bims-nenemi 2025-07-20 papers

bims-nenemi

Biomed News

on Neuroinflammation, neurodegeneration and mitochondria

Issue of 2025–07–20
fourteen papers selected by
Marco Tigano, Thomas Jefferson University

Drug Repurposing Screen Identifies an HRI Activating Compound that Promotes Adaptive Mitochondrial Remodeling in MFN2-deficient Cells.
Mitochondria-Targeting Abasic Site-Reactive Probe (mTAP) Enables the Manipulation of Mitochondrial DNA Levels.
Mitochondrial DNA: how does it leave mitochondria?
Why and how paternal mitochondrial DNA gets cut out of the inheritance.
Multi-site phosphorylation of BCL2L13 in a TBK1- and AMPK-dependent manner reveals new modes of mitophagy regulation.
Mitochondrial p32-mediated regulation of p-TBK1 affects the radiosensitivity of colorectal cancer.
Specialized high-capacity mitochondria fuel cell invasion.
POLR1A inhibits ferroptosis by regulating TFAM-mediated mitophagy and iron homeostasis.
Cell cycle- and dose-dependent effects on mitochondrial DNA copy number variation following irradiation.
Mitochondrial respiration is necessary for CD8+ T cell proliferation and cell fate.
Amyloid-β protein precursor modulates mitophagy and mitochondrial function through its cellular localization.
Laminar Shear Stress Increases the Cytosolic Pool of PINK1 in Endothelial Cells and Enhances Mitophagic Sensitivity Toward Dysfunctional Mitochondria.
mtKO: A dedicated guide RNA library for mitochondria research.
Respiratory complex III 2 assembles complex I via toxic intermediate in mitochondrial disease.

bioRxiv. 2025 Jun 26. pii: 2025.06.23.660251. [Epub ahead of print]

Drug Repurposing Screen Identifies an HRI Activating Compound that Promotes Adaptive Mitochondrial Remodeling in MFN2-deficient Cells.

Prerona Bora, Mashiat Zaman, Samantha Oviedo, Sergei Kutseikin, Nicole Madrazo, Prakhyat Mathur, Meera Pannikkat, Sophia Krasny, Alan Chu, Kristen A Johnson, Danielle A Grotjahn, Timothy E Shutt, R Luke Wiseman.

Pathogenic variants in the mitochondrial outer membrane GTPase MFN2 cause the peripheral neuropathy Charcot-Marie-Tooth Type 2A (CMT2A). These mutations disrupt MFN2-dependent regulation of diverse aspects of mitochondrial biology including organelle morphology, motility, mitochondrial-endoplasmic reticulum (ER) contacts (MERCs), and respiratory chain activity. However, no therapies currently exist to mitigate the mitochondrial dysfunction linked to genetic deficiencies in MFN2. Herein, we performed a drug repurposing screen to identify compounds that selectively activate the integrated stress response (ISR) - the predominant stress-responsive signaling pathway responsible for regulating mitochondrial morphology and function. This screen identified the compounds parogrelil and MBX-2982 as potent and selective activators of the ISR through the OMA1-DELE1-HRI signaling axis. We show that treatment with these compounds promotes adaptive, ISR-dependent remodeling of mitochondrial morphology and protects mitochondria against genetic and chemical insults. Moreover, we show that pharmacologic ISR activation afforded by parogrelil restores mitochondrial tubular morphology, promotes mitochondrial motility, rescues MERCs, and enhances mitochondrial respiration in MFN2 -deficient cells. These results demonstrate the potential for pharmacologic HRI activation as a viable strategy to mitigate mitochondrial dysfunction in CMT2A and other pathologies associated with MFN2 deficiency.

DOI: https://doi.org/10.1101/2025.06.23.660251
Angew Chem Int Ed Engl. 2025 Jul 15. e202502470

Mitochondria-Targeting Abasic Site-Reactive Probe (mTAP) Enables the Manipulation of Mitochondrial DNA Levels.

Anal Jana, Yu-Hsuan Chen, Linlin Zhao.

  Mitochondrial DNA (mtDNA) encodes essential genes for mitochondrial and cellular functions and acts as a cell signaling molecule in innate immune and inflammatory responses. Defects in mtDNA are implicated in a range of mitochondrial disorders and human diseases. Currently, no chemical strategy exists to prevent mtDNA loss under genotoxic stress. To address this, we developed a mitochondria-targeting probe (mTAP) that selectively reacts with key mtDNA repair intermediates-abasic (AP) sites. We confirmed that mTAP forms oxime conjugates exclusively with mitochondrial AP sites without conjugation with nuclear AP sites. Upon mTAP conjugation, DNA substrates containing AP sites were resistant to cleavage by AP endonuclease (APE1) and mitochondrial extracts. This conjugation significantly reduced the DNA-binding affinity of APE1 without affecting the DNA-binding activity of a mtDNA-packaging factor, mitochondrial transcription factor A (TFAM). Importantly, cellular experiments demonstrated that mTAP treatment alleviated the decrease in mtDNA and transcription product levels induced by mitochondrial AP site damage. Functional assays also demonstrated that mTAP treatment did not compromise mtDNA replication activity or increase the overall mtDNA damage level. These findings highlight the potential of mTAP as a valuable chemical tool to modulate mtDNA levels under genotoxic stress.

Keywords:  Abasic sites; DNA Repair; DNA damage; Mitochondrial DNA; Nucleic acid modifications

DOI:  https://doi.org/10.1002/anie.202502470
Trends Cell Biol. 2025 Jul 10. pii: S0962-8924(25)00146-1. [Epub ahead of print]

Mitochondrial DNA: how does it leave mitochondria?

Vladimir Gogvadze, Boris Zhivotovsky.

  In recent years, studies have reported the presence of mitochondrial DNA (mtDNA) in the cytosol. However, a certain number of publications on the mechanisms of mtDNA release contain uncertainties. mtDNA is located in the mitochondrial matrix and cannot be released through the same pathways as intermembrane space proteins. This forum article aims to examine the assumptions and elucidate the processes underlying this phenomenon.

Keywords:  Bcl-2 family proteins; inner mitochondrial membrane; mitochondria; mtDNA; outer mitochondrial membrane

DOI:  https://doi.org/10.1016/j.tcb.2025.06.005
Curr Opin Genet Dev. 2025 Jul 16. pii: S0959-437X(25)00073-5. [Epub ahead of print]94 102381

Why and how paternal mitochondrial DNA gets cut out of the inheritance.

Mayu Shimomura, Thomas R Hurd.

Mitochondrial DNA (mtDNA) is inherited maternally across animals, yet the evolutionary rationale behind this unusual mode of inheritance remains a longstanding mystery. Understanding the processes that prevent the transmission of paternal mtDNA and thus ensure maternal-only inheritance is crucial to uncovering the evolutionary significance of this widespread phenomenon. Historically, research has focused on mechanisms that act within eggs to destroy sperm mitochondria via autophagy and the ubiquitin-proteasome degradation system. However, recent discoveries across multiple animal species, including humans, reveal a surprising twist: paternal mtDNA is actively degraded within mitochondria independently of and prior to the complete breakdown of the organelle itself, often even prior to fertilization. Only a few studies have begun to illuminate the molecular machinery responsible for this early mtDNA elimination. In this review, we explore the emerging landscape of paternal mtDNA elimination mechanisms across species, highlighting newly discovered pathways, evolutionary implications, and open questions that are furthering our understanding of mitochondrial inheritance.

DOI: https://doi.org/10.1016/j.gde.2025.102381
bioRxiv. 2025 Jul 11. pii: 2025.07.10.663832. [Epub ahead of print]

Multi-site phosphorylation of BCL2L13 in a TBK1- and AMPK-dependent manner reveals new modes of mitophagy regulation.

Hoda Ahmed, Mack B Reynolds, Gary Kasof, Gerald S Shadel, Reuben Shaw.

Mitophagy is a selective autophagic process that eliminates damaged mitochondria via lysosomal degradation, playing a crucial role in maintaining cellular metabolic balance. Mitophagy can occur through two pathways: ubiquitin-dependent and ubiquitin-independent. Recently, we and others have shown that, upon mitochondrial stress, AMP-activated protein kinase (AMPK) contributes to Parkin-mediated, ubiquitin-dependent mitophagy. The ubiquitin-independent pathway involves multiple outer mitochondrial membrane (OMM) "mitophagy receptors" that contain LC3-interacting region (LIR) motifs, including BNIP3, NIX/ BNIP3L, FUNDC1, and BCL2L13. LIR motifs bind Atg8/LC3 family proteins, facilitating the recruitment of the autophagosome membrane to target damaged mitochondria for degradation. The kinase Unc-51 Like autophagy activating kinase 1 (ULK1) phosphorylates the serine preceding the LIR motif in BNIP3, NIX, and FUNDC1, enhancing their binding to LC3 and promoting mitophagy. However, while BCL2L13 has been identified as a ULK1 binding partner, its regulation by phosphorylation remains unclear. We utilized mass spectrometry (MS) to map phosphorylation sites in BCL2L13 following mitochondrial stress and developed phospho-specific antibodies against two sites, Ser261 and Ser275, which were induced after exposure to the mitochondrial uncoupler, CCCP. Endogenous BCL2L13 Ser261 and Ser275 were both phosphorylated in an AMPK-dependent manner in cells and tissues. As neither site matches the established AMPK substrate consensus motif, we sought to identify which kinases directly mediate their phosphorylation downstream of AMPK. Surprisingly, genetic studies revealed that ULK1 is not regulating either site, but instead, TBK1 is controlling Ser275. This work reveals that BCL2L13 is unique amongst mitophagy receptors in being activated by mitochondrial stress and innate immune stimuli in an AMPK- and TBK1-dependent manner.

DOI: https://doi.org/10.1101/2025.07.10.663832
Life Sci. 2025 Jul 12. pii: S0024-3205(25)00489-8. [Epub ahead of print] 123854

Mitochondrial p32-mediated regulation of p-TBK1 affects the radiosensitivity of colorectal cancer.

Yanjin Wu, Haitao Zhou, Jiahui Meng, Wenyue Zhao, Xiaotong Zhao, Yujia Hou, Qin Wang, Feng Wang, Qiang Liu, Yang Liu.

   PURPOSE: Many colorectal cancer (CRC) patients respond poorly to radiotherapy due to radioresistance. Understanding the molecular mechanisms underlying this resistance is crucial. It was demonstrated that p32, a mitochondrial protein translation regulator, is related to cancer development. However, its specific function and mechanism in CRC, has not yet been investigated. This study aims to explore the role of p32 in CRC and its impact on radiotherapy sensitivity.
METHODS: Cell viability was evaluated by MTT and EdU assay. Mitochondrial DNA (mtDNA) leakage was quantified by RT-qPCR. Radiosensitivity was indicated by cellular phosphorylation of H2AX (γH2AX) foci, phosphorylation of ataxia telangiectasia mutated (p-ATM) and phosphorylation of checkpoint kinase 2 (p-CHK2) levels, as well as by mice tumor model subjected to radiotherapy. Moreover, histological and transcriptomic analysis of p32 expression were performed in CRC patients.
RESULTS: In p32-KO cells, we observed reduced cell viability, damaged mitochondria, mtDNA leakage, and increased radiosensitivity. Furthermore, depletion of p32 induced the DNA damage response (DDR) by activating cytoplasmic DNA sensing cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING)-TANK-binding kinase 1 (TBK1), which was reversed by p32/TBK1 double knockout. Depletion of p32 also induced the mitochondrial fragmentation, induced mtDNA leakage through the mitochondrial permeability transition pore (mPTP), effects that could be mitigated by Mdivi-1 or Cyclosporin A (CsA).
CONCLUSIONS: Our study demonstrates that inhibiting p32 in CRC enhances radiosensitivity by causing mitochondrial dysfunction, increasing mitochondrial fission, inducing mtDNA leakage and activating the cGAS-STING-TBK1 pathway. These findings provide a potential therapeutic target for overcoming radioresistance in CRC.

Keywords:  Mitochondria dysfunction; Radiosensitivity; cGAS-STING-TBK1; mPTP; mtDNA

DOI:  https://doi.org/10.1016/j.lfs.2025.123854
bioRxiv. 2025 May 03. pii: 2025.05.02.651978. [Epub ahead of print]

Specialized high-capacity mitochondria fuel cell invasion.

Isabel W Kenny-Ganzert, Lena P Basta, Lianzijun Wang, Qiuyi Chi, Caitlin Su, Katherine S Morton, Joel N Meyer, David R Sherwood.

Cell invasion through basement membrane (BM) is energetically intensive, and how an invading cell produces high ATP levels to power invasion is understudied. By generating 20 endogenously tagged mitochondrial proteins, we identified a specialized mitochondrial subpopulation within the C. elegans anchor cell (AC) that localizes to the BM breaching site and generates elevated ATP to fuel invasion. These ETC-enriched high-capacity mitochondria are compositionally unique, harboring increased protein import machinery and dense cristae enriched with ETC components. High-capacity mitochondria emerge at the time of AC specification and depend on the AC pro-invasive transcriptional program. Finally, we show that netrin signaling through a Src kinase directs microtubule polarization, which facilitates metaxin adaptor complex dependent ETC-enriched mitochondrial trafficking to the AC invasive front. Our studies reveal that an invasive cell produces high ATP by generating and localizing high-capacity mitochondria. This might be common strategy used by other cells to meet energy demanding processes.

DOI: https://doi.org/10.1101/2025.05.02.651978
Redox Biol. 2025 Jul 08. pii: S2213-2317(25)00271-X. [Epub ahead of print]85 103758

POLR1A inhibits ferroptosis by regulating TFAM-mediated mitophagy and iron homeostasis.

Tuo Zhang, Yanyun Gao, Marcell Harhai, Alexis A Jourdain, Thomas M Marti, Erik Vassella, Zhang Yang, Qinghua Zhou, Patrick Dorn, Ren-Wang Peng.

  Evasion of programmed cell death (PCD) is a hallmark of cancer, yet the mechanisms underlying resistance to ferroptosis - an iron-dependent form of PCD triggered by excessive lipid peroxidation - remain incompletely understood. Here, we identify a previously unrecognized nucleolar-mitochondrial signaling axis that promotes ferroptosis resistance in pleural mesothelioma (PM) and potentially other cancers. This pathway involves RNA polymerase I (PolI) catalytic subunit A (POLR1A) and mitochondrial transcription factor A (TFAM), which together regulate mitophagy and intracellular iron metabolism to suppress ferroptosis. Mechanistically, POLR1A controls TFAM expression via the transcription factor ATF4, and this POLR1A-ATF4-TFAM axis inhibits mitophagy and limit mitophagy-dependent labile Fe2+ release, thereby preventing Fe2+-driven lipid peroxidation. Disruption of this pathway through POLR1A or TFAM inhibition leads to Fe2+ accumulation and increased sensitivity to ferroptosis inducers (FINs). Notably, CX-5461, a first-in-class RNA PolI inhibitor currently in clinical trials, synergizes with GPX4 blockade to induce ferroptotic cell death both in vitro and in vivo. This therapeutic synergy extends beyond PM, suggesting broader relevance in ferroptosis-resistant cancers. Together, our findings reveal a novel mechanism of ferroptosis evasion and establish a promising combinatorial strategy to overcome therapy resistance in cancer.

Keywords:  ATF4; Ferroptosis; Iron metabolism; POLR1A; TFAM; mitophagy

DOI:  https://doi.org/10.1016/j.redox.2025.103758
J Cell Sci. 2025 Jul 18. pii: jcs.263642. [Epub ahead of print]

Cell cycle- and dose-dependent effects on mitochondrial DNA copy number variation following irradiation.

Ryosuke Seino, Kai Nishikubo, Hisanori Fukunaga.

  Cell survival after irradiation depends on the cell cycle at the time of exposure. This has been thought to be due to cell cycle-dependent nuclear DNA damage repair mechanisms. Here we show the relationships between the exposed dose, the cell cycle phase at the time of exposure, and changes in mitochondrial DNA copy numbers (mtDNAcn) after irradiation. We used a fluorescent ubiquitination-based cell cycle indicator (FUCCI), which allows visualization of the cell cycle, and confirmed cell cycle synchronization in human cervical HeLa cells. In synchronous HeLa-FUCCI cells, the mtDNAcn changed with the progression of the cell cycle. Also, G1 phase-synchronized cells showed a dose-dependent increase of mtDNAcn at 48 h after X-ray exposure, while G2 cells showed a dose-dependent increase at 24 h. In addition, S phase-synchronized cells showed a dose-dependent increase at 24 and 48 h after irradiation. These results showed the cell cycle- and dose-dependent effects on mtDNAcn after irradiation, which may shed light on the emerging role of mitochondrial genome and in cell survival.

Keywords:  Cell cycle; DNA copy number variation; FUCCI; Mitochondrial DNA (mtDNA); Radiation

DOI:  https://doi.org/10.1242/jcs.263642
Nat Immunol. 2025 Jul 16.

Mitochondrial respiration is necessary for CD8+ T cell proliferation and cell fate.

Elizabeth M Steinert, Beatriz Furtado Bruza, Veronika D Danchine, Rogan A Grant, Karthik Vasan, Arjun Kharel, Yuqi Zhang, Weiguo Cui, Marten Szibor, Samuel E Weinberg, Navdeep S Chandel.

Mitochondrial electron transport chain (ETC) function is linked to the generation of ATP, signaling molecules including reactive oxygen species (ROS), pyrimidines and tricarboxylic acid cycle metabolites1. Mitochondrial electron transport is required for T cell proliferation2-4. However, which mitochondrial ETC functions are necessary for each dynamic state of CD8+ T cell responses is unknown. Here we report that impairing mitochondrial complex III function, which diminishes respiration, proton pumping linked to ATP production and superoxide production, decreases peripheral naive numbers, antigen-induced CD8+ T cell proliferation and memory formation. Acute stimulation of mitochondrial complex III-deficient CD8+ T cells induced an exhausted-like phenotype. Expression of Ciona intestinalis alternative oxidase (AOX) in mitochondrial complex III-deficient CD8+ T cells restores respiration without generating ROS or proton pumping, and rescues proliferation and the exhausted phenotype but not naive or memory formation. Thus, T cell development, proliferation and memory formation have distinct requirements for mitochondrial complex III ROS.

DOI: https://doi.org/10.1038/s41590-025-02202-x
J Alzheimers Dis. 2025 Jul 17. 13872877251360243

Amyloid-β protein precursor modulates mitophagy and mitochondrial function through its cellular localization.

Taylor A Strope, Benjamin Troutwine, Brittany M Hauger, Keith P Smith, Russell H Swerdlow, Heather M Wilkins.

  BackgroundAmyloid-β (Aβ) is generated from amyloid-β protein precursor (AβPP) via secretase enzymes. While AβPP processing and its localization are well understood, the function of AβPP is largely unknown. AβPP has been shown to localize to mitochondria, but the consequence of this is not understood.ObjectiveWe examined the consequences of modulating mitochondrial AβPP content on mitochondrial function.MethodsWe measured mitochondrial AβPP localization in postmortem human brain from non-demented and AD subjects. To understand the effects of mitochondrial localization of AβPP on mitochondria, we leveraged AβPP constructs with increased (D23A) or decreased (3 M) mitochondrial localization compared to a wild-type (WT) construct. We measured mitochondrial function including dynamics and mitophagy.ResultsWe observed increased AβPP mitochondrial localization in postmortem brain of sporadic AD subjects. Increased or decreased mitochondrial AβPP content led to reduced electron transport chain (ETC) activities, reduced ATP levels, increased mitochondrial superoxide production, hyperpolarized mitochondrial membrane potential, and increased mitochondrial calcium content. Reduced mitochondrial AβPP content reduced mitophagy flux, while increased mitochondrial AβPP content increased mitophagy flux. Increased or decreased mitochondrial AβPP content reduced mitochondrial biogenesis. We identified interactions between AβPP and mitophagy/autophagy proteins. We next examined if a specific motif in AβPP was responsible for alterations in mitochondrial function and mitophagy. Mitophagy flux was inhibited with expression of ΔCT AβPP, suggesting a role for the C-terminus of AβPP in mitophagy induction.ConclusionsOverall, these findings highlight a critical role of AβPP in mitochondrial physiology. Alterations to AβPP mitochondrial content can lead to mitochondrial dysfunction.

Keywords:  Alzheimer's disease; amyloid-β; amyloid-β protein precursor; mitochondria; mitophagy

DOI:  https://doi.org/10.1177/13872877251360243
bioRxiv. 2025 Jun 26. pii: 2025.06.21.660867. [Epub ahead of print]

Laminar Shear Stress Increases the Cytosolic Pool of PINK1 in Endothelial Cells and Enhances Mitophagic Sensitivity Toward Dysfunctional Mitochondria.

Soon-Gook Hong, Junchul Shin, Jason Saredy, Soo Young Choi, Hong Wang, Joon-Young Park.

Phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1) is an essential molecule in mitophagy process in mammalian cells. Mutation or deficiency of PINK1 has been closely related to several disease conditions. The purpose of this study was to determine PINK1 expression levels and subcellular localization under exercise-mimic laminar shear stress (LSS) condition in human aortic endothelial cells (HAECs) or in exercising mice, and its implication on endothelial homeostasis and cardiovascular disease (CVD) prevention. First, LSS significantly elevated both full-length PINK1 (FL-PINK1) mRNA and protein expressions in ECs. Mitochondrial fractionation assays and confocal microscopic analysis showed reduced FL-PINK1 accumulation on mitochondria with an increase in a cytosolic pool of FL-PINK1 under LSS. Mitophagy flux, determined by a mtKeima probe, decreased with intact mitochondrial morphology and membrane potential under LSS, suggesting that elevated cytosolic PINK1 is not utilized for immediate mitophagy inductions. However, increased cytosolic PINK1 seems to elevate mitophagic sensitivity toward dysfunctional mitochondria in pathological conditions. LSS-preconditioned ECs showed lower angiotensin II (AngII)-induced mtDNA lesions and displayed rapid Parkin recruitment and mitophagy induction in response to mitochondrial uncoupler (CCCP) treatment. Exercise-preconditioned mice, a physiological LSS-enhanced model, showed elevated PINK1 expression in ECs of the thoracic aorta compared to sedentary control. In addition, exercise enhanced AngII-induced mitophagy induction in ECs and reduced AngII-induced mtDNA lesion formation in the mouse aorta. Taken together, LSS increases a cytosolic pool of FL-PINK1, which may elevate the mitophagic sensitivity toward dysfunctional mitochondria in ECs.

DOI: https://doi.org/10.1101/2025.06.21.660867
Proc Natl Acad Sci U S A. 2025 Jul 22. 122(29): e2502285122

mtKO: A dedicated guide RNA library for mitochondria research.

Karambir Kaur, Javeria Zaheer, Fengchao Lang, Diego Luis Ribeiro, Meili Zhang, Hua Song, Wei Zhang, Raj Chari, Hussam Alkaissi, Chunzhang Yang.

  Mitochondria are multifunctional organelles central to both physiological and pathological processes. In malignant cancer cells, mitochondrial reprogramming establishes the metabolic foundation to meet cellular demands, which is particularly important in tumor cells with existing metabolic perturbations. To identify key mitochondrial pathways supporting cancer development, we developed mitochondria Knockout (mtKO), a robust and unbiased CRISPR screening platform to pinpoint critical mitochondria-associated pathways. The mtKO screen revealed that the mitochondrial antioxidant enzyme SOD2 is essential for cells harboring IDH1 mutations. Mechanistically, SOD2 activity determines the disease manifestation of IDH1-mutated cancers, through maintaining redox homeostasis and mitochondrial fitness. This study introduces a powerful functional genomic tool to identify mitochondrial-centered pathways and reveals the selective mitochondrial vulnerability in Krebs cycle-deficient cancers for future therapeutic intervention.

Keywords:  CRISPR screen; IDH1; SOD2; metabolism; mitochondria

DOI:  https://doi.org/10.1073/pnas.2502285122
bioRxiv. 2025 Jun 18. pii: 2025.06.17.660237. [Epub ahead of print]

Respiratory complex III 2 assembles complex I via toxic intermediate in mitochondrial disease.

Maria G Ayala-Hernandez, Anetzy Bermudez Torales, Hannah Camille Tan, Claire B Montgomery, Abhilash Padavannil, Gino Cortopassi, James A Letts.

Mutations in mitochondrial complex I can cause severe metabolic disease. Although no treatments are available for complex I deficiencies, chronic hypoxia improves lifespan and function in a mouse model of the severe mitochondrial disease Leigh syndrome caused by mutation of complex I subunit NDUFS4. To understand the molecular mechanism of NDUFS4 mutant pathophysiology and hypoxia rescue, we investigated the structure of complex I in respiratory supercomplexes isolated from NDUFS4 mutant mice. We identified complex I assembly intermediates bound to complex III 2 , proving the cooperative assembly model. Further, an accumulated complex I intermediate is structurally consistent with pathological oxygen-dependent reverse electron transfer, revealing unanticipated pathophysiology and hypoxia rescue mechanisms. Thus, the build-up of toxic intermediates and not simply decreases in complex I levels underlie mitochondrial disease.

DOI: https://doi.org/10.1101/2025.06.17.660237