bims-nenemi Biomed News
on Neuroinflammation, neurodegeneration and mitochondria
Issue of 2024–06–02
eleven papers selected by
Marco Tigano, Thomas Jefferson University
  1. Cells dispose of cytoplasmic mitochondrial DNA by nucleoid-phagy.
  2. Mitochondrial dysfunction heightens the integrated stress response to drive ALS pathogenesis.
  3. Mechanisms and pathologies of human mitochondrial DNA replication and deletion formation.
  4. Spatial detection of mitochondrial DNA and RNA in tissues.
  5. ANT2 functions as a translocon for mitochondrial cross-membrane translocation of RNAs.
  6. Mitochondrial RelA empowers mtDNA G-quadruplex formation for hypoxia adaptation in cancer cells.
  7. Multiple Unfolded Protein Response pathways cooperate to link cytosolic dsDNA release to Stimulator of Interferon Gene (STING) activation.
  8. Nanobodies as novel tools to monitor the mitochondrial fission factor Drp1.
  9. A mitochondrial-targeted activity-based sensing probe for ratiometric imaging of formaldehyde reveals key regulators of the mitochondrial one-carbon pool.
  10. Single-cell morphology encodes functional subtypes of senescence in aging human dermal fibroblasts.
  11. Pervasive nuclear envelope ruptures precede ECM signaling and disease onset without activating cGAS-STING in Lamin-cardiomyopathy mice.
  1. Nat Cell Biol. 2024 May 29.
    Cells dispose of cytoplasmic mitochondrial DNA by nucleoid-phagy.
      
    DOI:  https://doi.org/10.1038/s41556-024-01421-y
  2. bioRxiv. 2024 May 14. pii: 2024.05.13.594000. [Epub ahead of print]
    Mitochondrial dysfunction heightens the integrated stress response to drive ALS pathogenesis.
    Curran Landry, James Costanzo, Miguel Mitne-Neto, Mayana Zatz, Ashleigh Schaffer, Maria Hatzoglou, Alysson Muotri, Helen Cristina Miranda.
      Vesicle-associated membrane protein-associated protein-B (VAPB) is an ER membrane bound protein. VAPB P56S causes a dominant, familial form of amyotrophic lateral sclerosis (ALS), however, the mechanism through which this mutation causes motor neuron (MN) disease remains unknown. Using inducible wild type (WT) and VAPB P56S expressing iPSC-derived MNs we show that VAPB P56S, but not WT, protein decreased neuronal firing and mitochondrial-ER contact (MERC) with an associated age-dependent decrease in mitochondrial membrane potential (MMP); all typical characteristics of MN-disease. We further show that VAPB P56S expressing iPSC-derived MNs have enhanced age-dependent sensitivity to ER stress. We identified elevated expression of the master regulator of the Integrated Stress Response (ISR) marker ATF4 and decreased protein synthesis in the VAPB P56S iPSC-derived MNs. Chemical inhibition of ISR with the compound, ISRIB, rescued all MN disease phenotype in VAPB P56S MNs. Thus, our results not only support ISR inhibition as a potential therapeutic target for ALS patients, but also provides evidence to pathogenesis.
    DOI:  https://doi.org/10.1101/2024.05.13.594000
  3. Biochem J. 2024 Jun 05. 481(11): 683-715
    Mechanisms and pathologies of human mitochondrial DNA replication and deletion formation.
    Tiago M Bernardino Gomes, Amy E Vincent, Katja E Menger, James B Stewart, Thomas J Nicholls.
      Human mitochondria possess a multi-copy circular genome, mitochondrial DNA (mtDNA), that is essential for cellular energy metabolism. The number of copies of mtDNA per cell, and their integrity, are maintained by nuclear-encoded mtDNA replication and repair machineries. Aberrant mtDNA replication and mtDNA breakage are believed to cause deletions within mtDNA. The genomic location and breakpoint sequences of these deletions show similar patterns across various inherited and acquired diseases, and are also observed during normal ageing, suggesting a common mechanism of deletion formation. However, an ongoing debate over the mechanism by which mtDNA replicates has made it difficult to develop clear and testable models for how mtDNA rearrangements arise and propagate at a molecular and cellular level. These deletions may impair energy metabolism if present in a high proportion of the mtDNA copies within the cell, and can be seen in primary mitochondrial diseases, either in sporadic cases or caused by autosomal variants in nuclear-encoded mtDNA maintenance genes. These mitochondrial diseases have diverse genetic causes and multiple modes of inheritance, and show notoriously broad clinical heterogeneity with complex tissue specificities, which further makes establishing genotype-phenotype relationships challenging. In this review, we aim to cover our current understanding of how the human mitochondrial genome is replicated, the mechanisms by which mtDNA replication and repair can lead to mtDNA instability in the form of large-scale rearrangements, how rearranged mtDNAs subsequently accumulate within cells, and the pathological consequences when this occurs.
    Keywords:  DNA damage; DNA replication and recombination; mitochondrial dysfunction; mtDNA
    DOI:  https://doi.org/10.1042/BCJ20230262
  4. Front Cell Dev Biol. 2024 ;12 1346778
    Spatial detection of mitochondrial DNA and RNA in tissues.
    Michelle Giarmarco, Jordan Seto, Daniel Brock, Susan Brockerhoff.
       Background: Mitochondrial health has gained attention in a number of diseases, both as an indicator of disease state and as a potential therapeutic target. The quality and amount of mitochondrial DNA (mtDNA) and RNA (mtRNA) can be important indicators of mitochondrial and cell health, but are difficult to measure in complex tissues.
    Methods: mtDNA and mtRNA in zebrafish retina samples were fluorescently labeled using RNAscope™ in situ hybridization, then mitochondria were stained using immunohistochemistry. Pretreatment with RNase was used for validation. Confocal images were collected and analyzed, and relative amounts of mtDNA and mtRNA were reported. Findings regarding mtDNA were confirmed using qPCR.
    Results: Signals from probes detecting mtDNA and mtRNA were localized to mitochondria, and were differentially sensitive to RNase. This labeling strategy allows for quantification of relative mtDNA and mtRNA levels in individual cells. As a demonstration of the method in a complex tissue, single photoreceptors in zebrafish retina were analyzed for mtDNA and mtRNA content. An increase in mtRNA but not mtDNA coincides with proliferation of mitochondria at night in cones. A similar trend was measured in rods.
    Discussion: Mitochondrial gene expression is an important component of cell adaptations to disease, stress, or aging. This method enables the study of mtDNA and mtRNA in single cells of an intact, complex tissue. The protocol presented here uses commercially-available tools, and is adaptable to a range of species and tissue types.
    Keywords:  RNAscope; mitochondria; mtDNA; mtRNA; multiplex imaging; photoreceptor; spatial biology; zebrafish
    DOI:  https://doi.org/10.3389/fcell.2024.1346778
  5. Cell Res. 2024 May 29.
    ANT2 functions as a translocon for mitochondrial cross-membrane translocation of RNAs.
    Pengcheng Wang, Lixiao Zhang, Siyi Chen, Renjian Li, Peipei Liu, Xiang Li, Hongdi Luo, Yujia Huo, Zhirong Zhang, Yiqi Cai, Xu Liu, Jinliang Huang, Guangkeng Zhou, Zhe Sun, Shanwei Ding, Jiahao Shi, Zizhuo Zhou, Ruoxi Yuan, Liang Liu, Sipeng Wu, Geng Wang.
      Bidirectional transcription of mammalian mitochondrial DNA generates overlapping transcripts that are capable of forming double-stranded RNA (dsRNA) structures. Release of mitochondrial dsRNA into the cytosol activates the dsRNA-sensing immune signaling, which is a defense mechanism against microbial and viral attack and possibly cancer, but could cause autoimmune diseases when unchecked. A better understanding of the process is vital in therapeutic application of this defense mechanism and treatment of cognate human diseases. In addition to exporting dsRNAs, mitochondria also export and import a variety of non-coding RNAs. However, little is known about how these RNAs are transported across mitochondrial membranes. Here we provide direct evidence showing that adenine nucleotide translocase-2 (ANT2) functions as a mammalian RNA translocon in the mitochondrial inner membrane, independent of its ADP/ATP translocase activity. We also show that mitochondrial dsRNA efflux through ANT2 triggers innate immunity. Inhibiting this process alleviates inflammation in vivo, providing a potential therapeutic approach for treating autoimmune diseases.
    DOI:  https://doi.org/10.1038/s41422-024-00978-5
  6. Cell Chem Biol. 2024 May 21. pii: S2451-9456(24)00181-8. [Epub ahead of print]
    Mitochondrial RelA empowers mtDNA G-quadruplex formation for hypoxia adaptation in cancer cells.
    Gui-Xue Tang, Mao-Lin Li, Cui Zhou, Zhi-Shu Huang, Shuo-Bin Chen, Xiu-Cai Chen, Jia-Heng Tan.
      Mitochondrial DNA (mtDNA) G-quadruplexes (G4s) have important regulatory roles in energy metabolism, yet their specific functions and underlying regulatory mechanisms have not been delineated. Using a chemical-genetic screening strategy, we demonstrated that the JAK/STAT3 pathway is the primary regulatory mechanism governing mtDNA G4 dynamics in hypoxic cancer cells. Further proteomic analysis showed that activation of the JAK/STAT3 pathway facilitates the translocation of RelA, a member of the NF-κB family, to the mitochondria, where RelA binds to mtDNA G4s and promotes their folding, resulting in increased mtDNA instability, inhibited mtDNA transcription, and subsequent mitochondrial dysfunction. This binding event disrupts the equilibrium of energy metabolism, catalyzing a metabolic shift favoring glycolysis. Collectively, the results provide insights into a strategy employed by cancer cells to adapt to hypoxia through metabolic reprogramming.
    Keywords:  G-quadruplexes; NF-κB RelA; chemical genetic screen; energy metabolism; mitochondrial DNA
    DOI:  https://doi.org/10.1016/j.chembiol.2024.05.003
  7. bioRxiv. 2024 May 14. pii: 2024.05.10.593557. [Epub ahead of print]
    Multiple Unfolded Protein Response pathways cooperate to link cytosolic dsDNA release to Stimulator of Interferon Gene (STING) activation.
    Tiancheng Hu, Yiping Liu, Jeremy Fleck, Cason King, Elaine Schalk, Zhenyu Zhang, Andrew Mehle, Judith A Smith.
      The double-stranded DNA (dsDNA) sensor STING has been increasingly implicated in responses to "sterile" endogenous threats and pathogens without nominal DNA or cyclic di-nucleotide stimuli. Previous work showed an endoplasmic reticulum (ER) stress response, known as the unfolded protein response (UPR), activates STING. Herein, we sought to determine if ER stress generated a STING ligand, and to identify the UPR pathways involved. Induction of IFN-β expression following stimulation with the UPR inducer thapsigargin (TPG) or oxygen glucose deprivation required both STING and the dsDNA-sensing cyclic GMP-AMP synthase (cGAS). Furthermore, TPG increased cytosolic mitochondrial DNA, and immunofluorescence visualized dsDNA punctae in murine and human cells, providing a cGAS stimulus. N-acetylcysteine decreased IFN-β induction by TPG, implicating reactive oxygen species (ROS). However, mitoTEMPO, a mitochondrial oxidative stress inhibitor did not impact TPG-induced IFN. On the other hand, inhibiting the inositol requiring enzyme 1 (IRE1) ER stress sensor and its target transcription factor XBP1 decreased the generation of cytosolic dsDNA. iNOS upregulation was XBP1-dependent, and an iNOS inhibitor decreased cytosolic dsDNA and IFN-β, implicating ROS downstream of the IRE1-XBP1 pathway. Inhibition of the PKR-like ER kinase (PERK) pathway also attenuated cytoplasmic dsDNA release. The PERK-regulated apoptotic factor Bim was required for both dsDNA release and IFN-β mRNA induction. Finally, XBP1 and PERK pathways contributed to cytosolic dsDNA release and IFN-induction by the RNA virus, Vesicular Stomatitis Virus (VSV). Together, our findings suggest that ER stressors, including viral pathogens without nominal STING or cGAS ligands such as RNA viruses, trigger multiple canonical UPR pathways that cooperate to activate STING and downstream IFN-β via mitochondrial dsDNA release.
    DOI:  https://doi.org/10.1101/2024.05.10.593557
  8. Life Sci Alliance. 2024 Aug;pii: e202402608. [Epub ahead of print]7(8):
    Nanobodies as novel tools to monitor the mitochondrial fission factor Drp1.
    Theresa Froehlich, Andreas Jenner, Claudia Cavarischia-Rega, Funmilayo O Fagbadebo, Yannic Lurz, Desiree I Frecot, Philipp D Kaiser, Stefan Nueske, Armin M Scholz, Erik Schäffer, Ana J Garcia-Saez, Boris Macek, Ulrich Rothbauer.
      In cells, mitochondria undergo constant fusion and fission. An essential factor for fission is the mammalian dynamin-related protein 1 (Drp1). Dysregulation of Drp1 is associated with neurodegenerative diseases including Parkinson's, cardiovascular diseases and cancer, making Drp1 a pivotal biomarker for monitoring mitochondrial status and potential pathophysiological conditions. Here, we developed nanobodies (Nbs) as versatile binding molecules for proteomics, advanced microscopy and live cell imaging of Drp1. To specifically enrich endogenous Drp1 with interacting proteins for proteomics, we functionalized high-affinity Nbs into advanced capture matrices. Furthermore, we detected Drp1 by bivalent Nbs combined with site-directed fluorophore labelling in super-resolution STORM microscopy. For real-time imaging of Drp1, we intracellularly expressed fluorescently labelled Nbs, so-called chromobodies (Cbs). To improve the signal-to-noise ratio, we further converted Cbs into a "turnover-accelerated" format. With these imaging probes, we visualized the dynamics of endogenous Drp1 upon compound-induced mitochondrial fission in living cells. Considering the wide range of research applications, the presented Nb toolset will open up new possibilities for advanced functional studies of Drp1 in disease-relevant models.
    DOI:  https://doi.org/10.26508/lsa.202402608
  9. Chem Sci. 2024 May 29. 15(21): 8080-8088
    A mitochondrial-targeted activity-based sensing probe for ratiometric imaging of formaldehyde reveals key regulators of the mitochondrial one-carbon pool.
    Logan Tenney, Vanha N Pham, Thomas F Brewer, Christopher J Chang.
      Formaldehyde (FA) is both a highly reactive environmental genotoxin and an endogenously produced metabolite that functions as a signaling molecule and one-carbon (1C) store to regulate 1C metabolism and epigenetics in the cell. Owing to its signal-stress duality, cells have evolved multiple clearance mechanisms to maintain FA homeostasis, acting to avoid the established genotoxicity of FA while also redirecting FA-derived carbon units into the biosynthesis of essential nucleobases and amino acids. The highly compartmentalized nature of FA exposure, production, and regulation motivates the development of chemical tools that enable monitoring of transient FA fluxes with subcellular resolution. Here we report a mitochondrial-targeted, activity-based sensing probe for ratiometric FA detection, MitoRFAP-2, and apply this reagent to monitor endogenous mitochondrial sources and sinks of this 1C unit. We establish the utility of subcellular localization by showing that MitoRFAP-2 is sensitive enough to detect changes in mitochondrial FA pools with genetic and pharmacological modulation of enzymes involved in 1C and amino acid metabolism, including the pervasive, less active genetic mutant aldehyde dehydrogenase 2*2 (ALDH2*2), where previous, non-targeted versions of FA sensors are not. Finally, we used MitoRFAP-2 to comparatively profile basal levels of FA across a panel of breast cancer cell lines, finding that FA-dependent fluorescence correlates with expression levels of enzymes involved in 1C metabolism. By showcasing the ability of MitoRFAP-2 to identify new information on mitochondrial FA homeostasis, this work provides a starting point for the design of a broader range of chemical probes for detecting physiologically important aldehydes with subcellular resolution and a useful reagent for further studies of 1C biology.
    DOI:  https://doi.org/10.1039/d4sc01183j
  10. bioRxiv. 2024 May 16. pii: 2024.05.10.593637. [Epub ahead of print]
    Single-cell morphology encodes functional subtypes of senescence in aging human dermal fibroblasts.
    Pratik Kamat, Nico Macaluso, Chanhong Min, Yukang Li, Anshika Agrawal, Aaron Winston, Lauren Pan, Bartholomew Starich, Teasia Stewart, Pei-Hsun Wu, Jean Fan, Jeremy Walston, Jude M Phillip.
      Cellular senescence is an established driver of aging, exhibiting context-dependent phenotypes across multiple biological length-scales. Despite its mechanistic importance, profiling senescence within cell populations is challenging. This is in part due to the limitations of current biomarkers to robustly identify senescent cells across biological settings, and the heterogeneous, non-binary phenotypes exhibited by senescent cells. Using a panel of primary dermal fibroblasts, we combined live single-cell imaging, machine learning, multiple senescence induction conditions, and multiple protein-based senescence biomarkers to show the emergence of functional subtypes of senescence. Leveraging single-cell morphologies, we defined eleven distinct morphology clusters, with the abundance of cells in each cluster being dependent on the mode of senescence induction, the time post-induction, and the age of the donor. Of these eleven clusters, we identified three bona-fide senescence subtypes (C7, C10, C11), with C10 showing the strongest age-dependence across a cohort of fifty aging individuals. To determine the functional significance of these senescence subtypes, we profiled their responses to senotherapies, specifically focusing on Dasatinib + Quercetin (D+Q). Results indicated subtype-dependent responses, with senescent cells in C7 being most responsive to D+Q. Altogether, we provide a robust single-cell framework to identify and classify functional senescence subtypes with applications for next-generation senotherapy screens, and the potential to explain heterogeneous senescence phenotypes across biological settings based on the presence and abundance of distinct senescence subtypes.
    DOI:  https://doi.org/10.1101/2024.05.10.593637
  11. Cell Rep. 2024 May 29. pii: S2211-1247(24)00612-0. [Epub ahead of print]43(6): 114284
    Pervasive nuclear envelope ruptures precede ECM signaling and disease onset without activating cGAS-STING in Lamin-cardiomyopathy mice.
    Atsuki En, Hanumakumar Bogireddi, Briana Thomas, Alexis V Stutzman, Sachie Ikegami, Brigitte LaForest, Omar Almakki, Peter Pytel, Ivan P Moskowitz, Kohta Ikegami.
      Nuclear envelope (NE) ruptures are emerging observations in Lamin-related dilated cardiomyopathy, an adult-onset disease caused by loss-of-function mutations in Lamin A/C, a nuclear lamina component. Here, we test a prevailing hypothesis that NE ruptures trigger the pathological cGAS-STING cytosolic DNA-sensing pathway using a mouse model of Lamin cardiomyopathy. The reduction of Lamin A/C in cardio-myocyte of adult mice causes pervasive NE ruptures in cardiomyocytes, preceding inflammatory transcription, fibrosis, and fatal dilated cardiomyopathy. NE ruptures are followed by DNA damage accumulation without causing immediate cardiomyocyte death. However, cGAS-STING-dependent inflammatory signaling remains inactive. Deleting cGas or Sting does not rescue cardiomyopathy in the mouse model. The lack of cGAS-STING activation is likely due to the near absence of cGAS expression in adult cardiomyocytes at baseline. Instead, extracellular matrix (ECM) signaling is activated and predicted to initiate pro-inflammatory communication from Lamin-reduced cardiomyocytes to fibroblasts. Our work nominates ECM signaling, not cGAS-STING, as a potential inflammatory contributor in Lamin cardiomyopathy.
    Keywords:  CP: Cell biology; ECM; Lamin A/C; Lmna; cGAS STING; dilated cardiomyopathy; extracellular matrix; laminopathy; nuclear envelope rupture; nuclear lamina
    DOI:  https://doi.org/10.1016/j.celrep.2024.114284
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