrial helicase Twinkle fused with APEX2 has been successfully used to target mt-nucleoids, providing a tool to visualize these subcellular structures with high contrast and with the resolution of an electron microscope. In the presence of H2O2, APEX2 catalyzes the polymerization of DAB, generating a brown precipitate that can be visualized in specific regions of the mitochondrial matrix. Here, we provide a detailed protocol to generate murine cell lines expressing a transgenic variant of Twinkle, suitable to target and visualize mt-nucleoids. We also describe all the necessary steps to validate the cell lines prior to electron microscopy imaging and offer examples of anticipated results.

Keywords:  APEX2; Mitochondria; Nucleoid; TEM

DOI:  https://doi.org/10.1007/978-1-0716-2922-2_13
bioRxiv. 2023 Feb 15. pii: 2023.02.09.527880. [Epub ahead of print]

Regulatory principles of human mitochondrial gene expression revealed by kinetic analysis of the RNA life cycle.

Erik McShane, Mary Couvillion, Robert Ietswaart, Gyan Prakash, Brendan M Smalec, Iliana Soto, Autum R Baxter-Koenigs, Karine Choquet, L Stirling Churchman.

Mitochondria play critical roles in cellular metabolism, primarily by serving as the site of assembly and function of the oxidative phosphorylation (OXPHOS) machinery. The OXPHOS proteins are encoded by mitochondrial DNA (mtDNA) and nuclear DNA, which reside and are regulated within separate compartments. To unravel how the two gene expression systems collaborate to produce the OXPHOS complexes, the regulatory principles controlling the production of mtDNA-encoded proteins need to be elucidated. In this study, we performed a quantitative analysis of the mitochondrial messenger RNA (mt-mRNA) life cycle to determine which steps of gene expression experience strong regulatory control. Our analysis revealed that the high accumulation of mt-mRNA despite their rapid turnover was made possible by a 700-fold higher transcriptional output than nuclear-encoded OXPHOS genes. In addition, we observed that mt-mRNA processing and its association with the mitochondrial ribosome occur rapidly and that these processes are linked mechanistically. Based on these data, we developed a model of mtDNA expression that is predictive across human cell lines, revealing that differences in turnover and translation efficiency are the major contributors to mitochondrial-encoded protein synthesis. Applying this framework to a disease model of Leigh syndrome, French-Canadian type, we found that the disease-associated nuclear-encoded gene, LRPPRC , acts predominantly by stabilizing mt-mRNA. Our findings provide a comprehensive view of the intricate regulatory mechanisms governing mtDNA-encoded protein synthesis, highlighting the importance of quantitatively analyzing the mitochondrial RNA life cycle in order to decode the regulatory principles of mtDNA expression.

DOI: https://doi.org/10.1101/2023.02.09.527880
Methods Mol Biol. 2023 ;2615 381-395

Mitochondrial DNA Sequencing and Heteroplasmy Quantification by Next Generation Sequencing.

Andrea Legati, Daniele Ghezzi, Carlo Viscomi.

  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

DOI:  https://doi.org/10.1007/978-1-0716-2922-2_26
Bio Protoc. 2023 Feb 05. pii: e4602. [Epub ahead of print]13(3):

High-throughput Assessment of Mitochondrial Protein Synthesis in Mammalian Cells Using Mito-FUNCAT FACS.

Hironori Saito, Tatsuya Osaki, Yoshiho Ikeuchi, Shintaro Iwasaki.

  In addition to cytosolic protein synthesis, mitochondria also utilize another translation system that is tailored for mRNAs encoded in the mitochondrial genome. The importance of mitochondrial protein synthesis has been exemplified by the diverse diseases associated with in organello translation deficiencies. Various methods have been developed to monitor mitochondrial translation, such as the classic method of labeling newly synthesized proteins with radioisotopes and the more recent ribosome profiling. However, since these methods always assess the average cell population, measuring the mitochondrial translation capacity in individual cells has been challenging. To overcome this issue, we recently developed mito-fluorescent noncanonical amino acid tagging (FUNCAT) fluorescence-activated cell sorting (FACS), which labels nascent peptides generated by mitochondrial ribosomes with a methionine analog, L-homopropargylglycine (HPG), conjugates the peptides with fluorophores by an in situ click reaction, and detects the signal in individual cells by FACS equipment. With this methodology, the hidden heterogeneity of mitochondrial translation in cell populations can be addressed.

Keywords:   FACS ; FUNCAT ; Mitochondria ; Mitoribosome ; Translation

DOI:  https://doi.org/10.21769/BioProtoc.4602
Nat Commun. 2023 Feb 23. 14(1): 1009

TEFM variants impair mitochondrial transcription causing childhood-onset neurological disease.

Lindsey Van Haute, Emily O'Connor, Héctor Díaz-Maldonado, Benjamin Munro, Kiran Polavarapu, Daniella H Hock, Gautham Arunachal, Alkyoni Athanasiou-Fragkouli, Mainak Bardhan, Magalie Barth, Dominique Bonneau, Nicola Brunetti-Pierri, Gerarda Cappuccio, Nikeisha J Caruana, Natalia Dominik, Himanshu Goel, Guy Helman, Henry Houlden, Guy Lenaers, Karine Mention, David Murphy, Bevinahalli Nandeesh, Catarina Olimpio, Christopher A Powell, Veeramani Preethish-Kumar, Vincent Procaccio, Rocio Rius, Pedro Rebelo-Guiomar, Cas Simons, Seena Vengalil, Maha S Zaki, Alban Ziegler, David R Thorburn, David A Stroud, Reza Maroofian, John Christodoulou, Claes Gustafsson, Atchayaram Nalini, Hanns Lochmüller, Michal Minczuk, Rita Horvath.

Mutations in the mitochondrial or nuclear genomes are associated with a diverse group of human disorders characterized by impaired mitochondrial respiration. Within this group, an increasing number of mutations have been identified in nuclear genes involved in mitochondrial RNA biology. The TEFM gene encodes the mitochondrial transcription elongation factor responsible for enhancing the processivity of mitochondrial RNA polymerase, POLRMT. We report for the first time that TEFM variants are associated with mitochondrial respiratory chain deficiency and a wide range of clinical presentations including mitochondrial myopathy with a treatable neuromuscular transmission defect. Mechanistically, we show muscle and primary fibroblasts from the affected individuals have reduced levels of promoter distal mitochondrial RNA transcripts. Finally, tefm knockdown in zebrafish embryos resulted in neuromuscular junction abnormalities and abnormal mitochondrial function, strengthening the genotype-phenotype correlation. Our study highlights that TEFM regulates mitochondrial transcription elongation and its defect results in variable, tissue-specific neurological and neuromuscular symptoms.

DOI: https://doi.org/10.1038/s41467-023-36277-7
Chemistry. 2023 Feb 20. e202204021

Mitochondria-Targeted Gene Silencing Facilitated by Mito-CPDs.

Wenjie Lang, Wei Tan, Bizhong Zhou, Yuli Zhuang, Bei Zhang, Linye Jiang, Shao Q Yao, Jingyan Ge.

  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

DOI:  https://doi.org/10.1002/chem.202204021
Methods Mol Biol. 2023 ;2615 31-40

Coupling Differential Centrifugation with Exonuclease Treatment and Size Exclusion Chromatography (DIFSEC) for Purification of mtDNA from Mammalian Cells.

Andrew M Shaw, Payam A Gammage.

  Direct analysis of mtDNA using PCR-free methods is limited by the presence of persistent, contaminating nucleic acids originating from the nuclear genome, even following stringent mitochondrial isolations. Here we describe a method developed in our laboratory that couples existing, commercially available mtDNA isolation protocols with exonuclease treatment and size exclusion chromatography (DIFSEC). This protocol produces highly enriched mtDNA extracts from small-scale cell culture, with near-undetectable nuclear DNA contamination.

Keywords:  DNA; Gel filtration; Purification; Size exclusion chromatography; mtDNA

DOI:  https://doi.org/10.1007/978-1-0716-2922-2_3
Methods Mol Biol. 2023 ;2615 281-292

Detection of UV-Induced Deletions in Mitochondrial DNA.

Gabriele A Fontana, Hailey L Gahlon.

  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

DOI:  https://doi.org/10.1007/978-1-0716-2922-2_20
Methods Mol Biol. 2023 ;2611 269-282

Concomitant Sequencing of Accessible Chromatin and Mitochondrial Genomes in Single Cells Using mtscATAC-Seq.

Leif S Ludwig, Caleb A Lareau.

  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

DOI:  https://doi.org/10.1007/978-1-0716-2899-7_14
Oncogene. 2023 Feb 22.

Mitochondrial structure and function adaptation in residual triple negative breast cancer cells surviving chemotherapy treatment.

Mokryun L Baek, Junegoo Lee, Katherine E Pendleton, Mariah J Berner, Emily B Goff, Lin Tan, Sara A Martinez, Iqbal Mahmud, Tao Wang, Matthew D Meyer, Bora Lim, James P Barrish, Weston Porter, Philip L Lorenzi, Gloria V Echeverria.

Neoadjuvant chemotherapy (NACT) used for triple negative breast cancer (TNBC) eradicates tumors in ~45% of patients. Unfortunately, TNBC patients with substantial residual cancer burden have poor metastasis free and overall survival rates. We previously demonstrated mitochondrial oxidative phosphorylation (OXPHOS) was elevated and was a unique therapeutic dependency of residual TNBC cells surviving NACT. We sought to investigate the mechanism underlying this enhanced reliance on mitochondrial metabolism. Mitochondria are morphologically plastic organelles that cycle between fission and fusion to maintain mitochondrial integrity and metabolic homeostasis. The functional impact of mitochondrial structure on metabolic output is highly context dependent. Several chemotherapy agents are conventionally used for neoadjuvant treatment of TNBC patients. Upon comparing mitochondrial effects of conventional chemotherapies, we found that DNA-damaging agents increased mitochondrial elongation, mitochondrial content, flux of glucose through the TCA cycle, and OXPHOS, whereas taxanes instead decreased mitochondrial elongation and OXPHOS. The mitochondrial effects of DNA-damaging chemotherapies were dependent on the mitochondrial inner membrane fusion protein optic atrophy 1 (OPA1). Further, we observed heightened OXPHOS, OPA1 protein levels, and mitochondrial elongation in an orthotopic patient-derived xenograft (PDX) model of residual TNBC. Pharmacologic or genetic disruption of mitochondrial fusion and fission resulted in decreased or increased OXPHOS, respectively, revealing longer mitochondria favor oxphos in TNBC cells. Using TNBC cell lines and an in vivo PDX model of residual TNBC, we found that sequential treatment with DNA-damaging chemotherapy, thus inducing mitochondrial fusion and OXPHOS, followed by MYLS22, a specific inhibitor of OPA1, was able to suppress mitochondrial fusion and OXPHOS and significantly inhibit regrowth of residual tumor cells. Our data suggest that TNBC mitochondria can optimize OXPHOS through OPA1-mediated mitochondrial fusion. These findings may provide an opportunity to overcome mitochondrial adaptations of chemoresistant TNBC.

DOI: https://doi.org/10.1038/s41388-023-02596-8
Antioxidants (Basel). 2023 Jan 21. pii: 241. [Epub ahead of print]12(2):

Transcriptomic Profiling and Pathway Analysis of Mesenchymal Stem Cells Following Low Dose-Rate Radiation Exposure.

John E Slaven, Matthew Wilkerson, Anthony R Soltis, W Bradley Rittase, Dmitry T Bradfield, Michelle Bylicky, Lynnette Cary, Alena Tsioplaya, Roxane Bouten, Clifton Dalgard, Regina M Day.

  Low dose-rate radiation exposure can occur in medical imaging, as background from environmental or industrial radiation, and is a hazard of space travel. In contrast with high dose-rate radiation exposure that can induce acute life-threatening syndromes, chronic low-dose radiation is associated with Chronic Radiation Syndrome (CRS), which can alter environmental sensitivity. Secondary effects of chronic low dose-rate radiation exposure include circulatory, digestive, cardiovascular, and neurological diseases, as well as cancer. Here, we investigated 1-2 Gy, 0.66 cGy/h, 60Co radiation effects on primary human mesenchymal stem cells (hMSC). There was no significant induction of apoptosis or DNA damage, and cells continued to proliferate. Gene ontology (GO) analysis of transcriptome changes revealed alterations in pathways related to cellular metabolism (cholesterol, fatty acid, and glucose metabolism), extracellular matrix modification and cell adhesion/migration, and regulation of vasoconstriction and inflammation. Interestingly, there was increased hypoxia signaling and increased activation of pathways regulated by iron deficiency, but Nrf2 and related genes were reduced. The data were validated in hMSC and human lung microvascular endothelial cells using targeted qPCR and Western blotting. Notably absent in the GO analysis were alteration pathways for DNA damage response, cell cycle inhibition, senescence, and pro-inflammatory response that we previously observed for high dose-rate radiation exposure. Our findings suggest that cellular gene transcription response to low dose-rate ionizing radiation is fundamentally different compared to high-dose-rate exposure. We hypothesize that cellular response to hypoxia and iron deficiency are driving processes, upstream of the other pathway regulation.

Keywords:  RNAseq; gene regulation; human microvascular endothelial cells; low dose-rate; mesenchymal stem cells; radiation

DOI:  https://doi.org/10.3390/antiox12020241