bims-nenemi Biomed News
on Neuroinflammation, neurodegeneration and mitochondria
Issue of 2025–01–12
thirteen papers selected by
Marco Tigano, Thomas Jefferson University
  1. Mapping the Genetic Architecture of the Adaptive Integrated Stress Response in S. cerevisiae.
  2. Novel high-content and open-source image analysis tools for profiling mitochondrial morphology in neurological cell models.
  3. Mitochondria- and ER-associated actin are required for mitochondrial fusion.
  4. DHX15 and Rig-I Coordinate Apoptosis and Innate Immune Signaling by Antiviral RNase L.
  5. Mitochondrial damage causes inflammation via cGAS-STING signaling in ketamine-induced cystitis.
  6. Mapping mitochondrial morphology and function: COX-SBFSEM reveals patterns in mitochondrial disease.
  7. The integrated stress response drives MET oncogene overexpression in cancers.
  8. Regulation of mutant huntingtin mitochondrial toxicity by phosphomimetic mutations within its N-terminal region.
  9. A gated hydrophobic funnel within BAX binds long-chain alkenals to potentiate pro-apoptotic function.
  10. eIF2α Phosphorylation-ATF4 Axis-Mediated Transcriptional Reprogramming Mitigates Mitochondrial Impairment During ER Stress.
  11. The adaptor protein Miro1 modulates horizontal transfer of mitochondria in mouse melanoma models.
  12. Mitochondrial YME1L1 governs unoccupied protein translocase channels.
  13. Apoptotic priming in senescence predicts specific senolysis by quantitative analysis of mitochondrial dependencies.
  1. bioRxiv. 2024 Dec 22. pii: 2024.12.19.629525. [Epub ahead of print]
    Mapping the Genetic Architecture of the Adaptive Integrated Stress Response in S. cerevisiae.
    Rachel Baum, Jinyoung Kim, Ryan Y Muller, Nicholas Ingolia.
      The integrated stress response (ISR) is a conserved eukaryotic signaling pathway that responds to diverse stress stimuli to restore proteostasis. The strength and speed of ISR activation must be tuned properly to allow protein synthesis while maintaining proteostasis. Here, we describe how genetic perturbations change the dynamics of the ISR in budding yeast. We treated ISR dynamics, comprising timecourses of ISR activity across different levels of stress, as a holistic phenotype. We profiled changes in ISR dynamics across thousands of genetic perturbations in parallel using CRISPR interference with barcoded expression reporter sequencing (CiBER-seq). We treated cells with sulfometuron methyl, a titratable inhibitor of branched-amino acid synthesis, and measured expression of an ISR reporter. Perturbations to translation such as depletion of aminoacyl-tRNA synthetases or tRNA biogenesis factors reduced cell growth and caused a strikingly proportionate activation of the ISR activation. In contrast, impaired ribosome biogenesis reduced basal ISR activity and weakened ISR dynamics. Reduced ribosome capacity may lower the demand for amino acids and thereby explain these changes. Our work illustrates how CiBER-seq enables high-throughput measurements of complex and dynamic phenotypes that shed light on adaptive and homeostatic mechanisms.
    DOI:  https://doi.org/10.1101/2024.12.19.629525
  2. SLAS Discov. 2025 Jan 06. pii: S2472-5552(25)00001-2. [Epub ahead of print] 100208
    Novel high-content and open-source image analysis tools for profiling mitochondrial morphology in neurological cell models.
    Marcus Y Chin, David A Joy, Madhuja Samaddar, Anil Rana, Johann Chow, Takashi Miyamoto, Meredith Calvert.
      Mitochondria undergo dynamic morphological changes depending on cellular cues, stress, genetic factors, or disease. The structural complexity and disease-relevance of mitochondria have stimulated efforts to generate image analysis tools for describing mitochondrial morphology for therapeutic development. Using high-content analysis, we measured multiple morphological parameters and employed unbiased feature clustering to identify the most robust pair of texture metrics that described mitochondrial state. Here, we introduce a novel image analysis pipeline to enable rapid and accurate profiling of mitochondrial morphology in various cell types and pharmacological perturbations. We applied a high-content adapted implementation of our tool, MitoProfilerHC, to quantify mitochondrial morphology changes in i) a mammalian cell dose response study and ii) compartment-specific drug effects in primary neurons. Next, we expanded the usability of our pipeline by using napari, a Python-powered image analysis tool, to build an open-source version of MitoProfiler and validated its performance and applicability. In conclusion, we introduce MitoProfiler as both a high-content-based and an open-source method to accurately quantify mitochondrial morphology in cells, which we anticipate to greatly facilitate mechanistic discoveries in mitochondrial biology and disease.
    Keywords:  Mitochondria; high-content imaging; high-throughput screening; image analysis; mitochondrial morphology; napari plugin; neurons; open-source
    DOI:  https://doi.org/10.1016/j.slasd.2025.100208
  3. Nat Commun. 2025 Jan 07. 16(1): 451
    Mitochondria- and ER-associated actin are required for mitochondrial fusion.
    Priya Gatti, Cara Schiavon, Julien Cicero, Uri Manor, Marc Germain.
      Mitochondria are crucial for cellular metabolism and signalling. Mitochondrial activity is modulated by mitochondrial fission and fusion, which are required to properly balance metabolic functions, transfer material between mitochondria, and remove defective mitochondria. Mitochondrial fission occurs at mitochondria-endoplasmic reticulum (ER) contact sites, and requires the formation of actin filaments that drive mitochondrial constriction and the recruitment of the fission protein DRP1. The role of actin in mitochondrial fusion remains entirely unexplored. Here we show that preventing actin polymerisation on either mitochondria or the ER disrupts both fission and fusion. We show that fusion but not fission is dependent on Arp2/3, whereas both fission and fusion require INF2 formin-dependent actin polymerization. We also show that mitochondria-associated actin marks fusion sites prior to the fusion protein MFN2. Together, our work introduces a method for perturbing organelle-associated actin and demonstrates a previously unknown role for actin in mitochondrial fusion.
    DOI:  https://doi.org/10.1038/s41467-024-55758-x
  4. Viruses. 2024 Dec 13. pii: 1913. [Epub ahead of print]16(12):
    DHX15 and Rig-I Coordinate Apoptosis and Innate Immune Signaling by Antiviral RNase L.
    Barkha Ramnani, Trupti Devale, Praveen Manivannan, Aiswarya Haridas, Krishnamurthy Malathi.
      During virus infection, the activation of the antiviral endoribonuclease, ribonuclease L (RNase L), by a unique ligand 2'-5'-oilgoadenylate (2-5A) causes the cleavage of single-stranded viral and cellular RNA targets, restricting protein synthesis, activating stress response pathways, and promoting cell death to establish broad antiviral effects. The immunostimulatory dsRNA cleavage products of RNase L activity (RL RNAs) recruit diverse dsRNA sensors to activate signaling pathways to amplify interferon (IFN) production and activate inflammasome, but the sensors that promote cell death are not known. In this study, we found that DEAH-box polypeptide 15 (DHX15) and retinoic acid-inducible gene I (Rig-I) are essential for apoptosis induced by RL RNAs and require mitochondrial antiviral signaling (MAVS), c-Jun amino terminal kinase (JNK), and p38 mitogen-activated protein kinase (p38 MAPK) for caspase-3-mediated intrinsic apoptosis. In RNase L-activated cells, DHX15 interacts with Rig-I and MAVS, and cells lacking MAVS expression were resistant to apoptosis. RL RNAs induced the transcription of genes for IFN and proinflammatory cytokines by interferon regulatory factor 3 (IRF-3) and nuclear factor kB (NF-kB), while cells lacking both DHX15 and Rig-I showed a reduced induction of cytokines. However, apoptotic cell death is independent of both IRF-3 and NF-kB, suggesting that cytokine and cell death induction by RL RNAs are uncoupled. The RNA binding of both DHX15 and Rig-I is required for apoptosis induction, and the expression of both single proteins in cells lacking both DHX15 and Rig-I is insufficient to promote cell death by RL RNAs. Cell death induced by RL RNAs suppressed Coxsackievirus B3 (CVB3) replication, and inhibiting caspase-3 activity or cells lacking IRF-3 showed that the induction of apoptosis directly resulted in the CVB3 antiviral effect, and the effects were independent of the role of IRF-3.
    Keywords:  DHX15; RNase L; Rig-I; apoptosis; innate immune signaling
    DOI:  https://doi.org/10.3390/v16121913
  5. Inflamm Res. 2025 Jan 07. 74(1): 6
    Mitochondrial damage causes inflammation via cGAS-STING signaling in ketamine-induced cystitis.
    Jinji Chen, Shengsheng Liang, Cheng Li, Bowen Li, Mingdong He, Kezhen Li, Weijin Fu, Shenghua Li, Hua Mi.
       BACKGROUND: Mitochondrial dysfunction and damage can result in the release of mitochondrial DNA (mtDNA) into the cytoplasm, which subsequently activates the cGAS-STING pathway, promoting the onset of inflammatory diseases. Various factors, such as oxidative stress, viral infection, and drug toxicity, have been identified as inducers of mitochondrial damage. This study aims to investigate the role of mtDNA as a critical inflammatory mediator in the pathogenesis of ketamine (KET)-induced cystitis (KC) through the cGAS-STING pathway.
    METHODS: To investigate the role of the cGAS-STING pathway in KET-induced cystitis, we assessed the expression of cGAS and STING in rats with KET cystitis. Additionally, we evaluated STING expression in conditionally deficient Simian Virus-transformed Human Uroepithelial Cell Line 1 (SV-HUC-1) cells in vitro. Morphological changes in mitochondria were examined using transmission electron microscopy. We measured intracellular reactive oxygen species (ROS) production through flow cytometry and immunofluorescence techniques. Furthermore, alterations in associated inflammatory factors and cytokines were quantified using real-time quantitative PCR with fluorescence detection.
    RESULTS: We observed up-regulation of cGAS and STING expressions in the bladder tissue of rats in the KET group, stimulation with KET also led to increased cGAS and STING levels in SV-HUC-1 cells. Notably, the knockdown of STING inhibited the nuclear translocation of NF-κB p65 and IRF3, resulting in a decrease in the expression of inflammatory cytokines, including IL-6, IL-8, and CXCL10. Additionally, KET induced damage to the mitochondria of SV-HUC-1 cells, facilitating the release of mtDNA into the cytoplasm. This significant depletion of mtDNA inhibited the activation of cGAS-STING pathway, subsequently affecting the expression of NF-κB p65 and IRF3. Importantly, the reintroduction of mtDNA after STING knockdown partially restored the inflammatory response.
    CONCLUSION: Our findings confirmed the activation of the cGAS-STING pathway in KC rats and revealed mitochondrial damage in vitro. These results highlight the involvement of the cGAS-STING pathway in the pathogenesis of KC, suggesting its potential as a therapeutic target for intervention.
    Keywords:  Ketamine-induced cystitis; Mitochondrial damage; cGAS - STING signals
    DOI:  https://doi.org/10.1007/s00011-024-01973-7
  6. Commun Biol. 2025 Jan 09. 8(1): 24
    Mapping mitochondrial morphology and function: COX-SBFSEM reveals patterns in mitochondrial disease.
    Julie Faitg, Tracey Davey, Ross Laws, Conor Lawless, Helen Tuppen, Eric Fitton, Doug Turnbull, Amy E Vincent.
      Mitochondria play a crucial role in maintaining cellular health. It is interesting that the shape of mitochondria can vary depending on the type of cell, mitochondrial function, and other cellular conditions. However, there are limited studies that link functional assessment with mitochondrial morphology evaluation at high magnification, even fewer that do so in situ and none in human muscle biopsies. Therefore, we have developed a method which combines functional assessment of mitochondria through Cytochrome c Oxidase (COX) histochemistry, with a 3D electron microscopy (EM) technique, serial block-face scanning electron microscopy (SBFSEM). Here we apply COX-SBFSEM to muscle samples from patients with single, large-scale mtDNA deletions, a cause of mitochondrial disease. These deletions cause oxidative phosphorylation deficiency, which can be observed through changes in COX activity. One of the main advantages of combining 3D-EM with the COX reaction is the ability to look at how per-mitochondrion oxidative phosphorylation status is spatially distributed within muscle fibres. Here we show a robust spatial pattern in COX-positive and intermediate-fibres and that the spatial pattern is less clear in COX-deficient fibres.
    DOI:  https://doi.org/10.1038/s42003-024-07389-7
  7. EMBO J. 2025 Jan 07.
    The integrated stress response drives MET oncogene overexpression in cancers.
    Marina Cerqua, Marco Foiani, Carla Boccaccio, Paolo M Comoglio, Dogus M Altintas.
      Cancer cells rely on invasive growth to survive in a hostile microenvironment; this growth is characterised by interconnected processes such as epithelial-to-mesenchymal transition and migration. A master regulator of these events is the MET oncogene, which is overexpressed in the majority of cancers; however, since mutations in the MET oncogene are seen only rarely in cancers and are relatively infrequent, the mechanisms that cause this widespread MET overexpression remain obscure. Here, we show that the 5' untranslated region (5'UTR) of MET mRNA harbours two functional stress-responsive elements, conferring translational regulation by the integrated stress response (ISR), regulated by phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α) at serine 52. ISR activation by serum starvation, leucine deprivation, hypoxia, irradiation, thapsigargin or gemcitabine is followed by MET protein overexpression. We mechanistically link MET translation to the ISR by (i) mutation of the two uORFs within the MET 5'UTR, (ii) CRISPR/Cas9-mediated mutation of eIF2α (S52A), or (iii) the application of ISR pathway inhibitors. All of these interventions reduce stress-induced MET overexpression. Finally, we show that blocking stress-induced MET translation blunts MET-dependent invasive growth. These findings indicate that upregulation of the MET oncogene is a functional requirement linking integrated stress response to cancer progression.
    Keywords:  Integrated Stress Response; Invasive Growth; MET Oncogene
    DOI:  https://doi.org/10.1038/s44318-024-00338-4
  8. J Neurosci. 2025 Jan 08. pii: e1254242024. [Epub ahead of print]
    Regulation of mutant huntingtin mitochondrial toxicity by phosphomimetic mutations within its N-terminal region.
    Svitlana Yablonska, Colleen E Strohlein, Sergei V Baranov, Stacy M Yeh, Aashka Patel, Tanisha Singh, Abhishek Jauhari, JinHo Kim, Nicolas K Khattar, Fang Li, Xiaomin Wang, Yue-Fang Chang, C Y Daniel Lee, X William Yang, Diane L Carlisle, Robert M Friedlander.
      Huntington's disease (HD), a neurodegenerative disease, affects approximately 30,000 people in the United States, with 200,000 more at risk. Mitochondrial dysfunction caused by mutant huntingtin (mHTT) drives early HD pathophysiology. mHTT binds the translocase of mitochondrial inner membrane (TIM23) complex, inhibiting mitochondrial protein import and altering the mitochondrial proteome. The HTT N-terminal 17-amino acids sequence (N17) acts as a regulatory domain in HD pathogenesis; phosphomimetic modification of serine 13 and 16 of the N17 domain impacts subcellular localization, degradation, and ameliorates toxicity in mouse and cell models of HD. Using cellular and mouse (either sex) HD models, we investigated the mechanisms by which HTT phosphorylation affects intracellular localization. We demonstrate that introducing phosphomimetic mutations within the mHTT fragment N17 domain decreased TIM23 binding affinity and reduced inhibition of mHTT-mediated mitochondrial protein import. BACHD-SD mice expressing full-length mHTT harboring the same two N17 phosphomimetic mutations have an ameliorated HD-like phenotype as compared to mice expressing mHTT. Consistent with reduced toxicity in vivo, we found that the amount of full-length mHTT in brain mitochondria of BACHD-SD transgenic mice is less when the mHTT has two phosphomimetic mutations. To complement the relevance of the phosphomimic HTT findings, endogenous N17 phospho mHTT is less likely to translocate to the mitochondria compared to non-phosphorylated mHTT. We demonstrate that phosphorylation of mHTT at serines 13 and 16 is critical for negatively regulating mHTT mitochondrial targeting and that reducing mHTT mitochondrial localization and binding to TIM23 results in amelioration of mHTT-induced mitochondrial and neuronal toxicity.Significance Statement We establish the first 17 amino acids of mHTT as a mitochondrial targeting sequence driving mHTT accumulation in mitochondria and show that N-terminal phosphomimetic modifications reduce mitochondrial accumulation and toxicity. Utilizing super-resolution live imaging and expansion microscopy of isolated mitochondria and full-length mHTT immunoprecipitation from human cells, we demonstrate HTT intramitochondrial localization and interaction with the mitochondrial protein importing complex. mHTT N17 domain phosphomimetic mutations reduce binding affinity for the TIM23 subunit, preventing mHTT mitochondrial accumulation. This lessens mHTT inhibitory effect on mitochondrial protein import. Our study elucidates the molecular mechanism responsible for the BACHD-SD mouse ameliorated phenotype and underscores a critical need to identify enzymes responsible for regulating HTT phosphorylation to exploit their potential as therapeutic HD targets.
    DOI:  https://doi.org/10.1523/JNEUROSCI.1254-24.2024
  9. bioRxiv. 2024 Dec 23. pii: 2024.12.23.630122. [Epub ahead of print]
    A gated hydrophobic funnel within BAX binds long-chain alkenals to potentiate pro-apoptotic function.
    Jesse D Gelles, Yiyang Chen, Mark P A Luna-Vargas, Ariele Viacava Follis, Stella G Bayiokos, Jarvier N Mohammed, Tara M Sebastian, M Abdullah Al Noman, Ngoc Dung Pham, Yi Shi, Richard W Kriwacki, Jerry E Chipuk.
      Mitochondria maintain a biochemical environment that cooperates with BH3-only proteins (e.g., BIM) to potentiate BAX activation, the key event to initiate physiological and pharmacological forms of apoptosis. The sphingosine-1-phosphate metabolite 2-trans-hexadecenal (2t-hexadecenal) is one such component described to support BAX activation, but molecular mechanisms remain largely unknown. Here, we utilize complementary biochemical and biophysical techniques to reveal that 2t-hexadecenal non-covalently interacts with BAX, and cooperates with BIM to stimulate early-activation steps of monomeric BAX. Integrated structural and computational approaches reveal 2t-hexadecenal binds an undefined region - a hydrophobic cavity formed by core-facing residues of α5, α6, and gated by α8 - we now term the "BAX actuating funnel" (BAF). We define alkenal length and α8 mobility as critical determinants for 2t-hexadecenal synergy with BIM and BAX, and demonstrate that proline 168 allosterically regulates BAF function. Collectively, this work imparts detailed molecular insights advancing our fundamental knowledge of BAX regulation and identifies a regulatory region with implications for biological and therapeutic opportunities.
    DOI:  https://doi.org/10.1101/2024.12.23.630122
  10. Mol Cells. 2025 Jan 03. pii: S1016-8478(24)00201-2. [Epub ahead of print] 100176
    eIF2α Phosphorylation-ATF4 Axis-Mediated Transcriptional Reprogramming Mitigates Mitochondrial Impairment During ER Stress.
    Hien Thi Le, Jiyoung Yu, Hee Sung Ahn, Mi-Jeong Kim, In Gyeong Chae, Hyun-Nam Cho, Juhee Kim, Hye-Kyung Park, Hyuk Nam Kwon, Han-Jung Chae, Byoung Heon Kang, Jeong Kon Seo, Kyunggon Kim, Sung Hoon Back.
      Eukaryotic translation initiation factor 2α (eIF2α) phosphorylation, which regulates all three unfolded protein response pathways, helps maintain cellular homeostasis and overcome endoplasmic reticulum (ER) stress through transcriptional and translational reprogramming. However, transcriptional regulation of mitochondrial homeostasis by eIF2α phosphorylation during ER stress is not fully understood. Here, we report that the eIF2α phosphorylation-activating transcription factor 4 (ATF4) axis is required for expression of multiple transcription factors (TFs) including nuclear factor erythroid 2-related factor 2 (Nrf2) and their target genes responsible for mitochondrial homeostasis during ER stress. eIF2α phosphorylation-deficient (A/A) cells displayed dysregulated mitochondrial dynamics and mitochondrial DNA replication, decreased expression of oxidative phosphorylation complex proteins, and impaired mitochondrial functions during ER stress. ATF4 overexpression suppressed impairment of mitochondrial homeostasis in A/A cells during ER stress by promoting expression of downstream TFs and their target genes. Our findings underscore the importance of the eIF2α phosphorylation-ATF4 axis for maintaining mitochondrial homeostasis through transcriptional reprogramming during ER stress.
    Keywords:  ATF4; ER stress; Mitochondrial homeostasis; Nrf2; eIF2α phosphorylation
    DOI:  https://doi.org/10.1016/j.mocell.2024.100176
  11. Cell Rep. 2025 Jan 09. pii: S2211-1247(24)01505-5. [Epub ahead of print]44(1): 115154
    The adaptor protein Miro1 modulates horizontal transfer of mitochondria in mouse melanoma models.
    Jaromir Novak, Zuzana Nahacka, Gabriela L Oliveira, Petra Brisudova, Maria Dubisova, Sarka Dvorakova, Sona Miklovicova, Marketa Dalecka, Verena Puttrich, Lenka Grycova, Silvia Magalhaes-Novais, Catarina Mendes Correia, Jennifer Levoux, Ludek Stepanek, Jan Prochazka, David Svec, David Pajuelo Reguera, Guillermo Lopez-Domenech, Renata Zobalova, Radek Sedlacek, Mikkel G Terp, Payam A Gammage, Zdenek Lansky, Josef Kittler, Paulo J Oliveira, Henrik J Ditzel, Michael V Berridge, Anne-Marie Rodriguez, Stepana Boukalova, Jakub Rohlena, Jiri Neuzil.
      Recent research has shown that mtDNA-deficient cancer cells (ρ0 cells) acquire mitochondria from tumor stromal cells to restore respiration, facilitating tumor formation. We investigated the role of Miro1, an adaptor protein involved in movement of mitochondria along microtubules, in this phenomenon. Inducible Miro1 knockout (Miro1KO) mice markedly delayed tumor formation after grafting ρ0 cancer cells. Miro1KO mice with fluorescently labeled mitochondria revealed that this delay was due to hindered mitochondrial transfer from the tumor stromal cells to grafted B16 ρ0 cells, which impeded recovery of mitochondrial respiration and tumor growth. Miro1KO led to the perinuclear accumulation of mitochondria and impaired mobility of the mitochondrial network. In vitro experiments revealed decreased association of mitochondria with microtubules, compromising mitochondrial transfer via tunneling nanotubes (TNTs) in mesenchymal stromal cells. Here we show the role of Miro1 in horizontal mitochondrial transfer in mouse melanoma models in vivo and its involvement with TNTs.
    Keywords:  CP: Cancer; CP: Cell biology; Miro1; RHOT1; cancer; horizontal transfer of mitochondria; melanoma; mitochondria; tunneling nanotubes
    DOI:  https://doi.org/10.1016/j.celrep.2024.115154
  12. Nat Cell Biol. 2025 Jan 07.
    Mitochondrial YME1L1 governs unoccupied protein translocase channels.
    Meng-Chieh Hsu, Hiroki Kinefuchi, Linlin Lei, Reika Kikuchi, Koji Yamano, Richard J Youle.
      Mitochondrial protein import through the outer and inner membranes is key to mitochondrial biogenesis. Recent studies have explored how cells respond when import is impaired by a variety of different insults. Here, we developed a mammalian import blocking system using dihydrofolate reductase fused to the N terminus of the inner membrane protein MIC60. While stabilization of the dihydrofolate reductase domain by methotrexate inhibited endogenous mitochondrial protein import, it neither activated the transcription factor ATF4, nor was affected by ATAD1 expression or by VCP/p97 inhibition. On the other hand, notably, plugging the channel of translocase of the outer membrane) induced YME1L1, an ATP-dependent protease, to eliminate translocase of the inner membrane (TIM23) channel components TIMM17A and TIMM23. The data suggest that unoccupied TIM23 complexes expose a C-terminal degron on TIMM17A to YME1L1 for degradation. Import plugging caused a cell growth defect and loss of YME1L1 exacerbated the growth inhibition, showing the protective effect of YME1L1 activity. YME1L1 seems to play a crucial role in mitochondrial quality control to counteract precursor stalling in the translocase of the outer membrane complex and unoccupied TIM23 channels.
    DOI:  https://doi.org/10.1038/s41556-024-01571-z
  13. Cell Death Differ. 2025 Jan 06.
    Apoptotic priming in senescence predicts specific senolysis by quantitative analysis of mitochondrial dependencies.
    Julie A MacDonald, Gary A Bradshaw, Fleur Jochems, René Bernards, Anthony Letai.
      Cellular senescence contributes to a variety of pathologies associated with aging and is implicated as a cellular state in which cancer cells can survive treatment. Reported senolytic drug treatments act through varying molecular mechanisms, but heterogeneous efficacy across the diverse contexts of cellular senescence indicates a need for predictive biomarkers of senolytic activity. Using multi-parametric analyses of commonly reported molecular features of the senescent phenotype, we assayed a variety of models, including malignant and nonmalignant cells, using several triggers of senescence induction and found little univariate predictive power of these traditional senescence markers to identify senolytic drug sensitivity. We sought to identify novel drug targets in senescent cells that were insensitive to frequently implemented senolytic therapies, such as Navitoclax (ABT-263), using quantitative mass spectrometry to measure changes in the senescent proteome, compared to cells which acquire an acute sensitivity to ABT-263 with senescence induction. Inhibition of the antioxidant GPX4 or the Bcl-2 family member MCL-1 using small molecule compounds in combination with ABT-263 significantly increased the induction of apoptosis in some, but not all, previously insensitive senescent cells. We then asked if we could use BH3 profiling to measure differences in mitochondrial apoptotic priming in these models of cellular senescence and predict sensitivity to the senolytics ABT-263 or the combination of dasatinib and quercetin (D + Q). We found, despite being significantly less primed for apoptosis overall, the dependence of senescent mitochondria on BCL-XL was significantly correlated to senescent cell killing by both ABT-263 and D + Q, despite no significant changes in the gene or protein expression of BCL-XL. However, our data caution against broad classification of drugs as globally senolytic and instead provide impetus for context-specific senolytic targets and propose BH3 profiling as an effective predictive biomarker.
    DOI:  https://doi.org/10.1038/s41418-024-01431-1
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