bims-nenemi 2025-04-13 papers

bims-nenemi

Biomed News

on Neuroinflammation, neurodegeneration and mitochondria

Issue of 2025–04–13
twenty-six papers selected by
Marco Tigano, Thomas Jefferson University

Cooperative role of PACT and ADAR1 in preventing aberrant PKR activation by self-derived double-stranded RNA.
Mitochondria and cell death signalling.
Pulmonary mitochondrial DNA release and activation of the cGAS-STING pathway in Lethal Stx12 knockout mice.
The Volume of Mitochondria Inherited Impacts mtDNA Homeostasis in Budding Yeast.
Mitochondrial DNA released by senescent tumor cells enhances PMN-MDSC-driven immunosuppression through the cGAS-STING pathway.
Effective recognition of double-stranded RNA does not require activation of cellular inflammation.
Structural basis of BAK sequestration by MCL-1 in apoptosis.
Mitochondrial damage is associated with an early immune response in inclusion body myositis.
Small molecules restore mutant mitochondrial DNA polymerase activity.
ARMC1 partitions between distinct complexes and assembles MIRO with MTFR to control mitochondrial distribution.
GPAT4 sustains endoplasmic reticulum homeostasis in endocardial cells and safeguards heart development.
PACT prevents aberrant activation of PKR by endogenous dsRNA without sequestration.
CMPK2 promotes microglial activation through the cGAS-STING pathway in the neuroinflammatory mechanism.
Acute myeloid leukemia mitochondria hydrolyze ATP to support oxidative metabolism and resist chemotherapy.
RNA G-quadruplexes control mitochondria-localized mRNA translation and energy metabolism.
An unconventional autophagic pathway that inhibits ATP secretion during apoptotic cell death.
Knockdown of POLG Mimics the Neuronal Pathology of Polymerase-γ Spectrum Disorders in Human Neurons.
TMEM65 regulates and is required for NCLX-dependent mitochondrial calcium efflux.
The mitochondrial unfolded protein response inhibits pluripotency acquisition and mesenchymal-to-epithelial transition in somatic cell reprogramming.
Origin of yield stress and mechanical plasticity in model biological tissues.
MIRO1 Is Required for Dynamic Increases in Mitochondria-ER Contact Sites and Mitochondrial ATP During the Cell Cycle.
FLIPL permits apoptotic and inflammatory signaling and inhibits necroptosis in mice without Caspase-8 oligomerization.
Characterization of dsRNA binding proteins through solubility analysis identifies ZNF385A as a dsRNA homeostasis regulator.
Non-canonical STAT3 pathway induces alterations of mitochondrial dynamic proteins in the hippocampus of an LPS-induced murine neuroinflammation model.
Exploring the role of Mitoferrin-1 in Ferroptosis and mitochondrial damage in acute ischemic stroke.
A morphology and secretome map of pyroptosis.

Nat Commun. 2025 Apr 05. 16(1): 3246

Cooperative role of PACT and ADAR1 in preventing aberrant PKR activation by self-derived double-stranded RNA.

Lavanya Manjunath, Gisselle Santiago, Pedro Ortega, Ambrocio Sanchez, Sunwoo Oh, Alexander Garcia, Junyi Li, Dana Duong, Elodie Bournique, Alexis Bouin, Bert L Semler, Dheva Setiaputra, Rémi Buisson.

Double-stranded RNAs (dsRNAs) produced during viral infections are recognized by the innate immune sensor protein kinase R (PKR), triggering a host translation shutoff that inhibits viral replication and propagation. Given the harmful effects of uncontrolled PKR activation, cells must tightly regulate PKR to ensure that its activation occurs only in response to viral infections, not endogenous dsRNAs. Here, we use CRISPR-Translate, a FACS-based genome-wide CRISPR-Cas9 knockout screening method that exploits translation levels as a readout and identifies PACT as a key inhibitor of PKR during viral infection. We find that PACT-deficient cells hyperactivate PKR in response to different RNA viruses, raising the question of why cells need to limit PKR activity. Our results demonstrate that PACT cooperates with ADAR1 to suppress PKR activation from self-dsRNAs in uninfected cells. The simultaneous deletion of PACT and ADAR1 results in synthetic lethality, which can be fully rescued in PKR-deficient cells. We propose that both PACT and ADAR1 act as essential barriers against PKR, creating a threshold of tolerable levels to endogenous dsRNA in cells without activating PKR-mediated translation shutdown and cell death.

DOI: https://doi.org/10.1038/s41467-025-58412-2
Curr Opin Cell Biol. 2025 Apr 10. pii: S0955-0674(25)00048-1. [Epub ahead of print]94 102510

Mitochondria and cell death signalling.

Ella Hall-Younger, Stephen Wg Tait.

Mitochondria are essential organelles in the life and death of a cell. During apoptosis, mitochondrial outer membrane permeabilisation (MOMP) engages caspase activation and cell death. Under nonlethal apoptotic stress, some mitochondria undergo permeabilisation, termed minority MOMP. Nonlethal apoptotic signalling impacts processes including genome stability, senescence and innate immunity. Recent studies have shown that upon MOMP, mitochondria and consequent signalling can trigger inflammation. We discuss how this occurs, and how mitochondrial inflammation might be targeted to increase tumour immunogenicity. Finally, we highlight how mitochondria contribute to other types of cell death including pyroptosis and ferroptosis. Collectively, these studies reveal critical new insights into how mitochondria regulate cell death, highlighting that mitochondrial signals engaged under nonlethal apoptotic stress have wide-ranging biological functions.

DOI: https://doi.org/10.1016/j.ceb.2025.102510
Cell Commun Signal. 2025 Apr 08. 23(1): 174

Pulmonary mitochondrial DNA release and activation of the cGAS-STING pathway in Lethal Stx12 knockout mice.

Dan-Hua Liu, Fang Li, Run-Zhou Yang, Zhuanbin Wu, Xiao-Yan Meng, Sen-Miao Li, Wen-Xiu Li, Jia-Kang Li, Dian-Dian Wang, Rui-Yu Wang, Shu-Ang Li, Pei-Pei Liu, Jian-Sheng Kang.

  STX12 (syntaxin12 or syntaxin13), a member of the SNARE protein family, plays a crucial role in intracellular vesicle transport and membrane fusion. Our previous research demonstrated that Stx12 knockout mice exhibit perinatal lethality with iron deficiency anemia. Despite its importance, the comprehensive physiological and pathological mechanism of STX12 remains largely unknown. Here, we revealed that STX12 deficiency causes the depolarization of mitochondrial membrane potential in zebrafish embryos and mouse embryonic fibroblasts. Additionally, the loss of STX12 decreased the levels of mitochondrial complex subunits, accompanied by mitochondrial DNA (mtDNA) release and activated cGAS-STING pathway and Type I interferon pathway in the lung tissue of Stx12-/- mice. Additionally, we observed a substantial increase in cytokines and neutrophil infiltration within the lung tissues of Stx12 knockout mice, indicating severe inflammation, which could be a contributing factor for Stx12-/- mortality. Various interventions have failed to rescue the lethal phenotype, suggesting that systemic effects may contribute to lethality. Further research is warranted to elucidate potential intervention strategies. Overall, our findings uncover the critical role of STX12 in maintaining mitochondrial function and mtDNA stability in pulmonary cells, and reveal that STX12 depletion results in pulmonary mtDNA release and activates mtDNA-dependent innate immunity.

Keywords:  Inflammation; Mitochondria; STX12; cGAS-STING; mtDNA release

DOI:  https://doi.org/10.1186/s12964-025-02141-y
bioRxiv. 2025 Mar 26. pii: 2025.03.25.645216. [Epub ahead of print]

The Volume of Mitochondria Inherited Impacts mtDNA Homeostasis in Budding Yeast.

Michael W Ray, WeiTing Chen, Chengzhe Duan, Guadalupe Bravo, Kyle Krueger, Erica M Rosario, Alexis A Jacob, Laura L Lackner.

Most eukaryotic cells maintain mitochondria in well-distributed, reticular networks. The size of the mitochondrial network and copy number of its genome scale with cell size. However, while the size scaling features of mitochondria and their genome are interrelated, the fitness consequences of this interdependence are not well understood. We exploit the asymmetric cell division of budding yeast to test the hypothesis that mitochondrial scaling with cell size impacts mitochondrial DNA (mtDNA) function. We find that the volume of mitochondria inherited by daughter cells affects the ability of cells to maintain functional mtDNA; daughter cells that inherit a significantly reduced volume of mitochondria have an increased frequency of losing respiratory competence. In cells with such mitochondrial inheritance defects, mtDNA integrity can be maintained by upregulating mtDNA copy number. Collectively, these data support a bet-hedging model whereby the faithful inheritance of an adequate volume of mitochondria ensures enough mtDNA copies are transmitted to daughter cells to counteract pre-existing and/or inevitable mtDNA mutations.
Summary: Ray et al. demonstrate that the volume of mitochondria inherited impacts mtDNA homeostasis in the model system budding yeast. They propose a model by which inheritance of an adequate mitochondrial volume results in the transmission of sufficient mtDNA copies to counteract existing and/or inevitable mutations.

DOI: https://doi.org/10.1101/2025.03.25.645216
Immunity. 2025 Apr 08. pii: S1074-7613(25)00124-4. [Epub ahead of print]58(4): 811-825.e7

Mitochondrial DNA released by senescent tumor cells enhances PMN-MDSC-driven immunosuppression through the cGAS-STING pathway.

Ping Lai, Lei Liu, Nicolò Bancaro, Martina Troiani, Bianca Calì, Yuxin Li, Jingjing Chen, Prafull Kumar Singh, Rydell Alvarez Arzola, Giuseppe Attanasio, Nicolò Pernigoni, Emiliano Pasquini, Simone Mosole, Andrea Rinaldi, Jacopo Sgrignani, Shi Qiu, Pan Song, Yingrui Li, Maria Andrea Desbats, Azucena Rendón Ángel, Ricardo Pereira Mestre, Andrea Cavalli, Lucio Barile, Johann de Bono, Andrea Alimonti.

  Mitochondrial dysfunction is a hallmark of cellular senescence. Here, we investigated whether senescent cells release mitochondrial (mt)DNA into the extracellular space and its impact on innate immunity. We found that both primary senescent cells and tumor cells undergoing therapy-induced senescence actively released mtDNA into the extracellular environment. mtDNA released by senescent cells was packaged within extracellular vesicles and selectively transferred to polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) in the tumor microenvironment. Upon uptake, extracellular mtDNA enhanced the immunosuppressive activity of PMN-MDSCs via cGAS-STING-NF-κB signaling, thereby promoting tumor progression. While STING activation directly induced NF-κB signaling, it also activated PKR-like endoplasmic reticulum kinase (PERK), which further amplified NF-κB activity, in PMN-MDSCs. mtDNA release from senescent cells was mediated by voltage-dependent anion channels (VDACs), and pharmacological inhibition of VDAC reduced extracellular mtDNA levels, reversed PMN-MDSC-driven immunosuppression, and enhanced chemotherapy efficacy in prostate cancer mouse models. These findings suggest that targeting mtDNA release could reprogram the immunosuppressive tumor microenvironment, improving therapeutic outcomes for chemotherapy-treated patients.

Keywords:  DAMP; PMN-MDSCs; cGAS-STING pathway; immunosuppression; innate immunity; mtDNA; senescence; tumor microenvironment

DOI:  https://doi.org/10.1016/j.immuni.2025.03.005
Sci Adv. 2025 Apr 11. 11(15): eads6498

Effective recognition of double-stranded RNA does not require activation of cellular inflammation.

Karolina Drazkowska, Julia Cieslicka, Michal Kitowicz, Anna Pastucha, Lukasz Markiewicz, Wiktoria Szymanek, Krzysztof Goryca, Tomasz Kowalczyk, Dominik Cysewski, Andreas R Bausch, Pawel J Sikorski.

Excess double-stranded RNA (dsRNA) is present in the cytoplasm of human cells, usually following viral infections. Recognition of dsRNAs activates innate immune pathways, leading to cellular inflammation and inhibition of cell growth. Here, we show that an effective dsRNA response may occur without the onset of inflammation. Pro-inflammatory [RLR (retinoic acid-inducible gene I-like receptor)-dependent pathway] and cell growth inhibitory mechanisms [oligoadenylate synthetase (OAS)/ribonuclease L (RNase L)- and dsRNA-activated protein kinase (PKR)-dependent pathways] can act independently. We found that the 5' ends of dsRNA direct the onset of cellular inflammation, whereas the RNA duplex activates the OAS/RNase L and PKR pathways. Unexpectedly, three of the most common human RNA epitranscriptomic marks-i.e., N6-methyladenosine, 5-methylcytosine, and pseudouridine-had almost no influence on the immunogenicity of dsRNA; however, the presence of N6-methyladenosine inhibited the OAS/RNase L pathway. Our observations demonstrate how precisely innate immunity is fine tuned in cells to take appropriate countermeasures when a specific threat arises.

DOI: https://doi.org/10.1126/sciadv.ads6498
Mol Cell. 2025 Mar 28. pii: S1097-2765(25)00251-5. [Epub ahead of print]

Structural basis of BAK sequestration by MCL-1 in apoptosis.

Shagun Srivastava, Giridhar Sekar, Adedolapo Ojoawo, Anup Aggarwal, Elisabeth Ferreira, Emiko Uchikawa, Meek Yang, Christy R Grace, Raja Dey, Yi-Lun Lin, Cristina D Guibao, Seetharaman Jayaraman, Somnath Mukherjee, Anthony A Kossiakoff, Bin Dong, Alexander Myasnikov, Tudor Moldoveanu.

  Apoptosis controls cell fate, ensuring tissue homeostasis and promoting disease when dysregulated. The rate-limiting step in apoptosis is mitochondrial poration by the effector B cell lymphoma 2 (BCL-2) family proteins BAK and BAX, which are activated by initiator BCL-2 homology 3 (BH3)-only proteins (e.g., BIM) and inhibited by guardian BCL-2 family proteins (e.g., MCL-1). We integrated structural, biochemical, and pharmacological approaches to characterize the human prosurvival MCL-1:BAK complex assembled from their BCL-2 globular core domains. We reveal a canonical interaction with BAK BH3 bound to the hydrophobic groove of MCL-1 and disordered and highly dynamic BAK regions outside the complex interface. We predict similar conformations of activated effectors in complex with other guardians or effectors. The MCL-1:BAK complex is a major cancer drug target. We show that MCL-1 inhibitors are inefficient in neutralizing the MCL-1:BAK complex, requiring high doses to initiate apoptosis. Our study underscores the need to design superior clinical candidate MCL-1 inhibitors.

Keywords:  BCL-2 antagonist killer BAK; BCL-2 family proteins; BCL-2-like protein 11 BIM; BH3 mimetics; BH3-interacting domain death agonist BID; BH3-only initiator; MCL-1 inhibitors; NMR spectroscopy; Phorbol-12-myristate-13-acetate-induced protein 1 NOXA; X-ray crystallography; apoptosis; apoptosis resistance; cryo-electron microscopy; direct BAK activation; induced myeloid leukemia cell differentiation protein MCL-1; mitochondrial outer membrane permeabilization MOMP; mode II MCL-1:BAK sequestration; mode II neutralization; prodeath effector; prosurvival guardian

DOI:  https://doi.org/10.1016/j.molcel.2025.03.013
Brain. 2025 Apr 07. pii: awaf118. [Epub ahead of print]

Mitochondrial damage is associated with an early immune response in inclusion body myositis.

Felix Kleefeld, Emily Cross, Daniel Lagos, Sara Walli, Benedikt Schoser, Andreas Hentschel, Tobias Ruck, Christopher Nelke, Katrin Hahn, Denisa Hathazi, Andrew L Mammen, Maria Casal-Dominguez, Marta Gut, Ivo Glynne Gut, Simon Heath, Anne Schänzer, Hans-Hilmar Goebel, Iago Pinal-Fernandez, Andreas Roos, Corinna Preuße, Werner Stenzel, Rita Horvath.

  Polymyositis with mitochondrial pathology (PM-Mito) was first identified in 1997 as a subtype of idiopathic inflammatory myopathy. Recent findings demonstrated significant molecular similarities between PM-Mito and Inclusion Body Myositis (IBM), suggesting a trajectory from early to late IBM and prompting the inclusion of PM-Mito as an IBM precursor (early IBM) within the IBM spectrum. Both PM-Mito and IBM show mitochondrial abnormalities, suggesting mitochondrial disturbance is a critical element of IBM pathogenesis. The primary objective of this cross-sectional study was to characterize the mitochondrial phenotype in PM-Mito at histological, ultrastructural, and molecular levels and to study the interplay between mitochondrial dysfunction and inflammation. Skeletal muscle biopsies of 27 patients with PM-Mito and 27 with typical IBM were included for morphological and ultrastructural analysis. Mitochondrial DNA (mtDNA) copy number and deletions were assessed by qPCR and long-range PCR, respectively. In addition, full-length single-molecule sequencing of the mtDNA enabled precise mapping of deletions. Protein and RNA levels were studied using unbiased proteomic profiling, immunoblotting, and bulk RNA sequencing. Cell-free mtDNA (cf-mtDNA) was measured in the serum of IBM patients. We found widespread mitochondrial abnormalities in both PM-Mito and IBM, illustrated by elevated numbers of COX-negative and SDH-positive fibers and prominent ultrastructural abnormalities with disorganized and concentric cristae within enlarged and dysmorphic mitochondria. MtDNA copy numbers were significantly reduced, and multiple large-scale mtDNA deletions were already evident in PM-Mito, compared to healthy age-matched controls, similar to the IBM group. The canonical cGAS/STING inflammatory pathway was activated in PM-Mito and IBM, and we detected elevated levels of circulating cf-mtDNA indicative of mtDNA leakage. In PM-Mito and IBM, these findings were accompanied by dysregulation of proteins and transcripts linked to the mitochondrial membranes. In summary, we identified that mitochondrial dysfunction with multiple mtDNA deletions and depletion, disturbed mitochondrial ultrastructure, and defects of the inner mitochondrial membrane are features of PM-Mito and IBM, underlining the concept of an IBM-spectrum disease (IBM-SD). Notably, mitochondrial abnormalities precede tissue remodeling and infiltration by specific T-cell subpopulations (e.g., KLRG1+) characteristic of late IBM. The activation of inflammatory, DNA-sensing pathways might be related to mtDNA release, which would indicate a significant role of mitochondria-associated inflammation in the pathogenesis of IBM-SD. This study highlights the critical role of early mitochondrial abnormalities in the pathomechanism of IBM, which may lead to new approaches to therapy.

Keywords:  IBM-SD; PM-Mito; mitochondria; polymyositis with mitochondrial pathology

DOI:  https://doi.org/10.1093/brain/awaf118
Nature. 2025 Apr 09.

Small molecules restore mutant mitochondrial DNA polymerase activity.

Sebastian Valenzuela, Xuefeng Zhu, Bertil Macao, Mattias Stamgren, Carol Geukens, Paul S Charifson, Gunther Kern, Emily Hoberg, Louise Jenninger, Anja V Gruszczyk, Seoeun Lee, Katarina A S Johansson, Javier Miralles Fusté, Yonghong Shi, S Jordan Kerns, Laleh Arabanian, Gabriel Martinez Botella, Sofie Ekström, Jeremy Green, Andrew M Griffin, Carlos Pardo-Hernández, Thomas A Keating, Barbara Küppers-Munther, Nils-Göran Larsson, Cindy Phan, Viktor Posse, Juli E Jones, Xie Xie, Simon Giroux, Claes M Gustafsson, Maria Falkenberg.

Mammalian mitochondrial DNA (mtDNA) is replicated by DNA polymerase γ (POLγ), a heterotrimeric complex consisting of a catalytic POLγA subunit and two accessory POLγB subunits1. More than 300 mutations in POLG, the gene encoding the catalytic subunit, have been linked to severe, progressive conditions with high rates of morbidity and mortality, for which no treatment exists2. Here we report on the discovery and characterization of PZL-A, a first-in-class small-molecule activator of mtDNA synthesis that is capable of restoring function to the most common mutant variants of POLγ. PZL-A binds to an allosteric site at the interface between the catalytic POLγA subunit and the proximal POLγB subunit, a region that is unaffected by nearly all disease-causing mutations. The compound restores wild-type-like activity to mutant forms of POLγ in vitro and activates mtDNA synthesis in cells from paediatric patients with lethal POLG disease, thereby enhancing biogenesis of the oxidative phosphorylation machinery and cellular respiration. Our work demonstrates that a small molecule can restore function to mutant DNA polymerases, offering a promising avenue for treating POLG disorders and other severe conditions linked to depletion of mtDNA.

DOI: https://doi.org/10.1038/s41586-025-08856-9
Sci Adv. 2025 Apr 11. 11(15): eadu5091

ARMC1 partitions between distinct complexes and assembles MIRO with MTFR to control mitochondrial distribution.

Michael J McKenna, Felix Kraus, João P L Coelho, Muskaan Vasandani, Jiuchun Zhang, Benjamin M Adams, Joao A Paulo, J Wade Harper, Sichen Shao.

Maintaining an optimal mitochondrial distribution is critical to ensure an adequate supply of energy and metabolites to support important cellular functions. How cells balance dynamic mitochondrial processes to achieve homeostasis is incompletely understood. Here, we show that ARMC1 partitioning between distinct mitochondrial protein complexes is a key determinant of mitochondrial distribution. In one complex, the mitochondrial trafficking adaptor MIRO recruits ARMC1, which mediates the assembly of a mitochondrial fission regulator (MTFR). MTFR stability depends on ARMC1, and MIRO-MTFR complexes specifically antagonize retrograde mitochondrial movement. In another complex, DNAJC11 facilitates ARMC1 release from mitochondria. Disrupting MIRO-MTFR assembly fails to rescue aberrant mitochondrial distributions clustered in the perinuclear area observed with ARMC1 deletion, while disrupting ARMC1 interaction with DNAJC11 leads to excessive mitochondrially localized ARMC1 and distinct mitochondrial defects. Thus, the abundance and trafficking impact of MIRO-MTFR complexes require ARMC1, whose mito-cytoplasmic shuttling balanced by DNAJC11 tunes steady-state mitochondrial distributions.

DOI: https://doi.org/10.1126/sciadv.adu5091
Nat Commun. 2025 Apr 08. 16(1): 3345

GPAT4 sustains endoplasmic reticulum homeostasis in endocardial cells and safeguards heart development.

Tianyang Zhao, Kuipei Jin, Xiaodong Wang, Xiong Su, Youjun Wang, Mingming Gao, Wen Luo, Hongyuan Yang, Zhongzhou Yang.

The endocardium plays a pivotal role in governing myocardial development, and understanding the intrinsic regulatory insights will help apprehend pathological cardiomyopathy. Glycerol-3-phosphate acyltransferase 4 (GPAT4) is an endoplasmic reticulum (ER) membrane anchored protein. While the role of GPAT4 in glycerophospholipid biosynthesis is well established, its function in the ER is less explored. Here, we generate Gpat4 global and tissue-specific knockout mice and identify the essential role of GPAT4 in endocardial development. Deficiency of GPAT4 provokes endocardial ER stress response and enhances ER-mitochondrial (ER-mito) communications, leading to mitochondrial DNA (mtDNA) escape. As a result, the cGAS-STING pathway is triggered to stimulate type-I-interferon response, which affects heart development. Finally, abolishment of the cGAS-STING-type-I-interferon pathway rescues the heart defects of Gpat4 deletion mice. These findings uncover the pivotal role of GPAT4 in the maintenance of ER homeostasis during endocardial and heart development. Meanwhile, this study highlights the importance of the cGAS-STING pathway in cardiac organogenesis.

DOI: https://doi.org/10.1038/s41467-025-58722-5
Nat Commun. 2025 Apr 08. 16(1): 3325

PACT prevents aberrant activation of PKR by endogenous dsRNA without sequestration.

Sadeem Ahmad, Tao Zou, Jihee Hwang, Linlin Zhao, Xi Wang, Anton Davydenko, Ilana Buchumenski, Patrick Zhuang, Alyssa R Fishbein, Diego Capcha-Rodriguez, Aaron Orgel, Erez Y Levanon, Sua Myong, James Chou, Matthew Meyerson, Sun Hur.

The innate immune sensor PKR for double-stranded RNA (dsRNA) is critical for antiviral defense, but its aberrant activation by cellular dsRNA is linked to various diseases. The dsRNA-binding protein PACT plays a critical yet controversial role in this pathway. We show that PACT directly suppresses PKR activation by endogenous dsRNA ligands, such as inverted-repeat Alu RNAs, which robustly activate PKR in the absence of PACT. Instead of competing for dsRNA binding, PACT prevents PKR from scanning along dsRNA-a necessary step for PKR molecules to encounter and phosphorylate each other for activation. While PKR favors longer dsRNA for increased co-occupancy and scanning-mediated activation, longer dsRNA is also more susceptible to PACT-mediated regulation due to increased PACT-PKR co-occupancy. Unlike viral inhibitors that constitutively suppress PKR, this RNA-dependent mechanism allows PACT to fine-tune PKR activation based on dsRNA length and quantity, ensuring self-tolerance without sequestering most cellular dsRNA.

DOI: https://doi.org/10.1038/s41467-025-58433-x
Sci Rep. 2025 Apr 07. 15(1): 11807

CMPK2 promotes microglial activation through the cGAS-STING pathway in the neuroinflammatory mechanism.

Feng Gao, Zijian Zheng, Xinjie Liu, Jianwei Li.

  The activation of microglia and the resulting neuroinflammation play crucial regulatory roles in the pathogenesis and progression of neurological diseases, although the specific mechanisms remain incompletely understood. Cytidine monophosphate kinase 2 (CMPK2) is a key mitochondrial nucleotide kinase involved in cellular energy metabolism and nucleotide synthesis. Recent studies suggest that CMPK2 plays a role in microglial-mediated neuroinflammation; however, its specific impact on microglial activation remains unclear. In this study, we hypothesize that CMPK2 promotes microglial-mediated neuroinflammation by activating the cGAS-STING signaling pathway. To investigate this mechanism, we employed lipopolysaccharide (LPS)-treated microglial cells to investigate the detailed mechanisms by which CMPK2 regulates neuroinflammation. Our experimental results indicate that in the BV2 and mouse primary microglial neuroinflammation model, both CMPK2 protein and transcript levels were significantly elevated, accompanied by microglial activation phenotypes such as increased cell size, shortened processes, transformation to round or rod-like shapes, and elevated CD40 expression. Concurrently, there was an increase in pro-inflammatory cytokine levels and a decrease in anti-inflammatory cytokine levels. Further investigation revealed that in the microglial, the expression of cGAS and STING was elevated, along with an increase in oxidative products and inflammatory responses. CMA stimulation further intensified these changes, while cGAS knockdown mitigated them. Finally, we demonstrated that cGAS knockdown inhibited the oxidative stress, cell activation-related changes, and neuroinflammatory responses induced by CMPK2 overexpression in the BV2 neuroinflammation model. Molecular docking experiments showed that CMPK2 stably binds to cGAS at the protein level. These findings suggest that the cGAS-STING pathway mediates CMPK2-induced microglial activation. In summary, our study demonstrates that LPS-induced CMPK2 overactivity promotes microglial activation and neuroinflammatory through the cGAS-STING pathway.

Keywords:  CMPK2; Microglia; Neuroinflammatory; cGAS-STING Pathway

DOI:  https://doi.org/10.1038/s41598-025-97232-8
Sci Adv. 2025 Apr 11. 11(15): eadu5511

Acute myeloid leukemia mitochondria hydrolyze ATP to support oxidative metabolism and resist chemotherapy.

James T Hagen, McLane M Montgomery, Raphael T Aruleba, Brett R Chrest, Polina Krassovskaia, Thomas D Green, Emely A Pacheco, Miki Kassai, Tonya N Zeczycki, Cameron A Schmidt, Debajit Bhowmick, Su-Fern Tan, David J Feith, Charles E Chalfant, Thomas P Loughran, Darla Liles, Mark D Minden, Aaron D Schimmer, Md Salman Shakil, Matthew J McBride, Myles C Cabot, Joseph M McClung, Kelsey H Fisher-Wellman.

OxPhos inhibitors have struggled to show a clinical benefit because of their inability to distinguish healthy from cancerous mitochondria. Herein, we describe an actionable bioenergetic mechanism unique to acute myeloid leukemia (AML) mitochondria. Unlike healthy cells that couple respiration to ATP synthesis, AML mitochondria support inner-membrane polarization by consuming ATP. Matrix ATP consumption allows cells to survive bioenergetic stress. Thus, we hypothesized AML cells may resist chemotherapy-induced cell death by reversing the ATP synthase reaction. In support, BCL-2 inhibition with venetoclax abolished OxPhos flux without affecting mitochondrial polarization. In surviving AML cells, sustained mitochondrial polarization depended on matrix ATP consumption. Mitochondrial ATP consumption was further enhanced in AML cells made refractory to venetoclax, consequential to down-regulations in the endogenous F1-ATPase inhibitor ATP5IF1. Knockdown of ATP5IF1 conferred venetoclax resistance, while ATP5IF1 overexpression impaired F1-ATPase activity and heightened sensitivity to venetoclax. These data identify matrix ATP consumption as a cancer cell-intrinsic bioenergetic vulnerability actionable in the context of BCL-2 targeted chemotherapy.

DOI: https://doi.org/10.1126/sciadv.adu5511
Nat Commun. 2025 Apr 07. 16(1): 3292

RNA G-quadruplexes control mitochondria-localized mRNA translation and energy metabolism.

Leïla Dumas, Sauyeun Shin, Quentin Rigaud, Marie Cargnello, Beatriz Hernández-Suárez, Pauline Herviou, Nathalie Saint-Laurent, Marjorie Leduc, Morgane Le Gall, David Monchaud, Erik Dassi, Anne Cammas, Stefania Millevoi.

Cancer cells rely on mitochondria for their bioenergetic supply and macromolecule synthesis. Central to mitochondrial function is the regulation of mitochondrial protein synthesis, which primarily depends on the cytoplasmic translation of nuclear-encoded mitochondrial mRNAs whose protein products are imported into mitochondria. Despite the growing evidence that mitochondrial protein synthesis contributes to the onset and progression of cancer, and can thus offer new opportunities for cancer therapy, knowledge of the underlying molecular mechanisms remains limited. Here, we show that RNA G-quadruplexes (RG4s) regulate mitochondrial function by modulating cytoplasmic mRNA translation of nuclear-encoded mitochondrial proteins. Our data support a model whereby the RG4 folding dynamics, under the control of oncogenic signaling and modulated by small molecule ligands or RG4-binding proteins, modifies mitochondria-localized cytoplasmic protein synthesis. Ultimately, this impairs mitochondrial functions, affecting energy metabolism and consequently cancer cell proliferation.

DOI: https://doi.org/10.1038/s41467-025-58118-5
Nat Commun. 2025 Apr 10. 16(1): 3409

An unconventional autophagic pathway that inhibits ATP secretion during apoptotic cell death.

Elena Terraza-Silvestre, Raquel Villamuera, Julia Bandera-Linero, Michal Letek, Daniel Oña-Sánchez, Cristina Ramón-Barros, Clara Moyano-Jimeno, Felipe X Pimentel-Muiños.

Mobilisation of Damage-Associated Molecular Patterns (DAMPs) determines the immunogenic properties of apoptosis, but the mechanisms that control DAMP exposure are still unclear. Here we describe an unconventional autophagic pathway that inhibits the release of ATP, a critical DAMP in immunogenic apoptosis, from dying cells. Mitochondrial BAK activated by BH3-only molecules interacts with prohibitins and stomatin-1 through its latch domain, indicating the existence of an interactome specifically assembled by unfolded BAK. This complex engages the WD40 domain of the autophagic effector ATG16L1 to induce unconventional autophagy, and the resulting LC3-positive vesicles contain ATP. Functional interference with the pathway increases ATP release during cell death, reduces ATP levels remaining in the apoptotic bodies, and improves phagocyte activation. These results reveal that an unconventional component of the autophagic burst that often accompanies apoptosis sequesters intracellular ATP to prevent its release, thus favouring the immunosilent nature of apoptotic cell death.

DOI: https://doi.org/10.1038/s41467-025-58619-3
Cells. 2025 Mar 22. pii: 480. [Epub ahead of print]14(7):

Knockdown of POLG Mimics the Neuronal Pathology of Polymerase-γ Spectrum Disorders in Human Neurons.

Çağla Çakmak Durmaz, Felix Langerscheidt, Imra Mantey, Xinyu Xia, Hans Zempel.

  Impaired function of Polymerase-γ (Pol-γ) results in impaired replication of the mitochondrial genome (mtDNA). Pathogenic mutations in the POLG gene cause dysfunctional Pol-γ and dysfunctional mitochondria and are associated with a spectrum of neurogenetic disorders referred to as POLG spectrum disorders (POLG-SDs), which are characterized by neurologic dysfunction and premature death. Pathomechanistic studies and human cell models of these diseases are scarce. SH-SY5Y cells (SHC) are an easy-to-handle and low-cost human-derived neuronal cell model commonly used in neuroscientific research. Here, we aimed to study the effect of reduced Pol-γ function using stable lentivirus-based shRNA-mediated knockdown of POLG in SHC, in both the proliferating cells and SHC-derived neurons. POLG knockdown resulted in approximately 50% reductions in POLG mRNA and protein levels in naïve SHC, mimicking the residual Pol-γ activity observed in patients with common pathogenic POLG mutations. Knockdown cells exhibited decreased mtDNA content, reduced levels of mitochondrial-encoded proteins, and altered mitochondrial morphology and distribution. Notably, while chemical induction of mtDNA depletion via ddC could be rescued by the mitochondrial biosynthesis stimulators AICAR, cilostazol and resveratrol (but not MitoQ and formoterol) in control cells, POLG-knockdown cells were resistant to mitochondrial biosynthesis-mediated induction of mtDNA increase, highlighting the specificity of the model, and pathomechanistically hinting towards inefficiency of mitochondrial stimulation without sufficient Pol-γ activity. In differentiated SHC-derived human neurons, POLG-knockdown cells showed impaired neuronal differentiation capacity, disrupted cytoskeletal organization, and abnormal perinuclear clustering of mitochondria. In sum, our model not only recapitulates key features of POLG-SDs such as impaired mtDNA content, which cannot be rescued by mitochondrial biosynthesis stimulation, but also reduced ATP production, perinuclear clustering of mitochondria and impaired neuronal differentiation. It also offers a simple, cost-effective and human (and, as such, disease-relevant) platform for investigating disease mechanisms, one with screening potential for therapeutic approaches for POLG-related mitochondrial dysfunction in human neurons.

Keywords:  Polymerase-γ; SH-SY5Y; mitochondria; mtDNA; neurogenetics; neuronal differentiation

DOI:  https://doi.org/10.3390/cells14070480
Nat Metab. 2025 Apr 08.

TMEM65 regulates and is required for NCLX-dependent mitochondrial calcium efflux.

Joanne F Garbincius, Oniel Salik, Henry M Cohen, Carmen Choya-Foces, Adam S Mangold, Angelina D Makhoul, Anna E Schmidt, Dima Y Khalil, Joshua J Doolittle, Anya S Wilkinson, Emma K Murray, Michael P Lazaropoulos, Alycia N Hildebrand, Dhanendra Tomar, John W Elrod.

The balance between mitochondrial calcium (mCa2+) uptake and efflux is essential for ATP production and cellular homeostasis. The mitochondrial sodium-calcium exchanger, NCLX, is a critical route of mCa2+ efflux in excitable tissues, such as the heart and brain, and animal models support NCLX as a promising therapeutic target to limit pathogenic mCa2+ overload. However, the mechanisms that regulate NCLX activity are largely unknown. Using proximity biotinylation proteomic screening, we identify the mitochondrial inner membrane protein TMEM65 as an NCLX binding partner that enhances sodium (Na+)-dependent mCa2+ efflux. Mechanistically, acute pharmacological NCLX inhibition or genetic deletion of NCLX ablates the TMEM65-dependent increase in mCa2+ efflux, and loss-of-function studies show that TMEM65 is required for Na+-dependent mCa2+ efflux. In line with these findings, knockdown of Tmem65 in mice promotes mCa2+ overload in the heart and skeletal muscle and impairs both cardiac and neuromuscular function. Collectively, our results show that loss of TMEM65 function in excitable tissue disrupts NCLX-dependent mCa2+ efflux, causing pathogenic mCa2+ overload, cell death, and organ-level dysfunction. These findings demonstrate the essential role of TMEM65 in regulating NCLX-dependent mCa2+ efflux and suggest modulation of TMEM65 as a therapeutic strategy for a variety of diseases.

DOI: https://doi.org/10.1038/s42255-025-01250-9
Nat Metab. 2025 Apr 09.

The mitochondrial unfolded protein response inhibits pluripotency acquisition and mesenchymal-to-epithelial transition in somatic cell reprogramming.

Zhongfu Ying, Yanmin Xin, Zihuang Liu, Tianxin Tan, Yile Huang, Yingzhe Ding, Xuejun Hong, Qiuzhi Li, Chong Li, Jingyi Guo, Gaoshen Liu, Qi Meng, Shihe Zhou, Wenxin Li, Yao Yao, Ge Xiang, Linpeng Li, Yi Wu, Yang Liu, Miaohui Mu, Zifeng Ruan, Wenxi Liang, Junwei Wang, Yaofeng Wang, Baojian Liao, Yang Liu, Wuming Wang, Gang Lu, Dajiang Qin, Duanqing Pei, Wai-Yee Chan, Xingguo Liu.

The mitochondrial unfolded protein response (UPRmt), a mitochondria-to-nucleus retrograde pathway that promotes the maintenance of mitochondrial function in response to stress, plays an important role in promoting lifespan extension in Caenorhabditis elegans1,2. However, its role in mammals, including its contributions to development or cell fate decisions, remains largely unexplored. Here, we show that transient UPRmt activation occurs during somatic reprogramming in mouse embryonic fibroblasts. We observe a c-Myc-dependent, transient decrease in mitochondrial proteolysis, accompanied by UPRmt activation at the early phase of pluripotency acquisition. UPRmt impedes the mesenchymal-to-epithelial transition (MET) through c-Jun, thereby inhibiting pluripotency acquisition. Mechanistically, c-Jun enhances the expression of acetyl-CoA metabolic enzymes and reduces acetyl-CoA levels, thereby affecting levels of H3K9Ac, linking mitochondrial signalling to the epigenetic state of the cell and cell fate decisions. c-Jun also decreases the occupancy of H3K9Ac at MET genes, further inhibiting MET. Our findings reveal the crucial role of mitochondrial UPR-modulated MET in pluripotent stem cell plasticity. Additionally, we demonstrate that the UPRmt promotes cancer cell migration and invasion by enhancing epithelial-to-mesenchymal transition (EMT). Given the crucial role of EMT in tumour metastasis3,4, our findings on the connection between the UPRmt and EMT have important pathological implications and reveal potential targets for tumour treatment.

DOI: https://doi.org/10.1038/s42255-025-01261-6
Nat Commun. 2025 Apr 05. 16(1): 3260

Origin of yield stress and mechanical plasticity in model biological tissues.

Anh Q Nguyen, Junxiang Huang, Dapeng Bi.

During development and under normal physiological conditions, biological tissues are continuously subjected to substantial mechanical stresses. In response to large deformations, cells in a tissue must undergo multicellular rearrangements to maintain integrity and robustness. However, how these events are connected in time and space remains unknown. Here, using theoretical modeling, we study the mechanical plasticity of cell monolayers under large deformations. Our results suggest that the jamming-unjamming (solid-fluid) transition can vary significantly depending on the degree of deformation, implying that tissues are highly unconventional materials. We elucidate the origins of this behavior. We also demonstrate how large deformations are accommodated through a series of cellular rearrangements, similar to avalanches in non-living materials. We find that these 'tissue avalanches' are governed by stress redistribution and the spatial distribution of "soft" or vulnerable spots, which are more prone to undergo rearrangements. Finally, we propose a simple and experimentally accessible framework to infer tissue-level stress and predict avalanches based on static images.

DOI: https://doi.org/10.1038/s41467-025-58526-7
Cells. 2025 Mar 22. pii: 482. [Epub ahead of print]14(7):

MIRO1 Is Required for Dynamic Increases in Mitochondria-ER Contact Sites and Mitochondrial ATP During the Cell Cycle.

Benney T Endoni, Olha M Koval, Chantal Allamargot, Tara Kortlever, Lan Qian, Riley J Sadoski, Denise Juhr, Isabella M Grumbach.

  Mitochondria-ER contact sites (MERCS) are vital for mitochondrial dynamics, lipid exchange, Ca2+ homeostasis, and energy metabolism. We examined whether mitochondrial metabolism changes during the cell cycle depend on MERCS dynamics and are regulated by the outer mitochondrial protein mitochondrial rho GTPase 1 (MIRO1). Wound healing was assessed in mice with fibroblast-specific deletion of MIRO1. Wild-type and MIRO1-/- fibroblasts and vascular smooth muscle cells were evaluated for proliferation, cell cycle progression, number of MERCS, distance, and protein composition throughout the cell cycle. Restoration of MIRO1 mutants was used to test the role of MIRO1 domains; Ca2+ transients and mitochondrial metabolism were evaluated using biochemical, immunodetection, and fluorescence techniques. MERCS increased in number during G1/S compared with during G0, which was accompanied by a notable rise in protein-protein interactions involving VDAC1 and IP3R as well as GRP75 and MIRO1 by proximity-ligation assays. Split-GFP ER/mitochondrial contacts of 40 nm also increased. Mitochondrial Ca2+ concentration ([Ca2+]), membrane potential, and ATP levels correlated with the formation of MERCS during the cell cycle. MIRO1 deficiency blocked G1/S progression and the cell-cycle-dependent formation of MERCS and altered ER Ca2+ release and mitochondrial Ca2+ uptake. MIRO1 mutants lacking the Ca2+-sensitive EF hands or the transmembrane domain did not rescue cell proliferation or the formation of MERCS. MIRO1 controls an increase in the number of MERCS during cell cycle progression and increases mitochondrial [Ca2+], driving metabolic activity and proliferation through its EF hands.

Keywords:  Ca2+; ER; MAM; MERCS; MIRO1; cell cycle; fibroblasts; mitochondria; vascular smooth muscle cells

DOI:  https://doi.org/10.3390/cells14070482
Proc Natl Acad Sci U S A. 2025 Apr 15. 122(15): e2415992122

FLIPL permits apoptotic and inflammatory signaling and inhibits necroptosis in mice without Caspase-8 oligomerization.

Jeremy J P Shaw, Cliff Guy, Bart Tummers, Douglas R Green.

  Caspase-8 signaling has proapoptotic, antinecroptotic, and proinflammatory signaling roles dependent on interaction with the adapter molecule FADD, oligomerization, and autocleavage. Previously, a Caspase-8 binding partner cFLIPL (FLIP, encoded by Cflar) was shown to prevent Caspase-8-dependent apoptosis, but permit Caspase-8-dependent inhibition of necroptosis. We sought to explore the role of FLIP in Caspase-8-dependent apoptosis induction, necroptosis inhibition, and inflammatory signaling inhibition in vitro and in vivo. We provide evidence that in mice with a mutation that prevents Caspase-8 oligomerization (Casp8FGLG/FGLG), FLIP is necessary to inhibit necroptosis, promote apoptosis, regulate inflammation, and control lymphoproliferative disease. Unlike Casp8FGLG/FGLG mice, Casp8FGLG/FGLG,Cflar-/- mice do not survive embryogenesis, but ablation of Mlkl, required for necroptosis, allows their survival to adulthood. Further, unlike Casp8FGLG/FGLG,Mlkl-/- mice, Casp8FGLG/FGLG,Cflar-/-,Mlkl-/- mice display lymphoproliferative disease. We analyzed apoptosis, necroptosis, and inflammatory signaling in Casp8FGLG/FGLG mice with or without FLIP, gaining insights into the functions of the Caspase-8-FLIP heterodimer in vitro and in vivo.

Keywords:  Caspase-8; apoptosis; cFLIPL; necroptosis; pyroptosis

DOI:  https://doi.org/10.1073/pnas.2415992122
Nat Commun. 2025 Apr 11. 16(1): 3433

Characterization of dsRNA binding proteins through solubility analysis identifies ZNF385A as a dsRNA homeostasis regulator.

Na Jiang, Hekun Yang, Yi Lei, Weida Qin, Huifang Xiong, Kuan Chen, Kunrong Mei, Gongyu Li, Xin Mu, Ruibing Chen.

Double-stranded RNA (dsRNA) binding proteins (dsRBPs) play crucial roles in various cellular processes, especially in the innate immune response. Comprehensive characterization of dsRBPs is essential to understand the intricate mechanisms for dsRNA sensing and response. Traditional methods have predominantly relied on affinity purification, favoring the isolation of strong dsRNA binders. Here, we adopt the proteome integral solubility alteration (PISA) workflow for characterizing dsRBPs, resulting in the observation of 18 known dsRBPs and the identification of 200 potential dsRBPs. Next, we focus on zinc finger protein 385 A (ZNF385A) and discover that its knockout activates the transcription of interferon-β in the absence of immunogenic stimuli. The knockout of ZNF385A elevates the level of endogenous dsRNAs, especially transcripts associated with retroelements, such as short interspersed nuclear element (SINE), long interspersed nuclear element (LINE), and long terminal repeat (LTR). Moreover, loss of ZNF385A enhances the bioactivity of 5-Aza-2'-deoxycytidine (5-AZA-CdR) and tumor-killing effect of NK cells. Our findings greatly expand the dsRBP reservoir and contribute to the understanding of cellular dsRNA homeostasis.

DOI: https://doi.org/10.1038/s41467-025-58704-7
Neurochem Int. 2025 Apr 08. pii: S0197-0186(25)00052-X. [Epub ahead of print]186 105979

Non-canonical STAT3 pathway induces alterations of mitochondrial dynamic proteins in the hippocampus of an LPS-induced murine neuroinflammation model.

Périne Millot, Laurine Duquesne, Carine San, Baptiste Porte, Claire Pujol, Benoit Hosten, Jacques Hugon, Claire Paquet, François Mouton-Liger.

The activation of STAT3 is a crossroads of cellular regulation, induced in its canonical pathway by phosphorylation on a critical tyrosine residue (Y705). The existence of a STAT3 non-canonical signaling mechanisms, induced by phosphorylation at serine 727 (S727), has been recently identified in vitro. After cytoplasmic activation, non-canonical STAT3 could move to the level of mitochondria-endoplasmic reticulum contacts (MERCs). We have previously shown that LPS injections in mouse model induce STAT3 canonical pathway, leading to its nuclear translocation and to neuroinflammation. However, the effects of LPS on activation of the non-canonical pathway and its consequences on protein complexes of MERCs remain to be determined. In an in vivo LPS mouse model, we found that systemic inflammation induces in hippocampus the non-canonical STAT3 pathway. LPS-induced STAT3 affects specifically MERC protein BAP31, and that of a mitochondrial membrane protein known to interact with it, TOM40. These findings shed light on the role of STAT3 on mitochondrial - endoplasmic reticulum interaction under inflammatory conditions, offering new perspectives for targeting mitochondrial function and STAT3 activation in disease contexts.

DOI: https://doi.org/10.1016/j.neuint.2025.105979
Int Immunopharmacol. 2025 Apr 07. pii: S1567-5769(25)00615-0. [Epub ahead of print]155 114625

Exploring the role of Mitoferrin-1 in Ferroptosis and mitochondrial damage in acute ischemic stroke.

Ruijia You, Bin Sun, Jing Luo, Nan Shao, Wenwen Si.

  The pathogenesis of acute ischemic stroke (AIS) is complex, with limited therapeutic options available during the acute phase. Therefore, investigating the underlying mechanisms of AIS is critical. Ferroptosis has been implicated in AIS-induced damage; however, its precise molecular mechanisms remain elusive. In this study, we explored the role of Mitoferrin-1 (Mfrn1) in AIS using a combination of in vitro and in vivo models, including RNA sequencing, RNA interference (RNAi), Adeno-associated virus (AAV9) injection, gene overexpression, and ferroptosis detection. Our results demonstrated that Mfrn1 expression, mitochondrial iron levels, mitochondrial injury, and ferroptosis were significantly increased in AIS models. Knockdown of Mfrn1 attenuated ferroptosis and oxygen-glucose deprivation/reperfusion (OGD/R)-induced injury, whereas overexpression of Mfrn1 had the opposite effect. Similarly, silencing Mfrn1 decreased mitochondrial iron accumulation and injury, while its overexpression exacerbated both. In middle cerebral artery occlusion/reperfusion (MCAO/R) rats, silencing Mfrn1 suppressed ferroptosis, reduced AIS-related injury, lowered mitochondrial iron levels, and mitigated mitochondrial damage. These findings suggest that Mfrn1 exacerbates AIS damage by promoting mitochondrial iron accumulation and injury. This study highlights Mfrn1 as a potential therapeutic target for AIS.

Keywords:  Acute ischemic stroke; Ferroptosis; MCAO; Mitochondrial damage; Mitoferrin-1; OGD/R

DOI:  https://doi.org/10.1016/j.intimp.2025.114625
Mol Biol Cell. 2025 Apr 09. mbcE25030119

A morphology and secretome map of pyroptosis.

Michael J Lippincott, Jenna Tomkinson, Dave Bunten, Milad Mohammadi, Johanna Kastl, Johannes Knop, Ralf Schwandner, Jiamin Huang, Grant Ongo, Nathaniel Robichaud, Milad Dagher, Andrés Mansilla-Soto, Cynthia Saravia-Estrada, Masafumi Tsuboi, Carla Basualto-Alarcón, Gregory P Way.

Pyroptosis represents one type of Programmed Cell Death (PCD). It is a form of inflammatory cell death that is canonically defined by caspase-1 cleavage and Gasdermin-mediated membrane pore formation. Caspase-1 initiates the inflammatory response (through IL-1β processing), and the N-terminal cleaved fragment of Gasdermin D polymerizes at the cell periphery forming pores to secrete pro-inflammatory markers. Cell morphology also changes in pyroptosis, with nuclear condensation and membrane rupture. However, recent research challenges canon, revealing a more complex secretome and morphological response in pyroptosis, including overlapping molecular characterization with other forms of cell death, such as apoptosis. Here, we take a multimodal, systems biology approach to characterize pyroptosis. We treated human Peripheral Blood Mononuclear Cells (PBMCs) with 36 different combinations of stimuli to induce pyroptosis or apoptosis. We applied both secretome profiling (nELISA) and high-content fluorescence microscopy (Cell Painting). To differentiate apoptotic, pyroptotic and control cells, we used canonical secretome markers and modified our Cell Painting assay to mark the N-terminus of Gasdermin-D. We trained hundreds of machine learning (ML) models to reveal intricate morphology signatures of pyroptosis that implicate changes across many different organelles and predict levels of many pro-inflammatory markers. Overall, our analysis provides a detailed map of pyroptosis which includes overlapping and distinct connections with apoptosis revealed through a mechanistic link between cell morphology and cell secretome.

DOI: https://doi.org/10.1091/mbc.E25-03-0119