bims-nenemi Biomed News
on Neuroinflammation, neurodegeneration and mitochondria
Issue of 2024–04–14
ten papers selected by
Marco Tigano, Thomas Jefferson University
  1. Fatty acyl-coenzyme A activates mitochondrial division through oligomerization of MiD49 and MiD51.
  2. Turnover of PPP1R15A mRNA encoding GADD34 controls responsiveness and adaptation to cellular stress.
  3. A novel inhibitory BAK antibody enables assessment of non-activated BAK in cancer cells.
  4. Integrated stress response plasticity governs normal cell adaptation to chronic stress via the PP2A-TFE3-ATF4 pathway.
  5. Membrane damage by MBP-1 is mediated by pore formation and amplified by mtDNA.
  6. An Innovative Mitochondrial-targeted Gene Therapy for Cancer Treatment.
  7. Machine learning-based 3D segmentation of mitochondria in polarized epithelial cells.
  8. An in vitro CRISPR screen of cell-free DNA identifies apoptosis as the primary mediator of cell-free DNA release.
  9. Cytosolic RNA binding of the mitochondrial TCA cycle enzyme malate dehydrogenase (MDH2).
  10. Visualized Tracking and Multidimensional Assessing of Mitochondria-Associated Pyroptosis in Cancer Cells by a Small-Molecule Fluorescent Probe.
  1. Nat Cell Biol. 2024 Apr 09.
    Fatty acyl-coenzyme A activates mitochondrial division through oligomerization of MiD49 and MiD51.
    Ao Liu, Frieda Kage, Asan F Abdulkareem, Mac Pholo Aguirre-Huamani, Gracie Sapp, Halil Aydin, Henry N Higgs.
      Mitochondrial fission occurs in many cellular processes, but the regulation of fission is poorly understood. We show that long-chain acyl-coenzyme A (LCACA) activates two related mitochondrial fission proteins, MiD49 and MiD51, by inducing their oligomerization, which activates their ability to stimulate the DRP1 GTPase. The 1:1 stoichiometry of LCACA:MiD in the oligomer suggests interaction in the previously identified nucleotide-binding pocket, and a point mutation in this pocket reduces LCACA binding and LCACA-induced oligomerization for MiD51. In cells, this LCACA binding mutant does not assemble into puncta on mitochondria or rescue MiD49/51 knockdown effects on mitochondrial length and DRP1 recruitment. Furthermore, cellular treatment with BSA-bound oleic acid, which causes increased LCACA, promotes mitochondrial fission in an MiD49/51-dependent manner. These results suggest that LCACA is an endogenous ligand for MiDs, inducing mitochondrial fission and providing a potential mechanism for fatty-acid-induced mitochondrial division. Finally, MiD49 or MiD51 oligomers synergize with Mff, but not with actin filaments, in DRP1 activation, suggesting distinct pathways for DRP1 activation.
    DOI:  https://doi.org/10.1038/s41556-024-01400-3
  2. Cell Rep. 2024 Apr 10. pii: S2211-1247(24)00397-8. [Epub ahead of print]43(4): 114069
    Turnover of PPP1R15A mRNA encoding GADD34 controls responsiveness and adaptation to cellular stress.
    Vera Magg, Alessandro Manetto, Katja Kopp, Chia Ching Wu, Mohsen Naghizadeh, Doris Lindner, Lucy Eke, Julia Welsch, Stefan M Kallenberger, Johanna Schott, Volker Haucke, Nicolas Locker, Georg Stoecklin, Alessia Ruggieri.
      The integrated stress response (ISR) is a key cellular signaling pathway activated by environmental alterations that represses protein synthesis to restore homeostasis. To prevent sustained damage, the ISR is counteracted by the upregulation of growth arrest and DNA damage-inducible 34 (GADD34), a stress-induced regulatory subunit of protein phosphatase 1 that mediates translation reactivation and stress recovery. Here, we uncover a novel ISR regulatory mechanism that post-transcriptionally controls the stability of PPP1R15A mRNA encoding GADD34. We establish that the 3' untranslated region of PPP1R15A mRNA contains an active AU-rich element (ARE) recognized by proteins of the ZFP36 family, promoting its rapid decay under normal conditions and stabilization for efficient expression of GADD34 in response to stress. We identify the tight temporal control of PPP1R15A mRNA turnover as a component of the transient ISR memory, which sets the threshold for cellular responsiveness and mediates adaptation to repeated stress conditions.
    Keywords:  ARE; Brf1; CP: Cell biology; CP: Molecular biology; GADD34; PPP1R15A; TTP; ZFP36; integrated stress response; molecular memory; stress adaptation
    DOI:  https://doi.org/10.1016/j.celrep.2024.114069
  3. Cell Death Differ. 2024 Apr 06.
    A novel inhibitory BAK antibody enables assessment of non-activated BAK in cancer cells.
    Hema Preethi Subas Satish, Sweta Iyer, Melissa X Shi, Agnes W Wong, Karla C Fischer, Ahmad Z Wardak, Daisy Lio, Jason M Brouwer, Rachel T Uren, Peter E Czabotar, Michelle S Miller, Ruth M Kluck.
      BAX and BAK are pro-apoptotic members of the BCL2 family that are required to permeabilize the mitochondrial outer membrane. The proteins can adopt a non-activated monomeric conformation, or an activated conformation in which the exposed BH3 domain facilitates binding either to a prosurvival protein or to another activated BAK or BAX protein to promote pore formation. Certain cancer cells are proposed to have high levels of activated BAK sequestered by MCL1 or BCLXL, thus priming these cells to undergo apoptosis in response to BH3 mimetic compounds that target MCL1 or BCLXL. Here we report the first antibody, 14G6, that is specific for the non-activated BAK conformer. A crystal structure of 14G6 Fab bound to BAK revealed a binding site encompassing both the α1 helix and α5-α6 hinge regions of BAK, two sites involved in the unfolding of BAK during its activation. In mitochondrial experiments, 14G6 inhibited BAK unfolding triggered by three diverse BAK activators, supporting crucial roles for both α1 dissociation and separation of the core (α2-α5) and latch (α6-α9) regions in BAK activation. 14G6 bound the majority of BAK in several leukaemia cell lines, and binding decreased following treatment with BH3 mimetics, indicating only minor levels of constitutively activated BAK in those cells. In summary, 14G6 provides a new means of assessing BAK status in response to anti-cancer treatments.
    DOI:  https://doi.org/10.1038/s41418-024-01289-3
  4. Res Sq. 2024 Mar 28. pii: rs.3.rs-4013396. [Epub ahead of print]
    Integrated stress response plasticity governs normal cell adaptation to chronic stress via the PP2A-TFE3-ATF4 pathway.
    Analisa DiFeo, Rita A Avelar, Riya Gupta, Grace Carvette, Felipe da Veiga Leprevost, Jose Colina, Jessica Teitel, Alexey Nesvizhskii, Caitlin O'connor, Maria Hatzoglou, Shirish Shenolikar, Peter Arvan, Goutham Narla.
      The integrated stress response (ISR) regulates cell fate during conditions of stress by leveraging the cell's capacity to endure sustainable and efficient adaptive stress responses. Protein phosphatase 2A (PP2A) activity modulation has been shown to be successful in achieving both therapeutic efficacy and safety across various cancer models; however, the molecular mechanisms driving its selective antitumor effects remain unclear. Here, we show for the first time that ISR plasticity relies on PP2A activation to regulate drug response and dictate cellular fate under conditions of chronic stress. We demonstrate that genetic and chemical modulation of the PP2A leads to chronic proteolytic stress and triggers an ISR to dictate cell fate. More specifically, we uncovered that the PP2A-TFE3-ATF4 pathway governs ISR cell plasticity during endoplasmic reticular and cellular stress independent of the unfolded protein response. We further show that normal cells reprogram their genetic signatures to undergo ISR-mediated adaptation and homeostatic recovery thereby successfully avoiding toxicity following PP2A-mediated stress. Conversely, oncogenic specific cytotoxicity induced by chemical modulation of PP2A is achieved by activating chronic and irreversible ISR in cancer cells. Our findings propose that a differential response to chemical modulation of PP2A is determined by intrinsic ISR plasticity, providing a novel biological vulnerability to selectively induce cancer cell death and improve targeted therapeutic efficacy.
    DOI:  https://doi.org/10.21203/rs.3.rs-4013396/v1
  5. Cell Rep. 2024 Apr 06. pii: S2211-1247(24)00412-1. [Epub ahead of print]43(4): 114084
    Membrane damage by MBP-1 is mediated by pore formation and amplified by mtDNA.
    Lea Gigon, Philipp Müller, Beat Haenni, Ioan Iacovache, Maruša Barbo, Gordana Gosheva, Shida Yousefi, Alice Soragni, Christoph von Ballmoos, Benoît Zuber, Hans-Uwe Simon.
      Eosinophils play a crucial role in host defense while also contributing to immunopathology through the release of inflammatory mediators. Characterized by distinctive cytoplasmic granules, eosinophils securely store and rapidly release various proteins exhibiting high toxicity upon extracellular release. Among these, major basic protein 1 (MBP-1) emerges as an important mediator in eosinophil function against pathogens and in eosinophil-associated diseases. While MBP-1 targets both microorganisms and host cells, its precise mechanism remains elusive. We demonstrate that formation of small pores by MBP-1 in lipid bilayers induces membrane permeabilization and disrupts potassium balance. Additionally, we reveal that mitochondrial DNA (mtDNA) present in eosinophil extracellular traps (EETs) amplifies MBP-1 toxic effects, underscoring the pivotal role of mtDNA in EETs. Furthermore, we present evidence indicating that absence of CpG methylation in mtDNA contributes to the regulation of MBP-1-mediated toxicity. Taken together, our data suggest that the mtDNA scaffold within extracellular traps promotes MBP-1 toxicity.
    Keywords:  CP: Cell biology; CP: Molecular biology; DNA methylation; eosinophils; extracellular trap; major basic protein; mitochondrial DNA; pore formation
    DOI:  https://doi.org/10.1016/j.celrep.2024.114084
  6. bioRxiv. 2024 Mar 27. pii: 2024.03.24.584499. [Epub ahead of print]
    An Innovative Mitochondrial-targeted Gene Therapy for Cancer Treatment.
    Kai Chen, Patrick Ernst, Seulhee Kim, Yingnan Si, Tanvi Varadkar, Matthew D Ringel, Xiaoguang Margaret Liu, Lufang Zhou.
      Targeting cancer cell mitochondria holds great therapeutic promise, yet current strategies to specifically and effectively destroy cancer mitochondria in vivo are limited. Here, we introduce mLumiOpto, an innovative mitochondrial-targeted luminoptogenetics gene therapy designed to directly disrupt the inner mitochondrial membrane (IMM) potential and induce cancer cell death. We synthesize a blue light-gated channelrhodopsin (CoChR) in the IMM and co-express a blue bioluminescence-emitting Nanoluciferase (NLuc) in the cytosol of the same cells. The mLumiOpto genes are selectively delivered to cancer cells in vivo by using adeno-associated virus (AAV) carrying a cancer-specific promoter or cancer-targeted monoclonal antibody-tagged exosome-associated AAV. Induction with NLuc luciferin elicits robust endogenous bioluminescence, which activates mitochondrial CoChR, triggering cancer cell IMM permeability disruption, mitochondrial damage, and subsequent cell death. Importantly, mLumiOpto demonstrates remarkable efficacy in reducing tumor burden and killing tumor cells in glioblastoma or triple-negative breast cancer xenografted mouse models. These findings establish mLumiOpto as a novel and promising therapeutic strategy by targeting cancer cell mitochondria in vivo .
    DOI:  https://doi.org/10.1101/2024.03.24.584499
  7. Mitochondrion. 2024 Apr 08. pii: S1567-7249(24)00040-0. [Epub ahead of print] 101882
    Machine learning-based 3D segmentation of mitochondria in polarized epithelial cells.
    Nan W Hultgren, Tianli Zhou, David S Williams.
      Mitochondria are dynamic organelles that alter their morphological characteristics in response to functional needs. Therefore, mitochondrial morphology is an important indicator of mitochondrial function and cellular health. Reliable segmentation of mitochondrial networks in microscopy images is a crucial initial step for further quantitative evaluation of their morphology. However, 3D mitochondrial segmentation, especially in cells with complex network morphology, such as in highly polarized cells, remains challenging. To improve the quality of 3D segmentation of mitochondria in super-resolution microscopy images, we took a machine learning approach, using 3D Trainable Weka, an ImageJ plugin. We demonstrated that, compared with other commonly used methods, our approach segmented mitochondrial networks effectively, with improved accuracy in different polarized epithelial cell models, including differentiated human retinal pigment epithelial (RPE) cells. Furthermore, using several tools for quantitative analysis following segmentation, we revealed mitochondrial fragmentation in bafilomycin-treated RPE cells.
    Keywords:  3D segmentation; ImageJ; Machine learning algorithm; Mitochondrial morphology; Retinal pigment epithelium
    DOI:  https://doi.org/10.1016/j.mito.2024.101882
  8. Commun Biol. 2024 Apr 10. 7(1): 441
    An in vitro CRISPR screen of cell-free DNA identifies apoptosis as the primary mediator of cell-free DNA release.
    Brad A Davidson, Adam X Miranda, Sarah C Reed, Riley E Bergman, Justin D J Kemp, Anvith P Reddy, Morgan V Pantone, Ethan K Fox, R Dixon Dorand, Paula J Hurley, Sarah Croessmann, Ben Ho Park.
       ABTRACT: Clinical circulating cell-free DNA (cfDNA) testing is now routine, however test accuracy remains limited. By understanding the life-cycle of cfDNA, we might identify opportunities to increase test performance. Here, we profile cfDNA release across a 24-cell line panel and utilize a cell-free CRISPR screen (cfCRISPR) to identify mediators of cfDNA release. Our panel outlines two distinct groups of cell lines: one which releases cfDNA fragmented similarly to clinical samples and purported as characteristic of apoptosis, and another which releases larger fragments associated with vesicular or necrotic DNA. Our cfCRISPR screens reveal that genes mediating cfDNA release are primarily involved with apoptosis, but also identify other subsets of genes such as RNA binding proteins as potential regulators of cfDNA release. We observe that both groups of cells lines identified primarily produce cfDNA through apoptosis. These results establish the utility of cfCRISPR, genetically validate apoptosis as a major mediator of DNA release in vitro, and implicate ways to improve cfDNA assays.
    DOI:  https://doi.org/10.1038/s42003-024-06129-1
  9. RNA. 2024 Apr 12. pii: rna.079925.123. [Epub ahead of print]
    Cytosolic RNA binding of the mitochondrial TCA cycle enzyme malate dehydrogenase (MDH2).
    Michelle Noble, Aindrila Chatterjee, Thileepan Sekaran, Thomas Schwarzl, Matthias W Hentze.
      Several enzymes of intermediary metabolism have been identified to bind RNA in 2 cells, with potential consequences for the bound RNAs and/or the enzyme. In this 3 study, we investigate the RNA-binding activity of the mitochondrial enzyme malate 4 dehydrogenase 2 (MDH2), which functions in the tricarboxylic acid (TCA) cycle and 5 the malate-aspartate shuttle. We confirmed in cellulo RNA-binding of MDH2 using 6 orthogonal biochemical assays and performed enhanced crosslinking and 7 immunoprecipitation (eCLIP) to identify the cellular RNAs associated with endogenous 8 MDH2. Surprisingly, MDH2 preferentially binds cytosolic over mitochondrial RNAs, 9 although the latter are abundant in the milieu of the mature protein. Subcellular 10 fractionation followed by RNA-binding assays revealed that MDH2-RNA interactions 11 occur predominantly outside of mitochondria. We also found that a cytosolically-12 retained N-terminal deletion mutant of MDH2 is competent to bind RNA, indicating that 13 mitochondrial targeting is dispensable for MDH2-RNA interactions. MDH2 RNA 14 binding increased when cellular NAD+ levels (MDH2's co-factor) was 15 pharmacologically diminished, suggesting that the metabolic state of cells affects RNA 16 binding. Taken together, our data implicate an as yet unidentified function of MDH2 17 binding RNA in the cytosol.
    Keywords:  MDH2; RNA-binding proteins; metabolic enzymes
    DOI:  https://doi.org/10.1261/rna.079925.123
  10. Anal Chem. 2024 Apr 09.
    Visualized Tracking and Multidimensional Assessing of Mitochondria-Associated Pyroptosis in Cancer Cells by a Small-Molecule Fluorescent Probe.
    Wenhao Zhao, Chengyuan Wang, Yanzhe Zhu, Qi Wang, Xianyun Xu, Zonglong Shao, Man Chen, Yan Feng, Xiangming Meng.
      Pyroptosis is closely related to the development and treatment of various cancers; thus, comprehensive studies of the correlations between pyroptosis and its inductive or inhibitive factors can provide new ideas for the intervention and diagnosis of tumors. The dysfunction of mitochondria may induce pyroptosis in cancer cells, which can be reflected by the fluctuations of the microenvironmental parameters in mitochondria as well as the changes of mitochondrial DNA level and morphology, etc. To precisely track and assess the mitochondria-associated pyroptosis process, simultaneous visualization of changes in multiphysiological parameters in mitochondria is highly desirable. In this work, we reported a nonreaction-based, multifunctional small-molecule fluorescent probe Mito-DK with the capability of crosstalk-free response to polarity and mtDNA as well as mitochondrial morphology. Accurate assessment of mitochondria-associated pyroptosis induced by palmitic acid/H2O2 was achieved through monitoring changes in mitochondrial multiple parameters with the help of Mito-DK. In particular, the pyroptosis-inducing ability of an antibiotic doxorubicin and the pyroptosis-inhibiting capacity of an anticancer agent puerarin were evaluated by Mito-DK. These results provide new perspectives for visualizing mitochondria-associated pyroptosis and offer new approaches for screening pyroptosis-related anticancer agents.
    DOI:  https://doi.org/10.1021/acs.analchem.4c00318
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