bims-nenemi Biomed News
on Neuroinflammation, neurodegeneration and mitochondria
Issue of 2023‒08‒13
twenty papers selected by
Marco Tigano
Thomas Jefferson University
  1. NME6: ribonucleotide salvage sustains mitochondrial transcription.
  2. DELE1 oligomerization promotes integrated stress response activation.
  3. iNOS aggravates pressure overload-induced cardiac dysfunction via activation of the cytosolic-mtDNA-mediated cGAS-STING pathway.
  4. WDR77 inhibits prion-like aggregation of MAVS to limit antiviral innate immune response.
  5. Activation of ATF3 via the Integrated Stress Response Pathway Regulates Innate Immune and Autophagy Processes to Restrict Zika Virus.
  6. The RNA-binding protein ZFP36 strengthens innate antiviral signaling by targeting RIG-I for K63-linked ubiquitination.
  7. Mitochondrial Dysfunction Associated with mtDNA in Metabolic Syndrome and Obesity.
  8. FBXL4 mutations cause excessive mitophagy via BNIP3/BNIP3L accumulation leading to mitochondrial DNA depletion syndrome.
  9. ATAD3A: A Key Regulator of Mitochondria-Associated Diseases.
  10. RetroGREAT signaling: The lessons we learn from yeast.
  11. Refractive Index Imaging Reveals That Elimination of the ATP Synthase C Subunit Does Not Prevent the Adenine Nucleotide Translocase-Dependent Mitochondrial Permeability Transition.
  12. High-resolution kinetic characterization of the RIG-I-signaling pathway and the antiviral response.
  13. Mitochondrial integrated stress response controls lung epithelial cell fate.
  14. Prolonged STAT1 activation in neurons drives a pathological transcriptional response.
  15. Inhibition of METTL3 results in a cell-intrinsic interferon response that enhances anti-tumour immunity.
  16. Lactate limits CNS autoimmunity by stabilizing HIF-1α in dendritic cells.
  17. Editorial: Targeting DNA damage response to enhance antitumor innate immunity in radiotherapy.
  18. The complexities of investigating mitochondria dynamics in multiple sclerosis and mouse models of MS.
  19. Clearance phagocytosis by the retinal pigment epithelial during photoreceptor outer segment renewal: Molecular mechanisms and relation to retinal inflammation.
  20. Live-Cell High-Throughput Screen for Monitoring Autophagy Flux.
  1. EMBO J. 2023 Aug 07. e114990
    NME6: ribonucleotide salvage sustains mitochondrial transcription.
    Paulina H Wanrooij, Andrei Chabes.
      The building blocks for RNA and DNA are made in the cytosol, meaning mitochondria depend on the import and salvage of ribonucleoside triphosphates (rNTPs) and deoxyribonucleoside triphosphates (dNTPs) for the synthesis of their own genetic material. While extensive research has focused on mitochondrial dNTP homeostasis due to its defects being associated with various mitochondrial DNA (mtDNA) depletion and deletion syndromes, the investigation of mitochondrial rNTP homeostasis has received relatively little attention. In this issue of the EMBO Journal, Grotehans et al provide compelling evidence of a major role for NME6, a mitochondrial nucleoside diphosphate kinase, in the conversion of pyrimidine ribonucleoside diphosphates into the corresponding triphosphates. These data also suggest a significant physiological role for NME6, as its absence results in the depletion of mitochondrial transcripts and destabilization of the electron transport chain (Grotehans et al, 2023).
    DOI:  https://doi.org/10.15252/embj.2023114990
  2. Nat Struct Mol Biol. 2023 Aug 07.
    DELE1 oligomerization promotes integrated stress response activation.
    Jie Yang, Kelsey R Baron, Daniel E Pride, Anette Schneemann, Xiaoyan Guo, Wenqian Chen, Albert S Song, Giovanni Aviles, Martin Kampmann, R Luke Wiseman, Gabriel C Lander.
      Mitochondria are dynamic organelles that continually respond to cellular stress. Recent studies have demonstrated that mitochondrial stress is relayed from mitochondria to the cytosol by the release of a proteolytic fragment of DELE1 that binds to the eIF2α kinase HRI to initiate integrated stress response (ISR) signaling. We report the cryo-electron microscopy structure of the C-terminal cleavage product of human DELE1, which assembles into a high-order oligomer. The oligomer consists of eight DELE1 monomers that assemble with D4 symmetry via two sets of hydrophobic inter-subunit interactions. We identified the key residues involved in DELE1 oligomerization, and confirmed their role in stabilizing the octamer in vitro and in cells using mutagenesis. We further show that assembly-impaired DELE1 mutants are compromised in their ability to induce HRI-dependent ISR activation in cell culture models. Together, our findings provide molecular insights into the activity of DELE1 and how it signals to promote ISR activity following mitochondrial insult.
    DOI:  https://doi.org/10.1038/s41594-023-01061-0
  3. Theranostics. 2023 ;13(12): 4229-4246
    iNOS aggravates pressure overload-induced cardiac dysfunction via activation of the cytosolic-mtDNA-mediated cGAS-STING pathway.
    Yongzheng Guo, Yuehua You, Fei-Fei Shang, Xiaowen Wang, Bi Huang, Boying Zhao, Dingyi Lv, Shenglan Yang, Ming Xie, Lingwen Kong, Dingyuan Du, Suxin Luo, Xin Tian, Yong Xia.
      Background: Sterile inflammation contributes to the pathogenesis of cardiac dysfunction caused by various conditions including pressure overload in hypertension. Mitochondrial DNA (mtDNA) released from damaged mitochondria has been implicated in cardiac inflammation. However, the upstream mechanisms governing mtDNA release and how mtDNA activates sterile inflammation in pressure-overloaded hearts remain largely unknown. Here, we investigated the role of inducible NO synthase (iNOS) on pressure overload-induced cytosolic accumulation of mtDNA and whether mtDNA activated inflammation through the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway. Methods: To investigate whether the cGAS-STING cascade was involved in sterile inflammation and cardiac dysfunction upon pressure overload, cardiomyocyte-specific STING depletion mice and mice injected with adeno-associated virus-9 (AAV-9) to suppress the cGAS-STING cascade in the heart were subjected to transverse aortic constriction (TAC). iNOS null mice were used to determine the role of iNOS in cGAS-STING pathway activation in pressure-stressed hearts. Results: iNOS knockout abrogated mtDNA release and alleviated cardiac sterile inflammation resulting in improved cardiac function. Conversely, activating the cGAS-STING pathway blunted the protective effects of iNOS knockout. Moreover, iNOS activated the cGAS-STING pathway in isolated myocytes and this was prevented by depleting cytosolic mtDNA. In addition, disruption of the cGAS-STING pathway suppressed inflammatory cytokine transcription and modulated M1/M2 macrophage polarization, and thus mitigated cardiac remodeling and improved heart function. Finally, increased iNOS expression along with cytosolic mtDNA accumulation and cGAS-STING activation were also seen in human hypertensive hearts. Conclusion: Our findings demonstrate that mtDNA is released into the cytosol and triggers sterile inflammation through the cGAS-STING pathway leading to cardiac dysfunction after pressure overload. iNOS controls mtDNA release and subsequent cGAS activation in pressure-stressed hearts.
    Keywords:  Cardiac dysfunction; Sterile inflammation; cGAS; iNOS; mtDNA
    DOI:  https://doi.org/10.7150/thno.84049
  4. Nat Commun. 2023 Aug 10. 14(1): 4824
    WDR77 inhibits prion-like aggregation of MAVS to limit antiviral innate immune response.
    Jiaxin Li, Rui Zhang, Changwan Wang, Junyan Zhu, Miao Ren, Yingbo Jiang, Xianteng Hou, Yangting Du, Qing Wu, Shishi Qi, Lin Li, She Chen, Hui Yang, Fajian Hou.
      RIG-I-MAVS signaling pathway plays a crucial role in defending against pathogen infection and maintaining immune balance. Upon detecting viral RNA, RIG-I triggers the formation of prion-like aggregates of the adaptor protein MAVS, which then activates the innate antiviral immune response. However, the mechanisms that regulate the aggregation of MAVS are not yet fully understood. Here, we identified WDR77 as a MAVS-associated protein, which negatively regulates MAVS aggregation. WDR77 binds to MAVS proline-rich region through its WD2-WD3-WD4 domain and inhibits the formation of prion-like filament of recombinant MAVS in vitro. In response to virus infection, WDR77 is recruited to MAVS to prevent the formation of its prion-like aggregates and thus downregulate RIG-I-MAVS signaling in cells. WDR77 deficiency significantly potentiates the induction of antiviral genes upon negative-strand RNA virus infections, and myeloid-specific Wdr77-deficient mice are more resistant to RNA virus infection. Our findings reveal that WDR77 acts as a negative regulator of the RIG-I-MAVS signaling pathway by inhibiting the prion-like aggregation of MAVS to prevent harmful inflammation.
    DOI:  https://doi.org/10.1038/s41467-023-40567-5
  5. bioRxiv. 2023 Jul 27. pii: 2023.07.26.550716. [Epub ahead of print]
    Activation of ATF3 via the Integrated Stress Response Pathway Regulates Innate Immune and Autophagy Processes to Restrict Zika Virus.
    Pheonah Badu, Cara T Pager.
      Zika virus (ZIKV) is a re-emerging mosquito-borne flavivirus that can have devastating health consequences. The developmental and neurological effects from a ZIKV infection arise in part from the virus triggering cellular stress pathways and perturbing transcriptional programs. To date, the underlying mechanisms of transcriptional control directing viral restriction and virus-host interaction are understudied. Activating Transcription Factor 3 (ATF3) is a stress-induced transcriptional effector that modulates the expression of genes involved in a myriad of cellular processes, including inflammation and antiviral responses, to restore cellular homeostasis. While ATF3 is known to be upregulated during ZIKV infection, the mode by which ATF3 is activated and the specific role of ATF3 during ZIKV infection is unknown. In this study, we show via inhibitor and RNA interference approaches that ZIKV infection initiates the integrated stress response pathway to activate ATF4 which in turn induces ATF3 expression. Additionally, by using a CRISPR-Cas9 system to deplete ATF3, we found that ATF3 acts to limit ZIKV gene expression in A549 cells. In particular, the ATF3-dependent anti-ZIKV response occurred through regulation of innate immunity and autophagy pathways. We show that ATF3 differentially regulates the expression of innate immune response genes and suppresses the transcription of autophagy related genes to influence autophagic flux. Our study therefore highlights an important role for the integrated stress response pathway and ATF3 in establishing an antiviral effect during ZIKV infection.Importance: ZIKV is a re-emerging mosquito-borne flavivirus associated with congenital Zika syndrome in infants and Guillain Barré syndrome in adults. As a cytoplasmic virus, ZIKV co-opts host cellular mechanisms to support viral processes and consequently, reprograms the host transcriptional profile. Such viral-directed transcriptional changes and their proor anti-viral significance remain understudied. We previously showed that ATF3, a stress-induced transcription factor, is significantly upregulated in ZIKV infected mammalian cells, along with other cellular and immune response genes. Here, we specifically define the intracellular pathway responsible for ATF3 activation and elucidate the impact of ATF3 expression on ZIKV infection. Our data provides novel insights into the role of the integrated stress response pathway in stimulating ATF3 which differentially regulates the innate immune response and autophagy at the transcript level to antagonize ZIKV gene expression. This study establishes a framework that links viral-induced stress response to transcriptional regulation of host defense pathways and thus expands the depth of knowledge on virus-mediated transcriptional mechanisms during ZIKV infection which in turn will inform future therapeutic strategies.
    DOI:  https://doi.org/10.1101/2023.07.26.550716
  6. J Cell Physiol. 2023 Aug 10.
    The RNA-binding protein ZFP36 strengthens innate antiviral signaling by targeting RIG-I for K63-linked ubiquitination.
    Xue Jiang, Yanping Xiao, Wen Hou, Jingge Yu, Tian-Sheng He, Liang-Guo Xu.
      Innate immunity is the first line of defense against infections, which functions as a significant role in resisting pathogen invasion. Rapid immune response is initiated by pattern recognition receptors (PRRs) quickly distinguishing "self" and "non-self." Upon evolutionarily conserved pathogen-associated molecular pattern (PAMP) is recognized by PRRs, innate immune response against infection is triggered via an orchestration of molecular interaction, cytokines cascades, and immune cells. RIG-I plays a critical role in type I interferon (IFN-I) production by direct recognition of cytoplasmic double-stranded viral RNA. However, the activation mechanism of RIG-I is incompletely understood. In this study, we reported RNA-binding protein ZFP36 as a positive regulator of RIG-I-mediated IFN-I production. ZFP36 is a member of Zinc finger proteins (ZFPs) characterized by the zinc finger (ZnF) motif, which broadly involved gene transcription and signal transduction. However, its role in regulating antiviral innate immune signaling is still unclear. We found that ZFP36 associates with RIG-I and potentiates the FN-β production induced by SeV. Mechanistically, ZFP36 promotes K63-linked polyubiquitination of RIG-I, mostly at K154/K164/K172, thereby facilitating the activation of RIG-I during infection. While the mutant ZFP36 (C118S/C162S) failed to increase polyubiquitination of RIG-I and SeV induced FN-β. Our findings collectively demonstrated that ZFP36 acts as a positive regulator in antiviral innate immunity by targeting RIG-I for K63-linked ubiquitination, thus improving our understanding of the activation mechanism of RIG-I.
    Keywords:  RIG-I; ZFP36; cellular antiviral response; innate immunity
    DOI:  https://doi.org/10.1002/jcp.31088
  7. Int J Mol Sci. 2023 Jul 27. pii: 12012. [Epub ahead of print]24(15):
    Mitochondrial Dysfunction Associated with mtDNA in Metabolic Syndrome and Obesity.
    Natalia Todosenko, Olga Khaziakhmatova, Vladimir Malashchenko, Kristina Yurova, Maria Bograya, Maria Beletskaya, Maria Vulf, Natalia Gazatova, Larisa Litvinova.
      Metabolic syndrome (MetS) is a precursor to the major health diseases associated with high mortality in industrialized countries: cardiovascular disease and diabetes. An important component of the pathogenesis of the metabolic syndrome is mitochondrial dysfunction, which is associated with tissue hypoxia, disruption of mitochondrial integrity, increased production of reactive oxygen species, and a decrease in ATP, leading to a chronic inflammatory state that affects tissues and organ systems. The mitochondrial AAA + protease Lon (Lonp1) has a broad spectrum of activities. In addition to its classical function (degradation of misfolded or damaged proteins), enzymatic activity (proteolysis, chaperone activity, mitochondrial DNA (mtDNA)binding) has been demonstrated. At the same time, the spectrum of Lonp1 activity extends to the regulation of cellular processes inside mitochondria, as well as outside mitochondria (nuclear localization). This mitochondrial protease with enzymatic activity may be a promising molecular target for the development of targeted therapy for MetS and its components. The aim of this review is to elucidate the role of mtDNA in the pathogenesis of metabolic syndrome and its components as a key component of mitochondrial dysfunction and to describe the promising and little-studied AAA + LonP1 protease as a potential target in metabolic disorders.
    Keywords:  metabolic syndrome; mitochondrial dysfunction; mitochondrial protease Lonp1; mtDNA; obesity
    DOI:  https://doi.org/10.3390/ijms241512012
  8. Cell Death Differ. 2023 Aug 11.
    FBXL4 mutations cause excessive mitophagy via BNIP3/BNIP3L accumulation leading to mitochondrial DNA depletion syndrome.
    Yingji Chen, Dongyue Jiao, Yang Liu, Xiayun Xu, Yilin Wang, Xiaona Luo, Hexige Saiyin, Yao Li, Kun Gao, Yucai Chen, Shi-Min Zhao, Lixiang Ma, Chenji Wang.
      Mitochondria are essential organelles found in eukaryotic cells that play a crucial role in ATP production through oxidative phosphorylation (OXPHOS). Mitochondrial DNA depletion syndrome (MTDPS) is a group of genetic disorders characterized by the reduction of mtDNA copy number, leading to deficiencies in OXPHOS and mitochondrial functions. Mutations in FBXL4, a substrate-binding adaptor of Cullin 1-RING ubiquitin ligase complex (CRL1), are associated with MTDPS, type 13 (MTDPS13). Here, we demonstrate that, FBXL4 directly interacts with the mitophagy cargo receptors BNIP3 and BNIP3L, promoting their degradation through the ubiquitin-proteasome pathway via the assembly of an active CRL1FBXL4 complex. However, MTDPS13-associated FBXL4 mutations impair the assembly of an active CRL1FBXL4 complex. This results in a notable accumulation of BNIP3/3L proteins and robust mitophagy even at basal levels. Excessive mitophagy was observed in Knockin (KI) mice carrying a patient-derived FBXL4 mutation and cortical neurons (CNs)-induced from MTDPS13 patient human induced pluripotent stem cells (hiPSCs). In summary, our findings suggest that abnormal activation of BNIP3/BNIP3L-dependent mitophagy impairs mitochondrial homeostasis and underlies FBXL4-mutated MTDPS13.
    DOI:  https://doi.org/10.1038/s41418-023-01205-1
  9. Int J Mol Sci. 2023 Aug 07. pii: 12511. [Epub ahead of print]24(15):
    ATAD3A: A Key Regulator of Mitochondria-Associated Diseases.
    Liting Chen, Yuchang Li, Alexander Zambidis, Vassilios Papadopoulos.
      Mitochondrial membrane protein ATAD3A is a member of the AAA-domain-containing ATPases superfamily. It is important for the maintenance of mitochondrial DNA, structure, and function. In recent years, an increasing number of ATAD3A mutations have been identified in patients with neurological symptoms. Many of these mutations disrupt mitochondrial structure, function, and dynamics and are lethal to patients at a young age. Here, we summarize the current understanding of the relationship between ATAD3A and mitochondria, including the interaction of ATAD3A with mitochondrial DNA and mitochondrial/ER proteins, the regulation of ATAD3A in cholesterol mitochondrial trafficking, and the effect of known ATAD3A mutations on mitochondrial function. In the current review, we revealed that the oligomerization and interaction of ATAD3A with other mitochondrial/ER proteins are vital for its various functions. Despite affecting different domains of the protein, nearly all documented mutations observed in ATAD3A exhibit either loss-of-function or dominant-negative effects, potentially leading to disruption in the dimerization of ATAD3A; autophagy; mitophagy; alteration in mitochondrial number, size, and cristae morphology; and diminished activity of mitochondrial respiratory chain complexes I, IV, and V. These findings imply that ATAD3A plays a critical role in mitochondrial dynamics, which can be readily perturbed by ATAD3A mutation variants.
    Keywords:  ATAD3A; cancer; cholesterol; mitochondria; mitochondrial respiration; mtDNA; mutation; neurological diseases
    DOI:  https://doi.org/10.3390/ijms241512511
  10. IUBMB Life. 2023 Aug 10.
    RetroGREAT signaling: The lessons we learn from yeast.
    Thi Hoang Diu Bui, Karolina Labedzka-Dmoch.
      The mitochondrial retrograde signaling (RTG) pathway of communication from mitochondria to the nucleus was first studied in yeast Saccharomyces cerevisiae. It rewires cellular metabolism according to the mitochondrial state by reprogramming nuclear gene expression in response to mitochondrial triggers. The main players involved in retrograde signaling are the Rtg1 and Rtg3 transcription factors, and a set of positive and negative regulators, including the Rtg2, Mks1, Lst8, and Bmh1/2 proteins. Retrograde regulation is integrated with other processes, including stress response, osmoregulation, and nutrient sensing through functional crosstalk with cellular pathways such as high osmolarity glycerol or target of rapamycin signaling. In this review, we summarize metabolic changes observed upon retrograde stimulation and analyze the progress made to uncover the mechanisms underlying the integration of regulatory circuits. Comparisons of the evolutionary adaptations of the retrograde pathway that have occurred in the different yeast groups can help to fully understand the process.
    Keywords:  RTG signaling; mitochondria; retrograde response; yeast
    DOI:  https://doi.org/10.1002/iub.2775
  11. Cells. 2023 Jul 27. pii: 1950. [Epub ahead of print]12(15):
    Refractive Index Imaging Reveals That Elimination of the ATP Synthase C Subunit Does Not Prevent the Adenine Nucleotide Translocase-Dependent Mitochondrial Permeability Transition.
    Maria A Neginskaya, Sally E Morris, Evgeny V Pavlov.
      The mitochondrial permeability transition pore (mPTP) is a large, weakly selective pore that opens in the mitochondrial inner membrane in response to the pathological increase in matrix Ca2+ concentration. mPTP activation has been implicated as a key factor contributing to stress-induced necrotic and apoptotic cell death. The molecular identity of the mPTP is not completely understood. Both ATP synthase and adenine nucleotide translocase (ANT) have been described as important components of the mPTP. Using a refractive index (RI) imaging approach, we recently demonstrated that the removal of either ATP synthase or ANT eliminates the Ca2+-induced mPTP in experiments with intact cells. These results suggest that mPTP formation relies on the interaction between ATP synthase and ANT protein complexes. To gain further insight into this process, we used RI imaging to investigate mPTP properties in cells with a genetically eliminated C subunit of ATP synthase. These cells also lack ATP6, ATP8, 6.8PL subunits and DAPIT but, importantly, have a vestigial ATP synthase complex with assembled F1 and peripheral stalk domains. We found that these cells can still undergo mPTP activation, which can be blocked by the ANT inhibitor bongkrekic acid. These results suggest that ANT can form the pore independently from the C subunit but still requires the presence of other components of ATP synthase.
    Keywords:  ATP synthase; C subunit of ATP synthase; adenine nucleotide translocase; bongkrekic acid; cyclosporin A; holographic imaging; mitochondria; mitochondrial permeability transition; mitochondrial permeability transition pore; refractive index imaging
    DOI:  https://doi.org/10.3390/cells12151950
  12. Life Sci Alliance. 2023 Oct;pii: e202302059. [Epub ahead of print]6(10):
    High-resolution kinetic characterization of the RIG-I-signaling pathway and the antiviral response.
    Sandy S Burkart, Darius Schweinoch, Jamie Frankish, Carola Sparn, Sandra Wüst, Christian Urban, Marta Merlo, Vladimir G Magalhães, Antonio Piras, Andreas Pichlmair, Joschka Willemsen, Lars Kaderali, Marco Binder.
      RIG-I recognizes viral dsRNA and activates a cell-autonomous antiviral response. Upon stimulation, it triggers a signaling cascade leading to the production of type I and III IFNs. IFNs are secreted and signal to elicit the expression of IFN-stimulated genes, establishing an antiviral state of the cell. The topology of this pathway has been studied intensively, however, its exact dynamics are less understood. Here, we employed electroporation to synchronously activate RIG-I, enabling us to characterize cell-intrinsic innate immune signaling at a high temporal resolution. Employing IFNAR1/IFNLR-deficient cells, we could differentiate primary RIG-I signaling from secondary signaling downstream of the IFN receptors. Based on these data, we developed a comprehensive mathematical model capable of simulating signaling downstream of dsRNA recognition by RIG-I and the feedback and signal amplification by IFN. We further investigated the impact of viral antagonists on signaling dynamics. Our work provides a comprehensive insight into the signaling events that occur early upon virus infection and opens new avenues to study and disentangle the complexity of the host-virus interface.
    DOI:  https://doi.org/10.26508/lsa.202302059
  13. Nature. 2023 Aug 09.
    Mitochondrial integrated stress response controls lung epithelial cell fate.
    SeungHye Han, Minho Lee, Youngjin Shin, Regina Giovanni, Ram P Chakrabarty, Mariana M Herrerias, Laura A Dada, Annette S Flozak, Paul A Reyfman, Basil Khuder, Colleen R Reczek, Lin Gao, José Lopéz-Barneo, Cara J Gottardi, G R Scott Budinger, Navdeep S Chandel.
      Alveolar epithelial type 1 (AT1) cells are necessary to transfer oxygen and carbon dioxide between the blood and air. Alveolar epithelial type 2 (AT2) cells serve as a partially committed stem cell population, producing AT1 cells during postnatal alveolar development and repair after influenza A and SARS-CoV-2 pneumonia1-6. Little is known about the metabolic regulation of the fate of lung epithelial cells. Here we report that deleting the mitochondrial electron transport chain complex I subunit Ndufs2 in lung epithelial cells during mouse gestation led to death during postnatal alveolar development. Affected mice displayed hypertrophic cells with AT2 and AT1 cell features, known as transitional cells. Mammalian mitochondrial complex I, comprising 45 subunits, regenerates NAD+ and pumps protons. Conditional expression of yeast NADH dehydrogenase (NDI1) protein that regenerates NAD+ without proton pumping7,8 was sufficient to correct abnormal alveolar development and avert lethality. Single-cell RNA sequencing revealed enrichment of integrated stress response (ISR) genes in transitional cells. Administering an ISR inhibitor9,10 or NAD+ precursor reduced ISR gene signatures in epithelial cells and partially rescued lethality in the absence of mitochondrial complex I function. Notably, lung epithelial-specific loss of mitochondrial electron transport chain complex II subunit Sdhd, which maintains NAD+ regeneration, did not trigger high ISR activation or lethality. These findings highlight an unanticipated requirement for mitochondrial complex I-dependent NAD+ regeneration in directing cell fate during postnatal alveolar development by preventing pathological ISR induction.
    DOI:  https://doi.org/10.1038/s41586-023-06423-8
  14. J Neuroimmunol. 2023 Aug 02. pii: S0165-5728(23)00154-6. [Epub ahead of print]382 578168
    Prolonged STAT1 activation in neurons drives a pathological transcriptional response.
    Danielle N Clark, Shane M O'Neil, Li Xu, Justin T Steppe, Justin T Savage, Kavya Raghunathan, Anthony J Filiano.
      Neurons require physiological IFN-γ signaling to maintain central nervous system (CNS) homeostasis, however, pathological IFN-γ signaling can cause CNS pathologies. The downstream signaling mechanisms that cause these drastically different outcomes in neurons has not been well studied. We hypothesized that different levels of IFN-γ signaling in neurons results in differential activation of its downstream transcription factor, signal transducer and activator of transduction 1 (STAT1), causing varying outcomes. Using primary cortical neurons, we showed that physiological IFN-γ elicited brief and transient STAT1 activation, whereas pathological IFN-γ induced prolonged STAT1 activation, which primed the pathway to be more responsive to a subsequent IFN-γ challenge. This is an IFN-γ specific response, as other IFNs and cytokines did not elicit such STAT1 activation nor priming in neurons. Additionally, we did not see the same effect in microglia or astrocytes, suggesting this non-canonical IFN-γ/STAT1 signaling is unique to neurons. Prolonged STAT1 activation was facilitated by continuous janus kinase (JAK) activity, even in the absence of IFN-γ. Finally, although IFN-γ initially induced a canonical IFN-γ transcriptional response in neurons, pathological levels of IFN-γ caused long-term changes in synaptic pathway transcripts. Overall, these findings suggest that IFN-γ signaling occurs via non-canonical mechanisms in neurons, and differential STAT1 activation may explain how neurons have both homeostatic and pathological responses to IFN-γ signaling.
    Keywords:  Cytokine; Interferon-gamma; Neuroimmunology; Neuron; STAT1
    DOI:  https://doi.org/10.1016/j.jneuroim.2023.578168
  15. Cancer Discov. 2023 Aug 07. pii: CD-23-0007. [Epub ahead of print]
    Inhibition of METTL3 results in a cell-intrinsic interferon response that enhances anti-tumour immunity.
    Andrew A Guirguis, Yaara Ofir-Rosenfeld, Kathy Knezevic, Wesley Blackaby, David Hardick, Yih-Chih Chan, Ali Motazedian, Andrea Gillespie, Dane Vassiliadis, Enid Yn Lam, Kevin Tran, Byron Andrews, Michael E Harbour, Lina Vasiliauskaite, Claire J Saunders, Georgia Tsagkogeorga, Aleksandra Azevedo, Joanna Obacz, Ewa S Pilka, Marie Carkill, Laura MacPherson, Elanor N Wainwright, Brian Liddicoat, Benjamin J Blyth, Mark R Albertella, Oliver Rausch, Mark A Dawson.
      Therapies that enhance anti-tumour immunity have altered the natural history of many cancers. Consequently, leveraging non-overlapping mechanisms to increase immunogenicity of cancer cells remains a priority. Using a novel enzymatic inhibitor of the RNA methyltransferase, METTL3, we demonstrate a global decrease in N6-methyladenosine (m6A) results in double-stranded RNA formation and a profound cell-intrinsic interferon response. Through unbiased CRISPR screens, we establish dsRNA-sensing and interferon signalling are primary mediators that potentiate T-cell killing of cancer cells following METTL3 inhibition. We show in a range of immunocompetent mouse models that whilst METTL3 inhibition is equally efficacious to anti-PD1 therapy, the combination has far greater pre-clinical activity. Using SPLINTR barcoding, we demonstrate that anti-PD1 and METTL3 inhibition target distinct malignant clones and the combination of these therapies overcome clones insensitive to the single agents. These data provide the molecular and pre-clinical rationale for employing METTL3 inhibitors to promote anti-tumour immunity in the clinic.
    DOI:  https://doi.org/10.1158/2159-8290.CD-23-0007
  16. Nature. 2023 Aug 09.
    Lactate limits CNS autoimmunity by stabilizing HIF-1α in dendritic cells.
    Liliana M Sanmarco, Joseph M Rone, Carolina M Polonio, Gonzalo Fernandez Lahore, Federico Giovannoni, Kylynne Ferrara, Cristina Gutierrez-Vazquez, Ning Li, Anna Sokolovska, Agustin Plasencia, Camilo Faust Akl, Payal Nanda, Evelin S Heck, Zhaorong Li, Hong-Gyun Lee, Chun-Cheih Chao, Claudia M Rejano-Gordillo, Pedro H Fonseca-Castro, Tomer Illouz, Mathias Linnerbauer, Jessica E Kenison, Rocky M Barilla, Daniel Farrenkopf, Nikolas A Stevens, Gavin Piester, Elizabeth N Chung, Lucas Dailey, Vijay K Kuchroo, David Hava, Michael A Wheeler, Clary Clish, Roni Nowarski, Eduardo Balsa, Jose M Lora, Francisco J Quintana.
      Dendritic cells (DCs) have a role in the development and activation of self-reactive pathogenic T cells1,2. Genetic variants that are associated with the function of DCs have been linked to autoimmune disorders3,4, and DCs are therefore attractive therapeutic targets for such diseases. However, developing DC-targeted therapies for autoimmunity requires identification of the mechanisms that regulate DC function. Here, using single-cell and bulk transcriptional and metabolic analyses in combination with cell-specific gene perturbation studies, we identify a regulatory loop of negative feedback that operates in DCs to limit immunopathology. Specifically, we find that lactate, produced by activated DCs and other immune cells, boosts the expression of NDUFA4L2 through a mechanism mediated by hypoxia-inducible factor 1α (HIF-1α). NDUFA4L2 limits the production of mitochondrial reactive oxygen species that activate XBP1-driven transcriptional modules in DCs that are involved in the control of pathogenic autoimmune T cells. We also engineer a probiotic that produces lactate and suppresses T cell autoimmunity through the activation of HIF-1α-NDUFA4L2 signalling in DCs. In summary, we identify an immunometabolic pathway that regulates DC function, and develop a synthetic probiotic for its therapeutic activation.
    DOI:  https://doi.org/10.1038/s41586-023-06409-6
  17. Front Oncol. 2023 ;13 1257622
    Editorial: Targeting DNA damage response to enhance antitumor innate immunity in radiotherapy.
    Victoria Valvo, Emanuele Vitale, Marco Tigano, Rachel Evans, Meredith A Morgan, Qiang Zhang.
      
    Keywords:  DNA damage response; immunotherapy; innate immunity; radiotherapy; tumor microenvironment
    DOI:  https://doi.org/10.3389/fonc.2023.1257622
  18. Front Neurosci. 2023 ;17 1144896
    The complexities of investigating mitochondria dynamics in multiple sclerosis and mouse models of MS.
    Kelley C Atkinson, Marvellous Osunde, Seema K Tiwari-Woodruff.
      Multiple sclerosis (MS) is a demyelinating, degenerating disorder of the central nervous system (CNS) that is accompanied by mitochondria energy production failure. A loss of myelin paired with a deficit in energy production can contribute to further neurodegeneration and disability in patients in MS. Mitochondria are essential organelles that produce adenosine triphosphate (ATP) via oxidative phosphorylation in all cells in the CNS, including neurons, oligodendrocytes, astrocytes, and immune cells. In the context of demyelinating diseases, mitochondria have been shown to alter their morphology and undergo an initial increase in metabolic demand. This is followed by mitochondrial respiratory chain deficiency and abnormalities in mitochondrial transport that contribute to progressive neurodegeneration and irreversible disability. The current methodologies to study mitochondria are limiting and are capable of providing only a partial snapshot of the true mitochondria activity at a particular timepoint during disease. Mitochondrial functional studies are mostly performed in cell culture or whole brain tissue, which prevents understanding of mitochondrial pathology in distinct cell types in vivo. A true understanding of cell-specific mitochondrial pathophysiology of MS in mouse models is required. Cell-specific mitochondria morphology, mitochondria motility, and ATP production studies in animal models of MS will help us understand the role of mitochondria in the normal and diseased CNS. In this review, we present currently used methods to investigate mitochondria function in MS mouse models and discuss the current advantages and caveats with using each technique. In addition, we present recently developed mitochondria transgenic mouse lines expressing Cre under the control of CNS specific promoters to relate mitochondria to disease in vivo.
    Keywords:  EAE; cuprizone; demyelination; inflammation; mitochondria; multiple sclerosis; myelin; remyelination
    DOI:  https://doi.org/10.3389/fnins.2023.1144896
  19. Immunol Rev. 2023 Aug 09.
    Clearance phagocytosis by the retinal pigment epithelial during photoreceptor outer segment renewal: Molecular mechanisms and relation to retinal inflammation.
    Stephanie A Lieffrig, Gavin Gyimesi, Yingyu Mao, Silvia C Finnemann.
      Mammalian photoreceptor outer segment renewal is a highly coordinated process that hinges on timed cell signaling between photoreceptor neurons and the adjacent retinal pigment epithelial (RPE). It is a strictly rhythmic, synchronized process that underlies in part circadian regulation. We highlight findings from recently developed methods that quantify distinct phases of outer segment renewal in retinal tissue. At light onset, outer segments expose the conserved "eat-me" signal phosphatidylserine exclusively at their distal, most aged tip. A coordinated two-receptor efferocytosis process follows, in which ligands bridge outer segment phosphatidylserine with the RPE receptors αvβ5 integrin, inducing cytosolic signaling toward Rac1 and focal adhesion kinase/MERTK, and with MERTK directly, additionally inhibiting RhoA/ROCK and thus enabling F-actin dynamics favoring outer segment fragment engulfment. Photoreceptors and RPE persist for life with each RPE cell in the eye servicing dozens of overlying photoreceptors. Thus, RPE cells phagocytose more often and process more material than any other cell type. Mutant mice with impaired outer segment renewal largely retain functional photoreceptors and retinal integrity. However, when anti-inflammatory signaling in the RPE via MERTK or the related TYRO3 is lacking, catastrophic inflammation leads to immune cell infiltration that swiftly destroys the retina causing blindness.
    Keywords:  cell signaling; circadian rhythm; phagocytosis; photoreceptors; renewal; retinal pigment epithelial
    DOI:  https://doi.org/10.1111/imr.13264
  20. Methods Mol Biol. 2023 ;2706 215-224
    Live-Cell High-Throughput Screen for Monitoring Autophagy Flux.
    Sara Cano-Franco, Hung Ho-Xuan, Lorene Brunello, Alexandra Stolz.
      Autophagy is a cellular process implicated in the renewal of cellular components and the maintenance of cellular hemostasis and therefore associated with various types of diseases. In addition, autophagy belongs to the stress response pathways and is frequently activated by chemical compounds harboring characteristics of cell toxicity. High-throughput screens analyzing autophagy flux are therefore applied in both, the field of compound identification for targeting autophagy and compound characterization for analyzing compound toxicity. In this chapter, we describe a live-cell, fluorescent-based, high-throughput screening method in 384-well format for the fast and accurate measurement of autophagy flux over time suitable for academic research, pharmacological applications, and drug discovery.
    Keywords:  Autophagy; Fluorescence; GFP-LC3-RFP-LC3ΔG; Incucyte
    DOI:  https://doi.org/10.1007/978-1-0716-3397-7_16
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