bims-nenemi 2022-04-03 papers

bims-nenemi

Biomed News

on Neuroinflammation, neurodegeneration and mitochondria

Issue of 2022‒04‒03
nineteen papers selected by
Marco Tigano
Thomas Jefferson University

An In Vitro Approach to Study Mitochondrial Dysfunction: A Cybrid Model.
Starting the engine of the powerhouse: mitochondrial transcription and beyond.
ER-mitochondria communication is involved in NLRP3 inflammasome activation under stress conditions in the innate immune system.
DNA-PK inhibition and radiation promote anti-tumoral immunity through RNA Polymerase III in pancreatic cancer.
Analysis of Mitochondrial Dysfunction by Microplate Reader in hiPSC-Derived Neuronal Cell Models of Neurodegenerative Disorders.
Nicotinamide mononucleotide alleviates heat stress-induced oxidative stress and apoptosis in BMECs through reducing mitochondrial damage and endoplasmic reticulum stress.
Heteroplasmic mitochondrial DNA variants in cardiovascular diseases.
MnTE-2-PyP protects fibroblast mitochondria from hyperglycemia and radiation exposure.
Inhibition of cGAS-STING by JQ1 alleviates oxidative stress-induced retina inflammation and degeneration.
Robust and tunable signal processing in mammalian cells via engineered covalent modification cycles.
cGAS-STING mediates cytoplasmic mitochondrial-DNA-induced inflammatory signal transduction during accelerated senescence of pancreatic β-cells induced by metabolic stress.
Photocontrollable Probes for Mitochondrial Protein Profiling.
Cellular stress signaling and the unfolded protein response in retinal degeneration: mechanisms and therapeutic implications.
CRISPR Guide RNA Library Screens in Human Induced Pluripotent Stem Cells.
An inhibitory effect on the nuclear accumulation of phospho-STAT1 by its unphosphorylated form.
Ceramide-1-Phosphate Is Involved in Therapy-Induced Senescence.
Mitochondrial Displacement Loop Region SNPs Modify Sjögren's Syndrome Development by Regulating Cytokines Expression in Female Patients.
Cell death-induced immunogenicity enhances chemoimmunotherapeutic response by converting immune-excluded into T-cell inflamed bladder tumors.
Regulatory networks controlling mitochondrial quality control in skeletal muscle.

J Vis Exp. 2022 Mar 09.

An In Vitro Approach to Study Mitochondrial Dysfunction: A Cybrid Model.

Andrea Cavaliere, Silvia Marchet, Ivano Di Meo, Valeria Tiranti.

Deficiency of the mitochondrial respiratory chain complexes that carry out oxidative phosphorylation (OXPHOS) is the biochemical marker of human mitochondrial disorders. From a genetic point of view, the OXPHOS represents a unique example because it results from the complementation of two distinct genetic systems: nuclear DNA (nDNA) and mitochondrial DNA (mtDNA). Therefore, OXPHOS defects can be due to mutations affecting nuclear and mitochondrial encoded genes. The groundbreaking work by King and Attardi, published in 1989, showed that human cell lines depleted of mtDNA (named rho0) could be repopulated by exogenous mitochondria to obtain the so-called "transmitochondrial cybrids." Thanks to these cybrids containing mitochondria derived from patients with mitochondrial disorders (MDs) and nuclei from rho0 cells, it is possible to verify whether a defect is mtDNA- or nDNA-related. These cybrids are also a powerful tool to validate the pathogenicity of a mutation and study its impact at a biochemical level. This paper presents a detailed protocol describing cybrid generation, selection, and characterization.

DOI: https://doi.org/10.3791/63452
Biol Chem. 2022 Mar 31.

Starting the engine of the powerhouse: mitochondrial transcription and beyond.

Maria Miranda, Nina A Bonekamp, Inge Kühl.

  Mitochondria are central hubs for cellular metabolism, coordinating a variety of metabolic reactions crucial for human health. Mitochondria provide most of the cellular energy via their oxidative phosphorylation (OXPHOS) system, which requires the coordinated expression of genes encoded by both the nuclear (nDNA) and mitochondrial genomes (mtDNA). Transcription of mtDNA is not only essential for the biogenesis of the OXPHOS system, but also generates RNA primers necessary to initiate mtDNA replication. Like the prokaryotic system, mitochondria have no membrane-based compartmentalization to separate the different steps of mtDNA maintenance and expression and depend entirely on nDNA-encoded factors imported into the organelle. Our understanding of mitochondrial transcription in mammalian cells has largely progressed, but the mechanisms regulating mtDNA gene expression are still poorly understood despite their profound importance for human disease. Here, we review mechanisms of mitochondrial gene expression with a focus on the recent findings in the field of mammalian mtDNA transcription and disease phenotypes caused by defects in proteins involved in this process.

Keywords:   inhibitor of mitochondrial transcription; PPR proteins; mitochondria; mitochondrial disease; mitochondrial gene expression; mitochondrial transcription

DOI:  https://doi.org/10.1515/hsz-2021-0416
Cell Mol Life Sci. 2022 Mar 28. 79(4): 213

ER-mitochondria communication is involved in NLRP3 inflammasome activation under stress conditions in the innate immune system.

Ana Catarina Pereira, Jessica De Pascale, Rosa Resende, Susana Cardoso, Isabel Ferreira, Bruno Miguel Neves, Mylène A Carrascal, Mónica Zuzarte, Nuno Madeira, Sofia Morais, António Macedo, Anália do Carmo, Paula I Moreira, Maria Teresa Cruz, Cláudia F Pereira.

  Endoplasmic reticulum (ER) stress and mitochondrial dysfunction, which are key events in the initiation and/or progression of several diseases, are correlated with alterations at ER-mitochondria contact sites, the so-called "Mitochondria-Associated Membranes" (MAMs). These intracellular structures are also implicated in NLRP3 inflammasome activation which is an important driver of sterile inflammation, however, the underlying molecular basis remains unclear. This work aimed to investigate the role of ER-mitochondria communication during ER stress-induced NLRP3 inflammasome activation in both peripheral and central innate immune systems, by using THP-1 human monocytes and BV2 microglia cells, respectively, as in vitro models. Markers of ER stress, mitochondrial dynamics and mass, as well as NLRP3 inflammasome activation were evaluated by Western Blot, IL-1β secretion was measured by ELISA, and ER-mitochondria contacts were quantified by transmission electron microscopy. Mitochondrial Ca2+ uptake and polarization were analyzed with fluorescent probes, and measurement of aconitase and SOD2 activities monitored mitochondrial ROS accumulation. ER stress was demonstrated to activate the NLRP3 inflammasome in both peripheral and central immune cells. Studies in monocytes indicate that ER stress-induced NLRP3 inflammasome activation occurs by a Ca2+-dependent and ROS-independent mechanism, which is coupled with upregulation of MAMs-resident chaperones, closer ER-mitochondria contacts, as well as mitochondrial depolarization and impaired dynamics. Moreover, enhanced ER stress-induced NLRP3 inflammasome activation in the immune system was found associated with pathological conditions since it was observed in monocytes derived from bipolar disorder (BD) patients, supporting a pro-inflammatory status in BD. In conclusion, by demonstrating that ER-mitochondria communication plays a key role in the response of the innate immune cells to ER stress, this work contributes to elucidate the molecular mechanisms underlying NLRP3 inflammasome activation under stress conditions, and to disclose novel potential therapeutic targets for diseases associated with sterile inflammation.

Keywords:  Bipolar disorder (BD); Calcium; Endoplasmic reticulum (ER) stress; Mitochondria; Sterile inflammation; Unfolded protein response

DOI:  https://doi.org/10.1007/s00018-022-04211-7
Mol Cancer Res. 2022 Mar 29. pii: molcanres.MCR-21-0725-E.2021. [Epub ahead of print]

DNA-PK inhibition and radiation promote anti-tumoral immunity through RNA Polymerase III in pancreatic cancer.

Weiwei Wang, Matthew T McMillan, Xinyi Zhao, Zhuwen Wang, Long Jiang, David Karnak, Fatima Lima, Joshua D Parsels, Leslie A Parsels, Theodore S Lawrence, Timothy L Frankel, Meredith A Morgan, Michael D Green, Qiang Zhang.

Targeting the DNA damage response in combination with radiation enhances type I interferon (T1IFN)-driven innate immune signaling. It is not understood, however, whether DNA-dependent protein kinase (DNA-PK), the kinase critical for repairing the majority of radiation-induced DNA double-strand breaks in cancer cells, is immunomodulatory. We show that combining radiation with DNA-PK inhibition increases cytosolic double-stranded DNA and tumoral T1IFN signaling in a cGAS- and STING-independent, but an RNA POL III, RIG-I, and MAVS-dependent manner. Although DNA-PK inhibition and radiation also promote programmed death-ligand 1 (PD-L1) expression, the use of anti-PD-L1 in combination with radiation and DNA-PK inhibitor potentiates antitumor immunity in pancreatic cancer models. Our findings demonstrate a novel mechanism for the antitumoral immune effects of DNA-PK inhibitor and radiation that leads to increased sensitivity to anti-PD-L1 in poorly immunogenic pancreatic cancers. Implications: Our work nominates a novel therapeutic strategy as well as biomarkers of treatment resistance pertinent for future clinical trials combining M3814, radiation and αPD-L1 antibody in patients with pancreatic cancer.

DOI: https://doi.org/10.1158/1541-7786.MCR-21-0725
Methods Mol Biol. 2022 Mar 29.

Analysis of Mitochondrial Dysfunction by Microplate Reader in hiPSC-Derived Neuronal Cell Models of Neurodegenerative Disorders.

Tatiana R Rosenstock, Congxin Sun, Georgina Wynne Hughes, Katherine Winter, Sovan Sarkar.

  Mitochondria are responsible for many vital pathways governing cellular homeostasis, including cellular energy management, heme biosynthesis, lipid metabolism, cellular proliferation and differentiation, cell cycle regulation, and cellular viability. Electron transport and ADP phosphorylation coupled with proton pumping through the mitochondrial complexes contribute to the preservation of mitochondrial membrane potential (ΔΨm). Importantly, mitochondrial polarization is essential for reactive oxygen species (ROS) production and cytosolic calcium (Ca2+) handling. Thus, changes in mitochondrial oxidative phosphorylation (OXPHOS), ΔΨm, and ATP/ADP may occur in parallel or stimulate each other. Brain cells like neurons are heavily reliant on mitochondrial OXPHOS for its high-energy demands, and hence improper mitochondrial function is detrimental for neuronal survival. Indeed, several neurodegenerative disorders are associated with mitochondrial dysfunction. Modeling this disease-relevant phenotype in neuronal cells differentiated from patient-derived human induced pluripotent stem cells (hiPSCs) provide an appropriate cellular platform for studying the disease pathology and drug discovery. In this review, we describe high-throughput analysis of crucial parameters related to mitochondrial function in hiPSC-derived neurons. These methodologies include measurement of ΔΨm, intracellular Ca2+, oxidative stress, and ATP/ADP levels using fluorescence probes via a microplate reader. Benefits of such an approach include analysis of mitochondrial parameters on a large population of cells, simultaneous analysis of different cell lines and experimental conditions, and for drug screening to identify compounds restoring mitochondrial function.

Keywords:  Fluorescent dyes; Human induced pluripotent stem cells; Microplate reader; Mitochondria; Mitochondrial calcium; Mitochondrial dysfunction; Mitochondrial membrane potential; Mitochondrial superoxide; Neurodegenerative disease; Neuronal differentiation; Reactive oxygen species; hiPSC-derived neurons

DOI:  https://doi.org/10.1007/7651_2021_451
Ecotoxicol Environ Saf. 2022 Mar 28. pii: S0147-6513(22)00281-0. [Epub ahead of print]235 113441

Nicotinamide mononucleotide alleviates heat stress-induced oxidative stress and apoptosis in BMECs through reducing mitochondrial damage and endoplasmic reticulum stress.

Han-Fang Zeng, Jie Xu, Xin-Ling Wang, Shu-Jie Li, Zhao-Yu Han.

  Heat stress is directly correlated to mammary gland dysfunction in dairy cows, especially in summer. Abnormally high environmental temperature induces oxidative stress and apoptosis in bovine mammary epithelial cells (BMECs). Nicotinamide mononucleotide (NMN) has beneficial effects in maintaining the cellular physiological functions. In this study, we evaluate the protective effect of NMN on heat stress-induced apoptosis of BMECs and explore the potential underlying mechanisms. Our results showed that heat stress considerably decreased cell viability in BMECs, whereas pretreatment of BMECs with NMN (150 μM) for 24 h significantly alleviated the negative effects of heat stress on cells. NMN protected BMECs from heat stress-induced oxidative stress by inhibiting the excessive accumulation of reactive oxygen species (ROS) and increasing the activity of antioxidant enzymes. It also inhibited apoptosis by reducing the ratio of Bax/Bcl2 and blocking proteolytic the cleavage of Caspase-3 in heat stressed-BMECs. Importantly, NMN treatment could reduce mitochondrial damage through mediating the expression of mitochondrial fission and fusion-related genes, including Dynamin related protein 1 (Drp1), Mitochondrial fission 1 protein (Fis1), and Mitofusin1, 2 (MFN1, 2); and suppress endoplasmic reticulum stress through unfolded protein response regulator Glucose regulated protein 78 (GRP78), and downstream elements Recombinant activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP). Above all, our results demonstrate that NMN supplemention attenuates heat stress-induced oxidative stress and apoptosis in BMECs by maintaining mitochondrial fission and fusion, and regulating endoplasmic reticulum stress, which provides the convincing evidence that NMN has valuable potential in alleviating mammary gland injury of dairy cows caused by environmental heat stress.

Keywords:  Apoptosis; Bovine mammary epithelial cells; Endoplasmic reticulum stress; Heat stress; Mitochondria fission and fusion; Nicotinamide mononucleotide

DOI:  https://doi.org/10.1016/j.ecoenv.2022.113441
PLoS Genet. 2022 Apr 01. 18(4): e1010068

Heteroplasmic mitochondrial DNA variants in cardiovascular diseases.

Claudia Calabrese, Angela Pyle, Helen Griffin, Jonathan Coxhead, Rafiqul Hussain, Peter S Braund, Linxin Li, Annette Burgess, Patricia B Munroe, Louis Little, Helen R Warren, Claudia Cabrera, Alistair Hall, Mark J Caulfield, Peter M Rothwell, Nilesh J Samani, Gavin Hudson, Patrick F Chinnery.

Mitochondria are implicated in the pathogenesis of cardiovascular diseases (CVDs) but the reasons for this are not well understood. Maternally-inherited population variants of mitochondrial DNA (mtDNA) which affect all mtDNA molecules (homoplasmic) are associated with cardiometabolic traits and the risk of developing cardiovascular disease. However, it is not known whether mtDNA mutations only affecting a proportion of mtDNA molecules (heteroplasmic) also play a role. To address this question, we performed a high-depth (~1000-fold) mtDNA sequencing of blood DNA in 1,399 individuals with hypertension (HTN), 1,946 with ischemic heart disease (IHD), 2,146 with ischemic stroke (IS), and 723 healthy controls. We show that the per individual burden of heteroplasmic single nucleotide variants (mtSNVs) increases with age. The age-effect was stronger for low-level heteroplasmies (heteroplasmic fraction, HF, 5-10%), likely reflecting acquired somatic events based on trinucleotide mutational signatures. After correcting for age and other confounders, intermediate heteroplasmies (HF 10-95%) were more common in hypertension, particularly involving non-synonymous variants altering the amino acid sequence of essential respiratory chain proteins. These findings raise the possibility that heteroplasmic mtSNVs in the pathophysiology of hypertension.

DOI: https://doi.org/10.1371/journal.pgen.1010068
Redox Biol. 2022 Mar 24. pii: S2213-2317(22)00073-8. [Epub ahead of print]52 102301

MnTE-2-PyP protects fibroblast mitochondria from hyperglycemia and radiation exposure.

Arpita Chatterjee, Isin T Sakallioglu, Divya Murthy, Elizabeth A Kosmacek, Pankaj K Singh, J Tyson McDonald, Robert Powers, Rebecca E Oberley-Deegan.

  Radiation is a common anticancer therapy for prostate cancer, which transforms tumor-associated normal fibroblasts to myofibroblasts, resulting in fibrosis. Oxidative stress caused by radiation-mediated mitochondrial damage is one of the major contributors to fibrosis. As diabetics are oxidatively stressed, radiation-mediated reactive oxygen species cause severe treatment failure, treatment-related side effects, and significantly reduced survival for diabetic prostate cancer patients as compared to non-diabetic prostate cancer patients. Hyperglycemia and enhanced mitochondrial damage significantly contribute to oxidative damage and disease progression after radiation therapy among diabetic prostate cancer patients. Therefore, reduction of mitochondrial damage in normal prostate fibroblasts after radiation should improve the overall clinical state of diabetic prostate cancer patients. We previously reported that MnTE-2-PyP, a manganese porphyrin, reduces oxidative damage in irradiated hyperglycemic prostate fibroblasts by scavenging superoxide and activating NRF2. In the current study, we have investigated the potential role of MnTE-2-PyP to protect mitochondrial health in irradiated hyperglycemic prostate fibroblasts. This study revealed that hyperglycemia and radiation increased mitochondrial ROS via blocking the mitochondrial electron transport chain, altered mitochondrial dynamics, and reduced mitochondrial biogenesis. Increased mitochondrial damage preceeded an increase in myofibroblast differentiation. MnTE-2-PyP reduced myofibroblast differentiation, improved mitochondrial health by releasing the block on the mitochondrial electron transport chain, enhanced ATP production efficiency, and restored mitochondrial dynamics and metabolism in the irradiated-hyperglycemic prostate fibroblasts. Therefore, we are proposing that one of the mechanisms that MnTE-2-PyP protects prostate fibroblasts from irradiation and hyperglycemia-mediated damage is by protecting the mitochondrial health in diabetic prostate cancer patients.

Keywords:  Diabetes; Fibroblast metabolism; Manganese porphyrin; Mitochondria; ROS; Radiation

DOI:  https://doi.org/10.1016/j.redox.2022.102301
Cell Death Differ. 2022 Mar 28.

Inhibition of cGAS-STING by JQ1 alleviates oxidative stress-induced retina inflammation and degeneration.

Ming Zou, Qin Ke, Qian Nie, Ruili Qi, Xingfei Zhu, Wei Liu, Xuebin Hu, Qian Sun, Jia-Ling Fu, Xiangcheng Tang, Yizhi Liu, David Wan-Cheng Li, Lili Gong.

Atrophic ("dry") form of age-related macular degeneration (AMD) is a leading cause of vision loss characterized by macular retinal pigment epithelium (RPE) and the ensuing photoreceptor degeneration. cGAS-STING signaling is a key cytosolic DNA sensor system in innate immunity and have recently been shown promotes RPE degeneration. However, expression regulation and therapeutic potential of cGAS and STING are not explored in retina under dry AMD pathogenic conditions. Our analysis shows upregulated STING RNA and increased chromatin accessibility around cGAS and STING promoters in macular retinas from dry AMD patients. cGAS-STING activation was detected in oxidative stress-induced mouse retina degeneration, accompanied with cytosolic leakage of damaged DNA in photoreceptors. Pharmaceutical or genetic approaches indicates STING promotes retina inflammation and degeneration upon oxidative damage. Drug screening reveals that BRD4 inhibitor JQ1 reduces cGAS-STING activation, inflammation and photoreceptor degeneration in the injured retina. BRD4 inhibition epigenetically suppresses STING transcription, and promotes autophagy-dependent cytosolic DNA clearance. Together, our results show that activation of cGAS-STING in retina may present pivotal innate immunity response in GA pathogenesis, whereas inhibition of cGAS-STING signaling by JQ1 could serve as a potential therapeutic strategy.

DOI: https://doi.org/10.1038/s41418-022-00967-4
Nat Commun. 2022 Mar 31. 13(1): 1720

Robust and tunable signal processing in mammalian cells via engineered covalent modification cycles.

Ross D Jones, Yili Qian, Katherine Ilia, Benjamin Wang, Michael T Laub, Domitilla Del Vecchio, Ron Weiss.

Engineered signaling networks can impart cells with new functionalities useful for directing differentiation and actuating cellular therapies. For such applications, the engineered networks must be tunable, precisely regulate target gene expression, and be robust to perturbations within the complex context of mammalian cells. Here, we use bacterial two-component signaling proteins to develop synthetic phosphoregulation devices that exhibit these properties in mammalian cells. First, we engineer a synthetic covalent modification cycle based on kinase and phosphatase proteins derived from the bifunctional histidine kinase EnvZ, enabling analog tuning of gene expression via its response regulator OmpR. By regulating phosphatase expression with endogenous miRNAs, we demonstrate cell-type specific signaling responses and a new strategy for accurate cell type classification. Finally, we implement a tunable negative feedback controller via a small molecule-stabilized phosphatase, reducing output expression variance and mitigating the context-dependent effects of off-target regulation and resource competition. Our work lays the foundation for establishing tunable, precise, and robust control over cell behavior with synthetic signaling networks.

DOI: https://doi.org/10.1038/s41467-022-29338-w
FASEB J. 2022 May;36(5): e22266

cGAS-STING mediates cytoplasmic mitochondrial-DNA-induced inflammatory signal transduction during accelerated senescence of pancreatic β-cells induced by metabolic stress.

Huiqing Hu, Ruxing Zhao, Qin He, Chen Cui, Jia Song, Xinghong Guo, Nan Zang, Mengmeng Yang, Ying Zou, Jingwen Yang, Jinquan Li, Liming Wang, Longqing Xia, Lingshu Wang, Falian He, Xinguo Hou, Fei Yan, Li Chen.

  Type 2 diabetes mellitus (T2DM) is an age-related disease characterized by impaired pancreatic β cell function and insulin resistance. Recent studies have shown that the accumulation of senescent β cells under metabolic stress conditions leads to the progression of T2DM, while senolysis can improve the prognosis. However, the specific mechanism of β cell senescence is still unclear. In this study, we found that the increased load of senescence pancreatic β cells in both older mice and obese mice induced by high-fat diet (HFD) (DIO mice) was accompanied by activation of the Cyclic GMP-AMP synthase (cGAS) - stimulator of interferon genes (STING) pathway and using cGAS or STING small interfering RNA or STING inhibitor C176 to downregulate this pathway reduced the senescence-associated secretion profile (SASP) and senescence of Min6 cells treated with palmitic acid or hydrogen peroxide. C176 intervention in DIO mice also significantly reduced the inflammation and senescence of the islets, thereby protecting the function of pancreatic β cell and glucose metabolism. Our study further revealed that mitochondrial DNA (mtDNA) leakage under metabolic stress conditions was critical for the activation of the cGAS-STING pathway, which can be reversed by the mtDNA depleting agent ethidium bromide. Consistently, mtDNA leakage was more severe in older mice and was accelerated by a chronic HFD. In conclusion, we demonstrate that cytoplasmic mtDNA activates the cGAS-STING pathway to mediate SASP during the accelerated senescence of pancreatic β-cells induced by metabolic stress, and this process can be downregulated by the STING inhibitor C176.

Keywords:  SASP; cGAS-STING pathway; pancreatic β cell function; senescence; type 2 diabetes mellitus

DOI:  https://doi.org/10.1096/fj.202101988R
Chembiochem. 2022 Mar 28.

Photocontrollable Probes for Mitochondrial Protein Profiling.

Shuhong Guo, Chaonan Yuan, Wenjie Lang, Danqi Hong, Jian Liu, Jintao Huang, Jia Dong, Jingyan Ge.

  Mitochondrion is the core site of cell signaling, energy metabolism and biosynthesis. Here, taking advantage of activitybased probes, we synthesized two photocontrollable probes ( YGH-1 and YGH-2 ), composed of a mitochondrial localization moiety "triphenylphosphonium", a photo triggered group to achieve spatial and temporal controlled protein capture and an alkyne group to enrich the labeled protein. Proteomic validation was further carried out to facilitate identifications of mitochondrial proteomes in HeLa cells. The results showed that half of identified protein hits (~300) labeled by probes YGH-1 and YGH-2 belong to mitochondria, mostly localizing in mitochondrial matrix and inner mitochondrial membrane. Our research results provide a new tool for spatial and temporal analysis of subcellular proteome.

Keywords:  mitochondrial proteome; photocontrollable; photocross linker; quinone methide

DOI:  https://doi.org/10.1002/cbic.202200066
Mol Neurodegener. 2022 Mar 28. 17(1): 25

Cellular stress signaling and the unfolded protein response in retinal degeneration: mechanisms and therapeutic implications.

Todd McLaughlin, Andy Medina, Jacob Perkins, Maria Yera, Joshua J Wang, Sarah X Zhang.

  BACKGROUND: The retina, as part of the central nervous system (CNS) with limited capacity for self-reparation and regeneration in mammals, is under cumulative environmental stress due to high-energy demands and rapid protein turnover. These stressors disrupt the cellular protein and metabolic homeostasis, which, if not alleviated, can lead to dysfunction and cell death of retinal neurons. One primary cellular stress response is the highly conserved unfolded protein response (UPR). The UPR acts through three main signaling pathways in an attempt to restore the protein homeostasis in the endoplasmic reticulum (ER) by various means, including but not limited to, reducing protein translation, increasing protein-folding capacity, and promoting misfolded protein degradation. Moreover, recent work has identified a novel function of the UPR in regulation of cellular metabolism and mitochondrial function, disturbance of which contributes to neuronal degeneration and dysfunction. The role of the UPR in retinal neurons during aging and under disease conditions in age-related macular degeneration (AMD), retinitis pigmentosa (RP), glaucoma, and diabetic retinopathy (DR) has been explored over the past two decades. Each of the disease conditions and their corresponding animal models provide distinct challenges and unique opportunities to gain a better understanding of the role of the UPR in the maintenance of retinal health and function.METHOD: We performed an extensive literature search on PubMed and Google Scholar using the following keywords: unfolded protein response, metabolism, ER stress, retinal degeneration, aging, age-related macular degeneration, retinitis pigmentosa, glaucoma, diabetic retinopathy.
RESULTS AND CONCLUSION: We summarize recent advances in understanding cellular stress response, in particular the UPR, in retinal diseases, highlighting the potential roles of UPR pathways in regulation of cellular metabolism and mitochondrial function in retinal neurons. Further, we provide perspective on the promise and challenges for targeting the UPR pathways as a new therapeutic approach in age- and disease-related retinal degeneration.

Keywords:  Age related macular degeneration; Aging; Diabetic retinopathy; Endoplasmic reticulum stress; Glaucoma; Metabolism; Retinal degeneration; Retinitis pigmentosa; Unfolded protein response

DOI:  https://doi.org/10.1186/s13024-022-00528-w
Methods Mol Biol. 2022 Mar 29.

CRISPR Guide RNA Library Screens in Human Induced Pluripotent Stem Cells.

Yan Zhou, Qiang Fu, Huijun Shi, Guangqian Zhou.

  High-throughput CRISPR guide RNA (gRNA) library screen, that is, CRISPR/Cas9 screen, enables the unbiased identification of gene functions in a variety of biological processes. Typical pooled CRISPR/Cas9 screen couples a gRNA library and a guided Cas9 or dCas9 endonuclease to target specific gene loci, and then systematically uncover the causal link between candidate genes and observed cellular phenotypes via gRNA depletion or enrichment in screens. Here, we describe a detailed method of puromycin (PURO) concentration titration and lentiviral CRISPR gRNA library titration in Cas9 expressing monoclonal human iPSC line (Cas9+MNhiPSC) prior to performing the screens, conducting pooled CRISPR gRNA library screens in Cas9+MNhiPSC, genomic DNA extraction from the selected cell subpopulation and sequencing library preparation as well as next generation sequencing (NGS) to generate gRNA read counts. In CRISPR/Cas9 screen, we aim for 30% transduction efficiency (i.e., multiplicity of infection = 0.3) to ensure most of infected cells receive only one gRNA. The principles in this method can be applied to CRISPR perturbation (knockout, activation, repression or base editing) screens with other CRISPR gRNA libraries across many other cell models and other species.

Keywords:  CRISPR gRNA library screen; CRISPR gRNA library titration; CRISPR/Cas9; Human induced pluripotent stem cells (hiPSCs); Next generation sequencing (NGS); Puromycin (PURO) titration

DOI:  https://doi.org/10.1007/7651_2021_455
Cell Commun Signal. 2022 Mar 31. 20(1): 42

An inhibitory effect on the nuclear accumulation of phospho-STAT1 by its unphosphorylated form.

Priyanka Rajeev Menon, Julia Staab, Anke Gregus, Oliver Wirths, Thomas Meyer.

  BACKGROUND: Unphosphorylated signal transducer and activator of transcription 1 (U-STAT1) has been reported to elicit a distinct gene expression profile as compared to tyrosine-phosphorylated STAT1 (P-STAT1) homodimers. However, the impact of U-STAT1 on the IFNγ-induced immune response mediated by P-STAT1 is unknown. By generating a double mutant of STAT1 with mutation R602L in the Src-homology 2 (SH2) domain and Y701F in the carboxy-terminal transactivation domain mimicking U-STAT1, we investigated the effects of U-STAT1 on P-STAT1-mediated signal transduction.RESULTS: In this study, we discovered a novel activity of U-STAT1 that alters the nucleo-cytoplasmic distribution of cytokine-stimulated P-STAT1. While the dimerization-deficient mutant R602L/Y701F was not able to display cytokine-induced nuclear accumulation, it inhibited the nuclear accumulation of co-expressed IFNγ-stimulated wild-type P-STAT1. Disruption of the anti-parallel dimer interface in the R602L/Y701F mutant via additional R274W and T385A mutations did not rescue the impaired nuclear accumulation of co-expressed P-STAT1. The mutant U-STAT1 affected neither the binding of co-expressed P-STAT1 to gamma-activated sites in vitro, nor the transcription of reporter constructs and the activation of STAT1 target genes. However, the nuclear accumulation of P-STAT1 was diminished in the presence of mutant U-STAT1, which was not restored by mutations reducing the DNA affinity of mutant U-STAT1. Whereas single mutations in the amino-terminus of dimerization-deficient U-STAT1 similarly inhibited the nuclear accumulation of co-expressed P-STAT1, a complete deletion of the amino-terminus restored cytokine-stimulated nuclear accumulation of P-STAT1. Likewise, the disruption of a dimer-specific nuclear localization signal also rescued the U-STAT1-mediated inhibition of P-STAT1 nuclear accumulation.
CONCLUSION: Our data demonstrate a novel role of U-STAT1 in affecting nuclear accumulation of P-STAT1, such that a high intracellular concentration of U-STAT1 inhibits the detection of nuclear P-STAT1 in immunofluorescence assays. These observations hint at a possible physiological function of U-STAT1 in buffering the nuclear import of P-STAT1, while preserving IFNγ-induced gene expression. Based on these results, we propose a model of a hypothetical import structure, the assembly of which is impaired under high concentrations of U-STAT1. This mechanism maintains high levels of cytoplasmic STAT1, while simultaneously retaining signal transduction by IFNγ. Video Abstract.

Keywords:  Dimerization; Interferon-induced gene expression; JAK-STAT signalling; Nuclear accumulation; STAT1

DOI:  https://doi.org/10.1186/s12964-022-00841-3
ACS Chem Biol. 2022 Mar 30.

Ceramide-1-Phosphate Is Involved in Therapy-Induced Senescence.

Alec Millner, Logan Running, Nicole Colon-Rosa, Diana S Aga, Jonna Frasor, G Ekin Atilla-Gokcumen.

Sphingolipids are key signaling lipids and their dysregulation has been associated with various cellular processes. We have previously shown significant changes in sphingolipids in therapy-induced senescence, a state of cell cycle arrest as a response to chemotherapy, including the accumulation of ceramides, and provided evidence suggesting that ceramide processing is important for this process. Herein, we conducted a focused small molecule inhibitor screen targeting the sphingolipid pathway, which highlighted a new lipid regulator of therapy-induced senescence. Among the inhibitors tested, the inhibition of ceramide kinase by NVP-231 reduced the levels of senescent cells. Ceramide kinase knockdown exhibited similar effects, strongly supporting the involvement of ceramide kinase during this process. We showed that ceramide-1-phosphate was upregulated in therapy-induced senescence and that NVP-231 reduced ceramide-1-phosphate levels in different cell line models of therapy-induced senescence. Finally, ceramide-1-phosphate addition to NVP-231-treated cells reversed the effects of NVP-231 during senescence. Overall, our results identify a previously unknown lipid player in therapy-induced senescence and highlight a potential targetable enzyme to reduce the levels of therapy-induced senescent cells.

DOI: https://doi.org/10.1021/acschembio.2c00216
Front Genet. 2022 ;13 847521

Mitochondrial Displacement Loop Region SNPs Modify Sjögren's Syndrome Development by Regulating Cytokines Expression in Female Patients.

Yufei Zhao, Chenxing Peng, Jingjing Zhang, Ruixue Lai, Xiaoyun Zhang, Zhanjun Guo.

  Mitochondrial dysfunction could induce innate immune response with cytokines releasing to initiate Sjögren's syndrome (SS) onset. Single nucleotide polymorphisms (SNPs) in the mitochondrial displacement loop (D-loop) and mitochondrial DNA (mtDNA) copy number of female SS patients were evaluated for their association with SS in female patients. At the nucleotide site of 152, 16304, 16311 and 16362 in the D-loop, the frequencies for the minor alleles of 152C (p = 0.040, odds ratio [OR] = 0.504), 16304C (p = 0.045, OR = 0.406), 16311C (p = 0.045, OR = 0.406) and 16362C (p = 0.028, OR = 0.519) were significantly higher in the SS patients than those in the female controls, which indicated that 152,C, 16304C, 16311C, and 16362C allele in the D-loop of mtDNA were associated with the risk of SS. Meanwhile, the excessive SNPs were accumulated in D-loop region of SS patients (8.955 ± 2.028 versus 7.898 ± 1.987, p < 0.001, 95% confidence interval [CI]: 0.477-1.637) and mtDNA copy number increased in SS patients (1.509 ± 0.836 versus 1.221 ± 0.506, p = 0.006, 95% CI: 0.086-0.490) by a case-control analysis. The subsequent analysis showed that SS risk-related allele 16311C was associated with higher IL-2 levels (p = 0.010) at significantly statistical level whereas 152C associated with lower IL-10 levels (p = 0.058) at a borderline statistical levels. Our findings suggest that mitochondrial D-loop SNPs are predictors for SS risk, it might modify the SS development by regulating cytokine expression.

Keywords:  D-loop; MtDNA copy number; ROS; Sjögren's syndrome; cytokine; snps

DOI:  https://doi.org/10.3389/fgene.2022.847521
Nat Commun. 2022 Mar 28. 13(1): 1487

Cell death-induced immunogenicity enhances chemoimmunotherapeutic response by converting immune-excluded into T-cell inflamed bladder tumors.

Fotis Nikolos, Kazukuni Hayashi, Xen Ping Hoi, Mark Ellie Alonzo, Qianxing Mo, Armine Kasabyan, Hideki Furuya, Jane Trepel, Dolores Di Vizio, Jlenia Guarnerio, Dan Theodorescu, Charles Rosser, Andrea Apolo, Matthew Galsky, Keith Syson Chan.

Chemoimmunotherapy has recently failed to demonstrate significant clinical benefit in advanced bladder cancer patients; and the mechanism(s) underlying such suboptimal response remain elusive. To date, most studies have focused on tumor-intrinsic properties that render them "immune-excluded". Here, we explore an alternative, drug-induced mechanism that impedes therapeutic response via disrupting the onset of immunogenic cell death. Using two immune-excluded syngeneic mouse models of muscle-invasive bladder cancer (MIBC), we show that platinum-based chemotherapy diminishes CD8+ T cell tumor infiltration and constraines their antitumoral activity, despite expression of activation markers IFNγ and granzyme B. Mechanistically, chemotherapy induces the release of prostaglandin E2 (PGE2) from dying cancer cells, which is an inhibitory damage-associated molecular pattern (iDAMP) that hinderes dendritic cell maturation. Upon pharmaceutical blockade of PGE2 release, CD8+ T cells become tumoricidal and display an intraepithelial-infiltrating (or inflamed) pattern. This "iDAMP blockade" approach synergizes with chemotherapy and sensitizes bladder tumors towards anti-PD1 immune checkpoint inhibitor therapy. These findings provide a compelling rationale to evaluate this drug combination in future clinical trials.

DOI: https://doi.org/10.1038/s41467-022-29026-9
Am J Physiol Cell Physiol. 2022 Mar 30.

Regulatory networks controlling mitochondrial quality control in skeletal muscle.

Mikhaela B Slavin, Jonathan M Memme, Ashley N Oliveira, Neushaw Moradi, David A Hood.

The adaptive plasticity of mitochondria within skeletal muscle is regulated by signals converging on a myriad of regulatory networks that operate during conditions of increased (i.e. exercise) and decreased (inactivity, disuse) energy requirements. Notably, some of the initial signals that induce adaptive responses are common to both conditions, differing in their magnitude and temporal pattern, to produce vastly opposing mitochondrial phenotypes. In response to exercise, signaling to PGC-1α and other regulators ultimately produces an abundance of high quality mitochondria, leading to reduced mitophagy and a higher mitochondrial content. This is accompanied by the presence of an enhanced protein quality control system that consists of the protein import machinery as well chaperones and proteases termed the UPRmt. The UPRmt monitors intra-organelle proteostasis, and strives to maintain a mito-nuclear balance between nuclear- and mtDNA-derived gene products via retrograde signaling from the organelle to the nucleus. In addition, antioxidant capacity is improved, affording greater protection against oxidative stress. In contrast, chronic disuse conditions produce similar signaling but result in decrements in mitochondrial quality and content. Thus, the interactive cross-talk of the regulatory networks that control organelle turnover during wide variations in muscle use and disuse remain incompletely understood, despite our improving knowledge of the traditional regulators of organelle content and function. This brief review acknowledges existing regulatory networks and summarizes recent discoveries of novel biological pathways involved in determining organelle biogenesis, dynamics, mitophagy, protein quality control and antioxidant capacity, identifying ample protein targets for therapeutic intervention that determine muscle and mitochondrial health.

DOI: https://doi.org/10.1152/ajpcell.00065.2022