bims-nenemi Biomed News
on Neuroinflammation, neurodegeneration and mitochondria
Issue of 2022–09–18
fourteen papers selected by
Marco Tigano, Thomas Jefferson University
  1. OMA1-mediated integrated stress response protects against ferroptosis in mitochondrial cardiomyopathy.
  2. ER Stress-Induced Sphingosine-1-Phosphate Lyase Phosphorylation Potentiates the Mitochondrial Unfolded Protein Response.
  3. Activation of a type I interferon response associated with acute frataxin knockdown in iPSC-derived cardiomyocytes.
  4. Mitochondrial stress induces AREG expression and epigenomic remodeling through c-JUN and YAP-mediated enhancer activation.
  5. Mitochondrial dysfunction triggers actin polymerization necessary for rapid glycolytic activation.
  6. Lamin A/C impairments cause mitochondrial dysfunction by attenuating PGC1α and the NAMPT-NAD+ pathway.
  7. Mitochondrial DNA mediates immunoparalysis of dendritic cells in sepsis via STING signalling.
  8. Phosphoglycerate mutase 5 initiates inflammation in acute kidney injury by triggering mitochondrial DNA release by dephosphorylating the pro-apoptotic protein Bax.
  9. Actin cytoskeleton remodeling primes RIG-I-like receptor activation.
  10. HMGA2 drives the IGFBP1/AKT pathway to counteract the increase in P27KIP1 protein levels in mtDNA/RNA-less cancer cells.
  11. Tumburu L, Ghosh-Choudhary S, Seifuddin FT, et al. Circulating mitochondrial DNA is a proinflammatory DAMP in sickle cell disease. Blood. 2021;137(22):3116-3126.
  12. Particulate matter 2.5 promotes inflammation and cellular dysfunction via reactive oxygen species/p38 MAPK pathway in primary rat corneal epithelial cells.
  13. A systems biological analysis of the ATF4-GADD34-CHOP regulatory triangle upon endoplasmic reticulum stress.
  14. FACS-assisted CRISPR-Cas9 genome editing of human induced pluripotent stem cells.
  1. Cell Metab. 2022 Sep 08. pii: S1550-4131(22)00360-6. [Epub ahead of print]
    OMA1-mediated integrated stress response protects against ferroptosis in mitochondrial cardiomyopathy.
    Sofia Ahola, Pablo Rivera Mejías, Steffen Hermans, Srikanth Chandragiri, Patrick Giavalisco, Hendrik Nolte, Thomas Langer.
      Cardiomyopathy and heart failure are common manifestations in mitochondrial disease caused by deficiencies in the oxidative phosphorylation (OXPHOS) system of mitochondria. Here, we demonstrate that the cardiac-specific loss of the assembly factor Cox10 of the cytochrome c oxidase causes mitochondrial cardiomyopathy in mice, which is associated with OXPHOS deficiency, lysosomal defects, and an aberrant mitochondrial morphology. Activation of the mitochondrial peptidase Oma1 in Cox10-/- mice results in mitochondrial fragmentation and induction of the integrated stress response (ISR) along the Oma1-Dele1-Atf4 signaling axis. Ablation of Oma1 or Dele1 in Cox10-/- mice aggravates cardiomyopathy. ISR inhibition impairs the cardiac glutathione metabolism, limits the selenium-dependent accumulation of the glutathione peroxidase Gpx4, and increases lipid peroxidation in the heart, ultimately culminating in ferroptosis. Our results demonstrate a protective role of the Oma1-Dele1-mediated ISR in mitochondrial cardiomyopathy and link ferroptosis to OXPHOS deficiency and mitochondrial disease.
    Keywords:  Atf4; Dele1; Gpx4; Oma1; cardiomyopathy; ferroptosis; glutathione; integrated stress response; mitochondria; selenium
    DOI:  https://doi.org/10.1016/j.cmet.2022.08.017
  2. J Lipid Res. 2022 Sep 10. pii: S0022-2275(22)00112-2. [Epub ahead of print] 100279
    ER Stress-Induced Sphingosine-1-Phosphate Lyase Phosphorylation Potentiates the Mitochondrial Unfolded Protein Response.
    Asli D Yildirim, Mevlut Citir, Asli E Dogan, Zehra Veli, Zehra Yildirim, Ozlem Tufanli, Alexis Traynor Kaplan, Carsten Schultz, Ebru Erbay.
      The unfolded protein response (UPR) is an elaborate signaling network that evolved to maintain proteostasis in the endoplasmic reticulum (ER) and mitochondria (mt). These organelles are functionally and physically associated and consequently, their stress responses are often intertwined. It is unclear how these two adaptive stress responses are coordinated during ER stress. The inositol-requiring enzyme-1 (IRE1), a central ER stress sensor and proximal regulator of the UPRER, harbors dual kinase and endoribonuclease (RNase) activities. IRE1 RNase activity initiates the transcriptional layer of the UPRER, but IRE1's kinase substrate(s) and their functions are largely unknown. Here, we discovered that sphingosine 1-phosphate (S1P) lyase (SPL), the enzyme that degrades S1P, is a substrate for the mammalian IRE1 kinase. Our data show that IRE1-dependent SPL phosphorylation inhibits SPL's enzymatic activity, resulting in increased intracellular S1P levels. S1P has previously been shown to induce the activation of mitochondrial UPR (UPRmt) in nematodes. We determined that IRE1 kinase-dependent S1P induction during ER stress potentiates UPRmt signaling in mammalian cells. Phosphorylation of eukaryotic translation initiation factor 2α (eif2α) is recognized as a critical molecular event for UPRmt activation in mammalian cells. Our data further demonstrate that inhibition of the IRE1-SPL axis abrogates the activation of two eif2α kinases, namely double-stranded RNA-activated protein kinase (PKR) and PKR-like ER kinase (PERK) upon ER stress. These findings show that the IRE1-SPL axis plays a central role in coordinating the adaptive responses of both organelles to ER stress in mammalian cells.
    Keywords:  Adaptive Stress Response; Endoplasmic Reticulum; Endoribonuclease; Eukaryotic Translation Initiation Factor 2α; IRE1-SPL axis; Inositol-Requiring Enzyme-1; Kinase; Mitochondria; Proteostasis; Signaling Networks
    DOI:  https://doi.org/10.1016/j.jlr.2022.100279
  3. Dis Model Mech. 2022 Sep 15. pii: dmm.049497. [Epub ahead of print]
    Activation of a type I interferon response associated with acute frataxin knockdown in iPSC-derived cardiomyocytes.
    M Grazia Cotticelli, Shujuan Xia, Rachel Truitt, Nicolai M Doliba, Andrea V Rozo, John W Tobias, Taehee Lee, Justin Chen, Jill S Napierala, Marek Napierala, Wenli Yang, Robert B Wilson.
      Friedreich ataxia, the most common hereditary ataxia, is a neuro- and cardio-degenerative disorder caused, in most cases, by decreased expression of the mitochondrial protein frataxin. Cardiomyopathy is the leading cause of premature death. Frataxin functions in the biogenesis of iron-sulfur clusters, which are prosthetic groups that are found in proteins involved in many biological processes. To study the changes associated with decreased frataxin in human cardiomyocytes, we developed a novel isogenic model by acutely knocking down frataxin, post-differentiation, in cardiomyocytes derived from induced pluripotent stem cells. Transcriptome analysis of four biological replicates identified severe mitochondrial dysfunction and a type I interferon response as the pathways most affected by frataxin knockdown. We confirmed that in iPSC-derived cardiomyocytes, loss of frataxin leads to mitochondrial dysfunction. The type I interferon response was activated in multiple cell types following acute frataxin knockdown and was caused, at least in part, by release of mitochondrial DNA into the cytosol, activating the cGAS-STING sensor pathway.
    Keywords:  Cardiomyopathy; Friedreich; Innate Immunity; Interferon; mtDNA
    DOI:  https://doi.org/10.1242/dmm.049497
  4. Nucleic Acids Res. 2022 Sep 12. pii: gkac735. [Epub ahead of print]
    Mitochondrial stress induces AREG expression and epigenomic remodeling through c-JUN and YAP-mediated enhancer activation.
    Yuko Hino, Katsuya Nagaoka, Shinya Oki, Kan Etoh, Shinjiro Hino, Mitsuyoshi Nakao.
      Nucleus-mitochondria crosstalk is essential for cellular and organismal homeostasis. Although anterograde (nucleus-to-mitochondria) pathways have been well characterized, retrograde (mitochondria-to-nucleus) pathways remain to be clarified. Here, we found that mitochondrial dysfunction triggered a retrograde signaling via unique transcriptional and chromatin factors in hepatic cells. Our transcriptomic analysis revealed that the loss of mitochondrial transcription factor A led to mitochondrial dysfunction and dramatically induced expression of amphiregulin (AREG) and other secretory protein genes. AREG expression was also induced by various mitochondria stressors and was upregulated in murine liver injury models, suggesting that AREG expression is a hallmark of mitochondrial damage. Using epigenomic and informatic approaches, we identified that mitochondrial dysfunction-responsive enhancers of AREG gene were activated by c-JUN/YAP1/TEAD axis and were repressed by chromatin remodeler BRG1. Furthermore, while mitochondrial dysfunction-activated enhancers were enriched with JUN and TEAD binding motifs, the repressed enhancers possessed the binding motifs for hepatocyte nuclear factor 4α, suggesting that both stress responsible and cell type-specific enhancers were reprogrammed. Our study revealed that c-JUN and YAP1-mediated enhancer activation shapes the mitochondrial stress-responsive phenotype, which may shift from metabolism to stress adaptation including protein secretion under such stressed conditions.
    DOI:  https://doi.org/10.1093/nar/gkac735
  5. J Cell Biol. 2022 Nov 07. pii: e202201160. [Epub ahead of print]221(11):
    Mitochondrial dysfunction triggers actin polymerization necessary for rapid glycolytic activation.
    Rajarshi Chakrabarti, Tak Shun Fung, Taewook Kang, Pieti W Elonkirjo, Anu Suomalainen, Edward J Usherwood, Henry N Higgs.
      Mitochondrial damage represents a dramatic change in cellular homeostasis. One rapid response is perimitochondrial actin polymerization, termed acute damage-induced actin (ADA). The consequences of ADA are not understood. In this study, we show evidence suggesting that ADA is linked to rapid glycolytic activation upon mitochondrial damage in multiple cells, including mouse embryonic fibroblasts and effector CD8+ T lymphocytes. ADA-inducing treatments include CCCP, antimycin, rotenone, oligomycin, and hypoxia. The Arp2/3 complex inhibitor CK666 or the mitochondrial sodium-calcium exchanger (NCLX) inhibitor CGP37157 inhibits both ADA and the glycolytic increase within 5 min, supporting ADA's role in glycolytic stimulation. Two situations causing chronic reductions in mitochondrial ATP production, mitochondrial DNA depletion and mutation to the NDUFS4 subunit of complex 1 of the electron transport chain, cause persistent perimitochondrial actin filaments similar to ADA. CK666 treatment causes rapid mitochondrial actin loss and a drop in ATP in NDUFS4 knock-out cells. We propose that ADA is necessary for rapid glycolytic activation upon mitochondrial impairment, to re-establish ATP production.
    DOI:  https://doi.org/10.1083/jcb.202201160
  6. Nucleic Acids Res. 2022 Sep 13. pii: gkac741. [Epub ahead of print]
    Lamin A/C impairments cause mitochondrial dysfunction by attenuating PGC1α and the NAMPT-NAD+ pathway.
    Scott Maynard, Arnaldur Hall, Panagiotis Galanos, Salvatore Rizza, Tatsuro Yamamoto, Helena Hagner Gram, Sebastian H N Munk, Muhammad Shoaib, Claus Storgaard Sørensen, Vilhelm A Bohr, Mads Lerdrup, Apolinar Maya-Mendoza, Jiri Bartek.
      Mutations in the lamin A/C gene (LMNA) cause laminopathies such as the premature aging Hutchinson Gilford progeria syndrome (HGPS) and altered lamin A/C levels are found in diverse malignancies. The underlying lamin-associated mechanisms remain poorly understood. Here we report that lamin A/C-null mouse embryo fibroblasts (Lmna-/- MEFs) and human progerin-expressing HGPS fibroblasts both display reduced NAD+ levels, unstable mitochondrial DNA and attenuated bioenergetics. This mitochondrial dysfunction is associated with reduced chromatin recruitment (Lmna-/- MEFs) or low levels (HGPS) of PGC1α, the key transcription factor for mitochondrial homeostasis. Lmna-/- MEFs showed reduced expression of the NAD+-biosynthesis enzyme NAMPT and attenuated activity of the NAD+-dependent deacetylase SIRT1. We find high PARylation in lamin A/C-aberrant cells, further decreasing the NAD+ pool and consistent with impaired DNA base excision repair in both cell models, a condition that fuels DNA damage-induced PARylation under oxidative stress. Further, ATAC-sequencing revealed a substantially altered chromatin landscape in Lmna-/- MEFs, including aberrantly reduced accessibility at the Nampt gene promoter. Thus, we identified a new role of lamin A/C as a key modulator of mitochondrial function through impairments of PGC1α and the NAMPT-NAD+ pathway, with broader implications for the aging process.
    DOI:  https://doi.org/10.1093/nar/gkac741
  7. Cell Prolif. 2022 Sep 15. e13328
    Mitochondrial DNA mediates immunoparalysis of dendritic cells in sepsis via STING signalling.
    Qing Tu, Yi Li, Jiali Zhu, Long Guo, Chenchen Liu, Lu Liu, Yuan Yuan, Yun Zou, Feng Chen, Liangfang Yao, Jinbao Li.
       BACKGROUND: Mitochondrial DNA (mtDNA) is a potent activator for pro-inflammatory response. Dendritic cells (DCs) are immunosuppressed in sepsis, whether mtDNA mediates immunoparalysis in sepsis remains unknown.
    METHODS: The mRNAs were assessed by qPCR. Flow cytometry was used to measure the expression of costimulatory molecules and the proliferation of CD4+ T cells. Western blot and immunofluorescence staining were used to analyse the expression of proteins. Cytokine secretion was detected by ELISA. Histology of lung tissue was used to assess the inflammatory injury.
    RESULTS: Lipopolysaccharide-induced endotoxemia increased plasma mtDNA levels and immunoparalysis of spleen DCs, while hydrolysing mtDNA reversed immunoparalysis of spleen DCs in vivo. Moreover, cytoplasmic mtDNA of DCs was accumulated in endotoxemia and sepsis. mtDNA transfection into bone marrow-derived DCs (BMDCs) inhibited the expression of costimulatory molecules (e.g., CD40, CD80 and CD86) and the release of IL-12p70, while increasing the secretion of IL-10. Cytoplasmic mtDNA also inhibited the ability of BMDCs to promote the proliferation of CD4+ T cells. Mechanistic analysis revealed that STING signalling was required for mtDNA-mediated immunoparalysis of DCs in vivo and in vitro. Further studies showed deletion of STING reversed mtDNA-mediated immunoparalysis of DCs and improved the prognosis of endotoxemia and sepsis.
    CONCLUSION: Our results demonstrated that mtDNA promotes immunoparalysis of DCs, and contributes to sepsis-associated immunosuppression by activating STING signalling. Our study may provide new insights to elucidate the molecular pathogenesis of immunosuppressive DCs in sepsis.
    DOI:  https://doi.org/10.1111/cpr.13328
  8. Kidney Int. 2022 Sep 08. pii: S0085-2538(22)00698-6. [Epub ahead of print]
    Phosphoglycerate mutase 5 initiates inflammation in acute kidney injury by triggering mitochondrial DNA release by dephosphorylating the pro-apoptotic protein Bax.
    Jingyao Li, Xi'ang Sun, Ninghao Yang, Jiayun Ni, Hongyan Xie, Hengjiang Guo, Xin Wang, Li Zhou, Jun Liu, Sijia Chen, Xiaoxia Wang, Yingying Zhang, Chen Yu, Wei Zhang, Limin Lu.
      Acute kidney injury (AKI) is a worldwide public health problem characterized by excessive inflammation with no specific therapy in clinic. Inflammation is not only a feature of AKI but also an essential promoter for kidney deterioration. Phosphoglycerate mutase 5 (PGAM5) was up-regulated and positively correlated with kidney dysfunction in human biopsy samples and mouse kidneys with AKI. PGAM5 knockout in mice significantly alleviated ischemia/reperfusion-induced kidney injury, mitochondrial abnormality and production of inflammatory cytokines. Elevated PGAM5 was found to be mainly located in kidney tubular epithelial cells and was also related to inflammatory response. Knockdown of PGAM5 inhibited the hypoxia/reoxygenation-induced cytosolic release of mitochondrial DNA (mtDNA) and binding of mtDNA with the cellular DNA receptor cGAS in cultured cells. cGAS deficiency also attenuated the inflammation and kidney injury in AKI. Mechanistically, as a protein phosphatase, PGAM5 was able to dephosphorylate the pro-apoptotic protein Bax and facilitate its translocation to mitochondrial membranes, and then initiate increased mitochondrial membrane permeability and release of mtDNA. Leaked mtDNA recognized by cGAS then initiated its downstream-coupled STING pathway, a component of the innate immune system that functions to detect the presence of cytosolic DNA. Thus, our results demonstrated mtDNA release induced by PGAM5-mediated Bax dephosphorylation and the activation of cGAS-STING pathway as critical determinants of inflammation and kidney injury. Hence, targeting this axis may be useful for treating AKI.
    Keywords:  Bax; PGAM5; acute kidney injury; inflammation; ischemia reperfusion; mitochondrion
    DOI:  https://doi.org/10.1016/j.kint.2022.08.022
  9. Cell. 2022 Sep 15. pii: S0092-8674(22)01060-1. [Epub ahead of print]185(19): 3588-3602.e21
    Actin cytoskeleton remodeling primes RIG-I-like receptor activation.
    Dhiraj Acharya, Rebecca Reis, Meta Volcic, GuanQun Liu, May K Wang, Bing Shao Chia, Rayhane Nchioua, Rüdiger Groß, Jan Münch, Frank Kirchhoff, Konstantin M J Sparrer, Michaela U Gack.
      The current dogma of RNA-mediated innate immunity is that sensing of immunostimulatory RNA ligands is sufficient for the activation of intracellular sensors and induction of interferon (IFN) responses. Here, we report that actin cytoskeleton disturbance primes RIG-I-like receptor (RLR) activation. Actin cytoskeleton rearrangement induced by virus infection or commonly used reagents to intracellularly deliver RNA triggers the relocalization of PPP1R12C, a regulatory subunit of the protein phosphatase-1 (PP1), from filamentous actin to cytoplasmic RLRs. This allows dephosphorylation-mediated RLR priming and, together with the RNA agonist, induces effective RLR downstream signaling. Genetic ablation of PPP1R12C impairs antiviral responses and enhances susceptibility to infection with several RNA viruses including SARS-CoV-2, influenza virus, picornavirus, and vesicular stomatitis virus. Our work identifies actin cytoskeleton disturbance as a priming signal for RLR-mediated innate immunity, which may open avenues for antiviral or adjuvant design.
    Keywords:  MDA5; PP1; RIG-I; actin cytoskeleton; innate immunity; type-I interferon; viral infection
    DOI:  https://doi.org/10.1016/j.cell.2022.08.011
  10. Cancer Sci. 2022 Sep 14.
    HMGA2 drives the IGFBP1/AKT pathway to counteract the increase in P27KIP1 protein levels in mtDNA/RNA-less cancer cells.
    Tsuyoshi Maruyama, Koji Saito, Masato Higurashi, Fumihiro Ishikawa, Yohko Kohno, Kazunori Mori, Motoko Shibanuma.
      Recent comprehensive analyses of mtDNA and orthogonal RNA-sequencing data revealed that in numerous human cancers, mtDNA copy numbers and mtRNA amounts are significantly reduced, followed by low respiratory gene expression. Under such conditions (called mt-Low), cells encounter severe cell proliferation defects; thus, they must acquire countermeasures against this fatal disadvantage during malignant transformation. This study elucidated a countermeasure against the mt-Low condition-induced antiproliferative effects in hepatocellular carcinoma (HCC) cells. The mechanism relied on the architectural transcriptional regulator HMGA2, which was preferably expressed in HCC cells of the mt-Low type in vitro and in vivo. Detailed in vitro analyses suggest that HMGA2 regulates insulin-like growth factor binding protein 1 (IGFBP1) expression, leading to AKT activation, which then phosphorylates the cyclin-dependent kinase inhibitor (CKI), P27KIP1, and facilitates its ubiquitin-mediated degradation. Accordingly, intervention in the HMGA2 function by RNAi resulted in an increase in P27KIP1 levels and an induction of senescence-like cell proliferation inhibition in mt-Low-type HCC cells. Conclusively, the HMGA2/IGFBP1/AKT axis has emerged as a countermeasure against P27KIP1 CKI upregulation under mt-Low conditions, thereby circumventing cell proliferation inhibition and supporting the tumorigenic state. Notably, similar to in vitro cell lines, HMGA2 was likely to regulate IGFBP1 expression in HCC in vivo, thereby contributing to poor patient prognosis. Considering the significant number of cases under mt-Low or the threat of CKI upregulation cancer-wide, the axis is noteworthy as a vulnerability of cancer cells or target for tumor-agnostic therapy inducing irreversible cell proliferation inhibition via CKI upregulation in a large population with cancer.
    Keywords:  HMGA2; IGFBP1; P27KIP1; hepatocellular carcinoma; mitochondria-deficiency
    DOI:  https://doi.org/10.1111/cas.15582
  11. Blood. 2022 Sep 15. 140(11): 1327
    Tumburu L, Ghosh-Choudhary S, Seifuddin FT, et al. Circulating mitochondrial DNA is a proinflammatory DAMP in sickle cell disease. Blood. 2021;137(22):3116-3126.
      
    DOI:  https://doi.org/10.1182/blood.2021012839
  12. Cutan Ocul Toxicol. 2022 Sep 12. 1-15
    Particulate matter 2.5 promotes inflammation and cellular dysfunction via reactive oxygen species/p38 MAPK pathway in primary rat corneal epithelial cells.
    Da Hye Kim, Hyesook Lee, Hyun Hwangbo, So Young Kim, Seon Yeong Ji, Min Yeong Kim, Seh-Kwang Park, Sung-Ho Park, Mi-Young Kim, Gi-Young Kim, Jaehun Cheong, Soo-Wan Nam, Yung Hyun Choi.
       PURPOSE: Numerous studies have linked particulate matter 2.5 (PM2.5) to ocular surface diseases, but few studies have been conducted on the biological effect of PM2.5 on the cornea. The objective of the present study was to evaluate the harmful effect of PM2.5 on primary rat corneal epithelial cells (RCECs) in vitro and identify the toxic mechanism involved.
    MATERIALS AND METHODS: Primary cultured RCECs were characterized by pan-cytokeratin (CK) staining. In PM2.5-exposed RCECs, cell viability, microarray gene expression, inflammatory cytokine levels, mitochondrial damage, DNA double-strand break and signaling pathway were investigated.
    RESULTS: Exposure to PM2.5 induced cytotoxicity and morphological changes in RCECs. In addition, PM2.5 markedly up-regulated pro-inflammatory mediators but down-regulated the wound healing-related transforming growth factor-β. Furthermore, PM2.5 promoted mitochondrial reactive oxygen species (ROS) production and mediated cellular damage to mitochondria and DNA, whereas these cellular alterations induced by PM2.5 were markedly suppressed by a potential ROS scavenger. Noteworthy, removal of ROS selectively down-regulated the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and the activation of the nuclear factor-κB (NF-κB) p65 in PM2.5-stimulated cells. Additionally, SB203580, a p38 MAPK inhibitor, markedly suppressed these PM2.5-mediated cellular dysfunctions.
    CONCLUSIONS: Taken together, our findings show that PM2.5 can promote the ROS/p38 MAPK/NF-κB signaling pathway and lead to mitochondrial damage and DNA double-strand break, which is ultimately caused inflammation and cytotoxicity in RCECs. These findings indicate that the ROS/p38 MAPK/NF-κB signaling pathway is one mechanism involved in PM2.5-induced ocular surface disorders.
    Keywords:  NF-κB; Particulate matter 2.5; ROS; corneal epithelial cells; p38 MAPK
    DOI:  https://doi.org/10.1080/15569527.2022.2122489
  13. FEBS Open Bio. 2022 Sep 13.
    A systems biological analysis of the ATF4-GADD34-CHOP regulatory triangle upon endoplasmic reticulum stress.
    Margita Márton, Gábor Bánhegyi, Norbert Gyöngyösi, Eszter Éva Kálmán, Aladár Pettkó-Szandtner, Krisztina Káldi, Orsolya Kapuy.
      Endoplasmic reticulum stress-dependent accumulation of incorrectly folded proteins leads to activation of the unfolded protein response. The role of the UPR is to avoid cell damage and restore the homeostatic state by autophagy; however, excessive ER stress results in apoptosis. Here, we investigated the ER stress-dependent feedback loops inside one of the UPR branches by focusing on PERK-induced ATF4 and its two targets, called CHOP and GADD34. Our goal was to qualitatively describe the dynamic behaviour of the system by exploring the key regulatory motifs using both molecular and theoretical biological techniques. Using the HEK293T cell line as a model system, we confirmed that the life-or-death decision is strictly regulated. We investigated the dynamic characteristics of the crucial elements of the PERK pathway at both the RNA and protein level upon tolerable and excessive levels of ER stress. Of particular note, inhibition of GADD34 or CHOP resulted in various phenotypes upon high levels of ER stress. Our computer simulations suggest the existence of two new feedback loops inside the UPR. First, GADD34 seems to have a positive effect on ATF4 activity, while CHOP inhibits it. We claim that these newly described feedback loops ensure the fine-tuning of the ATF4-dependent stress response mechanism of the cell.
    Keywords:  ATF4-GADD34-CHOP; PERK pathway; endoplasmic reticulum stress; feedback loop; systems biology; unfolded protein response
    DOI:  https://doi.org/10.1002/2211-5463.13484
  14. STAR Protoc. 2022 Sep 16. pii: S2666-1667(22)00560-3. [Epub ahead of print]3(4): 101680
    FACS-assisted CRISPR-Cas9 genome editing of human induced pluripotent stem cells.
    Amandine Caillaud, Antoine Lévêque, Aurélie Thédrez, Aurore Girardeau, Robin Canac, Lise Bray, Manon Baudic, Julien Barc, Nathalie Gaborit, Guillaume Lamirault, Betty Gardie, Salam Idriss, Antoine Rimbert, Cédric Le May, Bertrand Cariou, Karim Si-Tayeb.
      This manuscript proposes an efficient and reproducible protocol for the generation of genetically modified human induced pluripotent stem cells (hiPSCs) by genome editing using CRISPR-Cas9 technology. Here, we describe the experimental strategy for generating knockout (KO) and knockin (KI) clonal populations of hiPSCs using single-cell sorting by flow cytometry. We efficiently achieved up to 15 kb deletions, molecular tag insertions, and single-nucleotide editing in hiPSCs. We emphasize the efficacy of this approach in terms of cell culture time. For complete details on the use and execution of this protocol, please refer to Canac et al. (2022) and Bray et al. (2022).
    Keywords:  CRISPR; Flow cytometry/Mass cytometry; Stem cells
    DOI:  https://doi.org/10.1016/j.xpro.2022.101680
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