bims-nenemi 2022-03-20 papers

bims-nenemi

Biomed News

on Neuroinflammation, neurodegeneration and mitochondria

Issue of 2022–03–20
eightteen papers selected by
Marco Tigano, Thomas Jefferson University

POLG mutations lead to abnormal mitochondrial remodeling during neural differentiation of human pluripotent stem cells via SIRT3/AMPK pathway inhibition.
TCA cycle off, ATF4 on for metabolic homeostasis.
Endoplasmic reticulum-unfolded protein response pathway modulates the cellular response to mitochondrial proteotoxic stress.
Lon upregulation contributes to cisplatin resistance by triggering NCLX-mediated mitochondrial Ca2+ release in cancer cells.
Disruption of Mitochondrial Quality Control Genes Promotes Caspase-Resistant Cell Survival Following Apoptotic Stimuli.
Differential mitochondrial and cellular responses between H vs. J mtDNA haplogroup-containing human RPE transmitochondrial cybrid cells.
Mitochondrial depolarization after acute ethanol treatment drives mitophagy in living mice.
Mitochondrial Dysfunction is a Driver of SP-2509 Drug Resistance in Ewing Sarcoma.
Type I Interferon Response Is Mediated by NLRX1-cGAS-STING Signaling in Brain Injury.
Neutrophil extracellular traps in hepatocellular carcinoma are enriched in oxidized mitochondrial DNA which is highly pro-inflammatory and pro-metastatic.
Mitochondrial Membrane Potential Assay.
DNA-PKcs interacts with and phosphorylates Fis1 to induce mitochondrial fragmentation in tubular cells during acute kidney injury.
PINK1/Parkin-mediated mitophagy is activated to protect against AFB1-induced kidney damage in mice.
Selective Cleavage at CCA Ends and Anticodon Loops of tRNAs by Stress-Induced RNases.
Activation of the UPR sensor ATF6α is regulated by its redox-dependent dimerization and ER retention by ERp18.
Structure of the BAK-activating antibody 7D10 bound to BAK reveals an unexpected role for the α1-α2 loop in BAK activation.
Deletion of Gdf15 reduces ER stress-induced beta cell apoptosis and diabetes.
Temporal and spatial topography of cell proliferation in cancer.

Cell Cycle. 2022 Mar 17. 1-16

POLG mutations lead to abnormal mitochondrial remodeling during neural differentiation of human pluripotent stem cells via SIRT3/AMPK pathway inhibition.

Anbin Chen, Cecilie Katrin Kristiansen, Lena Elise Høyland, Mathias Ziegler, Jian Wang, Gareth John Sullivan, Xingang Li, Laurence A Bindoff, Kristina Xiao Liang.

  We showed previously that POLG mutations cause major changes in mitochondrial function, including loss of mitochondrial respiratory chain (MRC) complex I, mitochondrial DNA (mtDNA) depletion and an abnormal NAD+/NADH ratio in both neural stem cells (NSCs) and astrocytes differentiated from induced pluripotent stem cells (iPSCs). In the current study, we looked at mitochondrial remodeling as stem cells transit pluripotency and during differentiation from NSCs to both dopaminergic (DA) neurons and astrocytes comparing the process in POLG-mutated and control stem cells. We saw that mitochondrial membrane potential (MMP), mitochondrial volume, ATP production and reactive oxygen species (ROS) changed in similar ways in POLG and control NSCs, but mtDNA replication, MRC complex I and NAD+ metabolism failed to remodel normally. In DA neurons differentiated from NSCs, we saw that POLG mutations caused failure to increase MMP and ATP production and blunted the increase in mtDNA and complex I. Interestingly, mitochondrial remodeling during astrocyte differentiation from NSCs was similar in both POLG-mutated and control NSCs. Further, we showed downregulation of the SIRT3/AMPK pathways in POLG-mutated cells, suggesting that POLG mutations lead to abnormal mitochondrial remodeling in early neural development due to the downregulation of these pathways. [Figure: see text].

Keywords:  DA neurons; Mitochondrial remodeling; NSCs; POLG; astrocytes; iPSCs

DOI:  https://doi.org/10.1080/15384101.2022.2044136
Trends Biochem Sci. 2022 Mar 12. pii: S0968-0004(22)00065-2. [Epub ahead of print]

TCA cycle off, ATF4 on for metabolic homeostasis.

Durga Mahor, Rishikesh Pandey, Vinay Bulusu.

  Tricarboxylic acid (TCA) cycle is a major hub for catabolic and anabolic reactions, yet cellular metabolic adaptations following its inhibition are largely unknown. Using multi-tiered omics approaches, Ryan et al. have shown convergent activation of the integrated stress response (ISR) through ATF4-mediated rewiring of cellular amino acid and redox metabolic pathways.

Keywords:  ATF4; TCA cycle; fumarate hydratase; glutathione synthesis; integrated stress response; succinate dehydrogenase

DOI:  https://doi.org/10.1016/j.tibs.2022.03.006
Cell Stress Chaperones. 2022 Mar 16.

Endoplasmic reticulum-unfolded protein response pathway modulates the cellular response to mitochondrial proteotoxic stress.

Rajasri Sarkar, Kannan Boosi Narayana Rao, Mainak Pratim Jha, Koyeli Mapa.

  Mitochondria and endoplasmic reticulum (ER) remain closely tethered by contact sites to maintain unhindered biosynthetic, metabolic, and signalling functions. Apart from its constituent proteins, contact sites localize ER-unfolded protein response (UPR) sensors like Ire1 and PERK, indicating the importance of ER-mitochondria communication during stress. In the mitochondrial sub-compartment-specific proteotoxic model of yeast, Saccharomyces cerevisiae, we show that an intact ER-UPR pathway is important in stress tolerance of mitochondrial intermembrane space (IMS) proteotoxic stress, while disrupting the pathway is beneficial during matrix stress. Deletion of IRE1 and HAC1 leads to accumulation of misfolding-prone proteins in mitochondrial IMS indicating the importance of intact ER-UPR pathway in enduring mitochondrial IMS proteotoxic stresses. Although localized proteotoxic stress within mitochondrial IMS does not induce ER-UPR, its artificial activation helps cells to better withstand the IMS proteotoxicity. Furthermore, overexpression of individual components of ER-mitochondria contact sites is found to be beneficial for general mitochondrial proteotoxic stress, in an Ire1-Hac1-independent manner.

Keywords:  ER stress; ER-mitochondria contact sites; Mito-UPR; Protein homeostasis; Proteotoxic stress; Unfolded protein response

DOI:  https://doi.org/10.1007/s12192-022-01264-2
Cell Death Dis. 2022 Mar 16. 13(3): 241

Lon upregulation contributes to cisplatin resistance by triggering NCLX-mediated mitochondrial Ca2+ release in cancer cells.

Vidhya Tangeda, Yu Kang Lo, Ananth Ponneri Babuharisankar, Han-Yu Chou, Cheng-Liang Kuo, Yung-Hsi Kao, Alan Yueh-Luen Lee, Jang-Yang Chang.

Mitochondria are the major organelles in sensing cellular stress and inducing the response for cell survival. Mitochondrial Lon has been identified as an important stress protein involved in regulating proliferation, metastasis, and apoptosis in cancer cells. However, the mechanism of retrograde signaling by Lon on mitochondrial DNA (mtDNA) damage remains to be elucidated. Here we report the role of Lon in the response to cisplatin-induced mtDNA damage and oxidative stress, which confers cancer cells on cisplatin resistance via modulating calcium levels in mitochondria and cytosol. First, we found that cisplatin treatment on oral cancer cells caused oxidative damage of mtDNA and induced Lon expression. Lon overexpression in cancer cells decreased while Lon knockdown sensitized the cytotoxicity towards cisplatin treatment. We further identified that cisplatin-induced Lon activates the PYK2-SRC-STAT3 pathway to stimulate Bcl-2 and IL-6 expression, leading to the cytotoxicity resistance to cisplatin. Intriguingly, we found that activation of this pathway is through an increase of intracellular calcium (Ca2+) via NCLX, a mitochondrial Na+/Ca2+ exchanger. We then verified that NCLX expression is dependent on Lon levels; Lon interacts with and activates NCLX activity. NCLX inhibition increased the level of mitochondrial calcium and sensitized the cytotoxicity to cisplatin in vitro and in vivo. In summary, mitochondrial Lon-induced cisplatin resistance is mediated by calcium release into cytosol through NCLX, which activates calcium-dependent PYK2-SRC-STAT3-IL-6 pathway. Thus, our work uncovers the novel retrograde signaling by mitochondrial Lon on resistance to cisplatin-induced mtDNA stress, indicating the potential use of Lon and NCLX inhibitors for better clinical outcomes in chemoresistant cancer patients.

DOI: https://doi.org/10.1038/s41419-022-04668-1
J Biol Chem. 2022 Mar 15. pii: S0021-9258(22)00275-7. [Epub ahead of print] 101835

Disruption of Mitochondrial Quality Control Genes Promotes Caspase-Resistant Cell Survival Following Apoptotic Stimuli.

Yulia Kushnareva, Vivian Moraes, Julian Suess, Bjoern Peters, Donald D Newmeyer, Tomomi Kuwana.

  In cells undergoing cell-intrinsic apoptosis, mitochondrial outer membrane permeabilization (MOMP) typically marks an irreversible step in the cell death process. However, in some cases a subpopulation of treated cells can exhibit a sublethal response, termed "minority MOMP". In this phenomenon, the affected cells survive, despite a low level of caspase activation and subsequent limited activation of the endonuclease CAD (DFFB). Consequently, these cells can experience DNA damage, increasing the probability of oncogenesis. However, little is known about the minority MOMP response. To discover genes that affect the MOMP response in individual cells, we conducted an imaging-based phenotypic siRNA screen. We identified multiple candidate genes whose downregulation increased the heterogeneity of MOMP within single cells, among which were genes related to mitochondrial dynamics and mitophagy that participate in the mitochondrial quality control (MQC) system. Furthermore, to test the hypothesis that functional MQC is important for reducing the frequency of minority MOMP, we developed an assay to measure the clonogenic survival of caspase-engaged cells. We found that cells deficient in various MQC genes were indeed prone to aberrant post-MOMP survival. Our data highlight the important role of proteins involved in mitochondrial dynamics and mitophagy in preventing apoptotic dysregulation and oncogenesis.

Keywords:  apoptosis; mitochondrial dynamics; mitochondrial heterogeneity; mitochondrial outer membrane permeabilization; mitochondrial quality control; mitophagy; oncogenesis; siRNA screen

DOI:  https://doi.org/10.1016/j.jbc.2022.101835
Exp Eye Res. 2022 Mar 10. pii: S0014-4835(22)00094-X. [Epub ahead of print] 109013

Differential mitochondrial and cellular responses between H vs. J mtDNA haplogroup-containing human RPE transmitochondrial cybrid cells.

Ana Rubin Panvini, Anzor Gvritishvili, Hannah Galvan, Sonali Nashine, Shari R Atilano, M Cristina Kenney, Joyce Tombran-Tink.

  Mitochondrial dysfunction is associated with several retinal degenerative diseases including Age-related Macular Degeneration (AMD). Human mitochondrial DNA (mtDNA) haplogroups are inherited from a common ancestral clan and are defined by specific sets of genetic differences. The purpose of this study was to determine and compare the effects of mtDNA haplogroups H and J on transcriptome regulation and cellular resilience to oxidative stress in human RPE cytoplasmic hybrid (cybrid) cell lines in vitro. ARPE-19 cybrid cell lines containing mtDNA haplogroups H and J were created by fusing platelets obtained from normal individuals containing H and J haplogroups with mitochondria-deficient (Rho0) ARPE-19 cell lines. These cybrids were exposed to oxidative stress using 300 μM hydrogen peroxide (H2O2), following which mitochondrial structural dynamics was studied at varying time points using the mitochondrial markers - TOMM20 (Translocase of Outer Mitochondrial Membrane 20) and Mitotracker. To evaluate mitochondrial function, levels of ROS, ΔΨm and [Ca2+]m were measured using flow cytometry, and ATP levels were measured using luminescence. The H and J cybrid cell transcriptomes were compared using RNAseq to determine how changes in mtDNA regulate gene expression. Inflammatory and angiogenic markers were measured using Luminex assay to understand how these mtDNAs influenced cellular response to oxidative stress. Actin filaments' morphology was examined using confocal microscopy. Following exposure to H2O2 stress, the J cybrids showed increased mitochondrial swelling and perinuclear localization, disturbed fission and fusion, increased calcium uptake (p < 0.05), and higher secreted levels of TNF-α and VEGF (p < 0.001), compared to the H cybrids. Calcium uptake by J cybrids was reduced using an IP3R inhibitor. Thirteen genes involved in mitochondrial complex I and V function, fusion/fission events cellular energy homeostasis, antioxidant defenses, and inflammatory responses, were significantly downregulated with log2 fold changes ranging between -1.5 and -5.1. Actin levels were also significantly reduced in stressed J cybrids (p ≤ 0.001) and disruption in actin filaments was observed. Thirty-eight genes involved in mitochondrial and cellular support functions, were upregulated with log2 fold changes of +1.5 to +5.9 in J cybrids compared to H cybrids. Our results demonstrate significant structural and functional differences between mtDNA haplogroups H and J -containing cybrid cells. Our study suggests that the J mtDNA haplogroup can alter the transcriptome to increase cellular susceptibility to stress and retinal degenerations.

Keywords:  AMD; Age-related macular degeneration; Mitochondrial DNA haplogroups; mtDNA H haplogroup; mtDNA J haplogroup

DOI:  https://doi.org/10.1016/j.exer.2022.109013
Autophagy. 2022 Mar 16. 1-15

Mitochondrial depolarization after acute ethanol treatment drives mitophagy in living mice.

Devadoss J Samuvel, Li Li, Yasodha Krishnasamy, Monika Gooz, Kenji Takemoto, Patrick M Woster, John J Lemasters, Zhi Zhong.

  Ethanol increases hepatic mitophagy driven by unknown mechanisms. Type 1 mitophagy sequesters polarized mitochondria for nutrient recovery and cytoplasmic remodeling. In Type 2, mitochondrial depolarization (mtDepo) initiates mitophagy to remove the damaged organelles. Previously, we showed that acute ethanol administration produces reversible hepatic mtDepo. Here, we tested the hypothesis that ethanol-induced mtDepo initiates Type 2 mitophagy. GFP-LC3 transgenic mice were gavaged with ethanol (2-6 g/kg) with and without pre-treatment with agents that decrease or increase mtDepo-Alda-1, tacrolimus, or disulfiram. Without ethanol, virtually all hepatocytes contained polarized mitochondria with infrequent autophagic GFP-LC3 puncta visualized by intravital microscopy. At ~4 h after ethanol treatment, mtDepo occurred in an all-or-none fashion within individual hepatocytes, which increased dose dependently. GFP-LC3 puncta increased in parallel, predominantly in hepatocytes with mtDepo. Mitochondrial PINK1 and PRKN/parkin also increased. After covalent labeling of mitochondria with MitoTracker Red (MTR), GFP-LC3 puncta encircled MTR-labeled mitochondria after ethanol treatment, directly demonstrating mitophagy. GFP-LC3 puncta did not associate with fat droplets visualized with BODIPY558/568, indicating that increased autophagy was not due to lipophagy. Before ethanol administration, rhodamine-dextran (RhDex)-labeled lysosomes showed little association with GFP-LC3. After ethanol treatment, TFEB (transcription factor EB) translocated to nuclei, and lysosomal mass increased. Many GFP-LC3 puncta merged with RhDex-labeled lysosomes, showing autophagosomal processing into lysosomes. After ethanol treatment, disulfiram increased, whereas Alda-1 and tacrolimus decreased mtDepo, and mitophagy changed proportionately. In conclusion, mtDepo after acute ethanol treatment induces mitophagic sequestration and subsequent lysosomal processing.Abbreviations : AcAld, acetaldehyde; ADH, alcohol dehydrogenase; ALDH, aldehyde dehydrogenase; ALD, alcoholic liver disease; Alda-1, N-(1,3-benzodioxol-5-ylmethyl)-2,6-dichlorobenzamide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; LAMP1, lysosomal-associated membrane protein 1; LMNB1, lamin B1; MAA, malondialdehyde-acetaldehyde adducts; MAP1LC3/LC3, microtubule-associated protein 1 light chain 3; MPT, mitochondrial permeability transition; mtDAMPS, mitochondrial damage-associated molecular patterns; mtDepo, mitochondrial depolarization; mtDNA, mitochondrial DNA; MTR, MitoTracker Red; PI, propidium iodide; PINK1, PTEN induced putative kinase 1; PRKN, parkin; RhDex, rhodamine dextran; TFEB, transcription factor EB; Tg, transgenic; TMRM, tetramethylrhodamine methylester; TOMM20, translocase of outer mitochondrial membrane 20; VDAC, voltage-dependent anion channel.

Keywords:  Acetaldehyde; Alda-1; alcoholic liver disease; mitochondrial depolarization; mitophagy; tacrolimus

DOI:  https://doi.org/10.1080/15548627.2022.2046457
Mol Cancer Res. 2022 Mar 17. pii: molcanres.0027.2022. [Epub ahead of print]

Mitochondrial Dysfunction is a Driver of SP-2509 Drug Resistance in Ewing Sarcoma.

E John Tokarsky, Jesse C Crow, Lillian M Guenther, John Sherman, Cenny Taslim, Gabriela Alexe, Kathleen I Pishas, Galen Rask, Blake S Justis, Ana Kasumova, Kimberly Stegmaier, Stephen L Lessnick, Emily R Theisen.

Expression of the fusion oncoprotein EWS/FLI causes Ewing sarcoma, an aggressive pediatric tumor characterized by widespread epigenetic deregulation. These epigenetic changes are targeted by novel lysine specific demethylase-1 (LSD1) inhibitors, which are currently in early phase clinical trials. Single agent targeted therapy often induces resistance, and successful clinical development requires knowledge of resistance mechanisms, enabling the design of effective combination strategies. Here, we used a genome-scale CRISPR-Cas9 loss-of-function screen to identify genes whose knockout (KO) conferred resistance to the LSD1 inhibitor SP-2509 in Ewing sarcoma cell lines. Multiple genes required for mitochondrial electron transport chain (ETC) complexes III and IV function were hits in our screen. We validated this finding using genetic and chemical approaches including CRISPR KO, ETC inhibitors, and mitochondrial depletion. Further global transcriptional profiling revealed that altered complex III/IV function disrupted the oncogenic program mediated by EWS/FLI and LSD1 and blunted the transcriptomic response to SP-2509. Implications: These findings demonstrate that mitochondrial dysfunction modulates SP-2509 efficacy and suggest that new therapeutic strategies combining LSD1 with agents which prevent mitochondrial dysfunction may benefit patients with this aggressive malignancy.

DOI: https://doi.org/10.1158/1541-7786.MCR-22-0027
Front Mol Neurosci. 2022 ;15 852243

Type I Interferon Response Is Mediated by NLRX1-cGAS-STING Signaling in Brain Injury.

Lauren E Fritsch, Jing Ju, Erwin Kristobal Gudenschwager Basso, Eman Soliman, Swagatika Paul, Jiang Chen, Alexandra M Kaloss, Elizabeth A Kowalski, Taylor C Tuhy, Rachana Deven Somaiya, Xia Wang, Irving Coy Allen, Michelle H Theus, Alicia M Pickrell.

   Background: Inflammation is a significant contributor to neuronal death and dysfunction following traumatic brain injury (TBI). Recent evidence suggests that interferons may be a key regulator of this response. Our studies evaluated the role of the Cyclic GMP-AMP Synthase-Stimulator of Interferon Genes (cGAS-STING) signaling pathway in a murine model of TBI.
Methods: Male, 8-week old wildtype, STING knockout (-/-), cGAS -/-, and NLRX1 -/- mice were subjected to controlled cortical impact (CCI) or sham injury. Histopathological evaluation of tissue damage was assessed using non-biased stereology, which was complemented by analysis at the mRNA and protein level using qPCR and western blot analysis, respectively.
Results: We found that STING and Type I interferon-stimulated genes were upregulated after CCI injury in a bi-phasic manner and that loss of cGAS or STING conferred neuroprotection concomitant with a blunted inflammatory response at 24 h post-injury. cGAS -/- animals showed reduced motor deficits 4 days after injury (dpi), and amelioration of tissue damage was seen in both groups of mice up to 14 dpi. Given that cGAS requires a cytosolic damage- or pathogen-associated molecular pattern (DAMP/PAMP) to prompt downstream STING signaling, we further demonstrate that mitochondrial DNA is present in the cytosol after TBI as one possible trigger for this pathway. Recent reports suggest that the immune modulator NLR containing X1 (NLRX1) may sequester STING during viral infection. Our findings show that NLRX1 may be an additional regulator that functions upstream to regulate the cGAS-STING pathway in the brain.
Conclusions: These findings suggest that the canonical cGAS-STING-mediated Type I interferon signaling axis is a critical component of neural tissue damage following TBI and that mtDNA may be a possible trigger in this response.

Keywords:  STING; brain injury; cGAS; inflammation; innate immunity

DOI:  https://doi.org/10.3389/fnmol.2022.852243
J Cancer. 2022 ;13(4): 1261-1271

Neutrophil extracellular traps in hepatocellular carcinoma are enriched in oxidized mitochondrial DNA which is highly pro-inflammatory and pro-metastatic.

Lu-Yu Yang, Xiao-Tian Shen, Hao-Ting Sun, Wen-Wei Zhu, Ju-Bo Zhang, Lu Lu.

  Background: Neutrophil extracellular traps (NETs) are net like extracellular structure formed by neutrophils in response to certain stimulation. It works as inflammatory regulator and metastasis promoter in cancer. Mitochondrial-(mt)DNA is a circular, mitochondria derived double strain molecule, which is involved in NETs formation. Its role in NETs induced inflammatory alteration in hepatocellular carcinoma (HCC) remained unexplored. Method: We evaluated the mitochondrial reactive oxygen species (mitoROS) level in peripheral neutrophils from HCC patients and the oxidative level of mtDNA in derived NETs. The association between the NETs and oxidized mtDNA was assessed to reveal their relevance. A function assay was applied to uncover how the oxidation state of mtDNA directed the metastasis promoting inflammation state in HCC cells in a NETs protein dependent manner. Finally, using animal models, we explored the potential of a therapy strategy against NETs-drove metastasis by targeting the oxidized mtDNA with metformin. Results: Neutrophils in HCC patients contained high level of mitoROS level, and formed NETs that were enriched in oxidized mtDNA in a mitoROS dependent manner. NETs and oxidized mtDNA were clinically relevant. Bound with NETs protein, oxidized mtDNA is more capable of triggering the metastasis-promoting inflammatory mediators in HepG2 cells. Targeting the oxidized mtDNA with metformin attenuated the metastasis-promoting inflammatory state and hereby undermine the metastasis capacity of HCC. Conclusion: HCC is capable to stimulate NETs enriched in oxidized mtDNA, which are highly pro-inflammatory and pro-metastatic. Oxidized mtDNA in NETs may serve as a potential anti-metastatic target by metformin therapy.

Keywords:  Neutrophil extracellular traps; hepatocellular carcinoma; metastasis; oxidized mitochondrial DNA

DOI:  https://doi.org/10.7150/jca.64170
Methods Mol Biol. 2022 ;2474 11-19

Mitochondrial Membrane Potential Assay.

Srilatha Sakamuru, Jinghua Zhao, Matias S Attene-Ramos, Menghang Xia.

  Mitochondrial function, a key indicator of cell health, can be assessed through monitoring changes in mitochondrial membrane potential (MMP). Cationic fluorescent dyes are commonly used tools to assess MMP. We used a water-soluble mitochondrial membrane potential indicator (m-MPI) to detect changes in MMP in various types of cells, such as HepG2, HepaRG, and AC16 cells. A homogenous cell-based MMP assay has been optimized and performed in a 1536-well plate format, which can be used to screen several compound libraries for mitochondrial toxicity by evaluating the effects of chemical compounds on MMP.

Keywords:  1536-well plate; Mesoxalonitrile 4-trifluoromethoxyphenylhydrazone (FCCP); Mitochondrial membrane potential (MMP); Mitochondrial membrane potential indicator (m-MPI); Mitochondrial toxicity

DOI:  https://doi.org/10.1007/978-1-0716-2213-1_2
Sci Signal. 2022 Mar 15. 15(725): eabh1121

DNA-PKcs interacts with and phosphorylates Fis1 to induce mitochondrial fragmentation in tubular cells during acute kidney injury.

Shiyuan Wang, Hang Zhu, Ruibing Li, David Mui, Sam Toan, Xing Chang, Hao Zhou.

The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) regulates cell death. We sought to determine whether DNA-PKcs played a role in the tubular damage that occurs during acute kidney injury (AKI) induced by LPS injection (to mimic sepsis), cisplatin administration, or renal ischemia/reperfusion injury. Although DNA-PKcs normally localizes to the nucleus, we detected cytoplasmic DNA-PKcs in mouse kidney tissues and urinary sediments of human patients with septic AKI. Increased cytoplasmic amounts of DNA-PKcs correlated with renal dysfunction. Tubule cell-specific DNA-PKcs deletion attenuated AKI-mediated tubular cell death and changes in the abundance of various proteins with mitochondrial functions or roles in apoptotic pathways. DNA-PKcs interacted with Fis1 and phosphorylated it at Thr34 in its TQ motif, which increased the affinity of Fis1 for Drp1 and induced mitochondrial fragmentation. Knockin mice expressing a nonphosphorylatable T34A mutant exhibited improved renal function and histological features and reduced mitochondrial fragmentation upon induction of AKI. Phosphorylation of Thr34 in Fis1 was detectable in urinary sediments of human patients with septic AKI and correlated with renal dysfunction. Our findings provide insight into the role of cytoplasmic DNA-PKcs and phosphorylated Fis1 in AKI development.

DOI: https://doi.org/10.1126/scisignal.abh1121
Chem Biol Interact. 2022 Mar 15. pii: S0009-2797(22)00089-8. [Epub ahead of print] 109884

PINK1/Parkin-mediated mitophagy is activated to protect against AFB1-induced kidney damage in mice.

Yuping Wang, Miao Song, Qi Wang, Chen Guo, Jian Zhang, Xuliang Zhang, Yilong Cui, Zheng Cao, Yanfei Li.

  Aflatoxin B1 (AFB1) is a toxic food pollutant that has extensive deleterious impacts on the kidney. Oxidative stress represents the primary mechanism of AFB1 nephrotoxicity and can also cause mitochondrial damage. Damaged mitochondria can trigger apoptosis leading to kidney injury. PINK1/Parkin-mediated mitophagy can alleviate mitochondrial injury to maintain cellular homeostasis, however, its role in AFB1-induced kidney damage is unknown. To investigate the effect of PINK1/Parkin-mediated mitophagy on kidney impairment triggered by AFB1, 40 male wild-type (WT) C57BL/6N mice were first assigned to 4 groups and orally exposed to AFB1 at 0, 0.5, 0.75, and 1 mg/kg body weight (BW) for 28 days. The results revealed that AFB1 induced kidney damage, oxidative stress, mitochondrial damage, apoptosis and activated PINK1/Parkin-mediated mitophagy with a dose-dependent effect. Then, 20 male WT C57BL/6N mice and 20 male Parkin knockout (Parkin-/-) C57BL/6N mice were assigned to 4 groups and orally exposed to AFB1 at 0, 1, 0, and 1 mg/kg BW for 28 days. The results revealed that Parkin-/- suppressed mitophagy and exacerbated kidney damage, oxidative stress, mitochondrial damage, and apoptosis under AFB1 exposure. The aforementioned evidences demonstrate that PINK1/Parkin-mediated mitophagy is activated by AFB1 and protects against kidney damage in mice.

Keywords:  Aflatoxin B(1); Apoptosis; Kidney damage; Mitophagy; Oxidative stress; Parkin

DOI:  https://doi.org/10.1016/j.cbi.2022.109884
Front Mol Biosci. 2022 ;9 791094

Selective Cleavage at CCA Ends and Anticodon Loops of tRNAs by Stress-Induced RNases.

Yasutoshi Akiyama, Shawn M Lyons, Marta M Fay, Yoshihisa Tomioka, Takaaki Abe, Paul J Anderson, Pavel Ivanov.

  Stress-induced tRNA cleavage has been implicated in various cellular processes, where tRNA fragments play diverse regulatory roles. Angiogenin (ANG), a member of the RNase A superfamily, induces cleavage of tRNAs resulting in the formation of tRNA-derived stress-induced RNAs (tiRNAs) that contribute to translational reprogramming aiming at cell survival. In addition to cleaving tRNA anticodon loops, ANG has been shown to cleave 3'-CCA termini of tRNAs in vitro, although it is not known whether this process occurs in cells. It has also been suggested that tiRNAs can be generated independently of ANG, although the role of other stress-induced RNases in tRNA cleavage is poorly understood. Using gene editing and biochemical approaches, we examined the involvement of ANG in stress-induced tRNA cleavage by focusing on its cleavage of CCA-termini as well as anticodon loops. We show that ANG is not responsible for CCA-deactivation under sodium arsenite (SA) treatment in cellulo, and although ANG treatment significantly increases 3'-tiRNA levels in cells, the majority of 3'-tiRNAs retain their 3'-CCA termini. Instead, other RNases can cleave CCA-termini in cells, although with low efficiency. Moreover, in the absence of ANG, other RNases are able to promote the production of tiRNAs in cells. Depletion of RNH1 (an endogenous inhibitor of RNase A superfamily) promotes constitutively-produced tiRNAs and CCA-deactivated tRNAs in cells. Interestingly, SA treatment in RNH1-depleted cells did not increase the amount of tiRNAs or CCA-deactivated tRNAs, suggesting that RNase A superfamily enzymes are largely responsible for SA-induced tRNA cleavage. We show that interplay between stress-induced RNases cause targeting tRNAs in a stress-specific manner in cellulo.

Keywords:  CCA-terminus; RNase A superfamily; angiogenin; stress response; tRNAs

DOI:  https://doi.org/10.3389/fmolb.2022.791094
Proc Natl Acad Sci U S A. 2022 Mar 22. 119(12): e2122657119

Activation of the UPR sensor ATF6α is regulated by its redox-dependent dimerization and ER retention by ERp18.

Ojore Benedict Valentine Oka, Arvin Shedrach Pierre, Marie Anne Pringle, Wanida Tungkum, Zhenbo Cao, Bethany Fleming, Neil John Bulleid.

  SignificanceMembrane and secretory proteins are synthesized in the endoplasmic reticulum (ER). Perturbations to ER function disrupts protein folding, causing misfolded proteins to accumulate, a condition known as ER stress. Cells adapt to stress by activating the unfolded protein response (UPR), which ultimately restores proteostasis. A key player in the UPR response is ATF6α, which requires release from ER retention and modulation of its redox status during activation. Here, we report that ER stress promotes formation of a specific ATF6α dimer, which is preferentially trafficked to the Golgi for processing. We show that ERp18 regulates ATF6α by mitigating its dimerization and trafficking to the Golgi and identify redox-dependent oligomerization of ATF6α as a key mechanism regulating its function during the UPR.

Keywords:  ATF6; ER stress; proteostasis; unfolded protein response

DOI:  https://doi.org/10.1073/pnas.2122657119
Cell Death Differ. 2022 Mar 12.

Structure of the BAK-activating antibody 7D10 bound to BAK reveals an unexpected role for the α1-α2 loop in BAK activation.

Adeline Y Robin, Michelle S Miller, Sweta Iyer, Melissa X Shi, Ahmad Z Wardak, Daisy Lio, Nicholas A Smith, Brian J Smith, Richard W Birkinshaw, Peter E Czabotar, Ruth M Kluck, Peter M Colman.

Pro-apoptotic BAK and BAX are activated by BH3-only proteins to permeabilise the outer mitochondrial membrane. The antibody 7D10 also activates BAK on mitochondria and its epitope has previously been mapped to BAK residues in the loop connecting helices α1 and α2 of BAK. A crystal structure of the complex between the Fv fragment of 7D10 and the BAK mutant L100A suggests a possible mechanism of activation involving the α1-α2 loop residue M60. M60 mutants of BAK have reduced stability and elevated sensitivity to activation by BID, illustrating that M60, through its contacts with residues in helices α1, α5 and α6, is a linchpin stabilising the inert, monomeric structure of BAK. Our data demonstrate that BAK's α1-α2 loop is not a passive covalent connector between secondary structure elements, but a direct restraint on BAK's activation.

DOI: https://doi.org/10.1038/s41418-022-00961-w
Endocrinology. 2022 Mar 15. pii: bqac030. [Epub ahead of print]

Deletion of Gdf15 reduces ER stress-induced beta cell apoptosis and diabetes.

Guanlan Xu, Junqin Chen, Seong Ho Jo, Truman B Grayson, Sasanka Ramanadham, Akio Koizumi, Emily L Germain-Lee, Se-Jin Lee, Anath Shalev.

  Endoplasmic reticulum (ER) stress contributes to pancreatic beta cell apoptosis in diabetes, but the factors involved are still not fully elucidated. Growth differentiation factor 15 (GDF15) is a stress response gene and has been reported to be increased and play an important role in various diseases. However, the role of GDF15 in beta cells in the context of ER stress and diabetes is still unclear. In this study, we have discovered that GDF15 promotes ER stress-induced beta cell apoptosis and that downregulation of GDF15 has beneficial effects on beta cell survival in diabetes. Specifically, we found that GDF15 is induced by ER stress in beta cells and human islets, and that the transcription factor C/EBPβ is involved in this process. Interestingly, ER stress-induced apoptosis was significantly reduced in INS-1 cells with Gdf15 knockdown and in isolated Gdf15 knockout mouse islets. In vivo, we found that Gdf15 deletion attenuates streptozotocin-induced diabetes by preserving beta cells and insulin levels. Moreover, deletion of Gdf15 significantly delayed diabetes development in spontaneous ER stress-prone Akita mice. Thus, our findings suggest that GDF15 contributes to ER stress-induced beta cell apoptosis and that inhibition of GDF15 may represent a novel strategy to promote beta cell survival and treat diabetes.

Keywords:  Beta cells; Diabetes; ER stress; apoptosis and GDF15

DOI:  https://doi.org/10.1210/endocr/bqac030
Nat Cell Biol. 2022 Mar;24(3): 316-326

Temporal and spatial topography of cell proliferation in cancer.

Giorgio Gaglia, Sheheryar Kabraji, Danae Rammos, Yang Dai, Ana Verma, Shu Wang, Caitlin E Mills, Mirra Chung, Johann S Bergholz, Shannon Coy, Jia-Ren Lin, Rinath Jeselsohn, Otto Metzger, Eric P Winer, Deborah A Dillon, Jean J Zhao, Peter K Sorger, Sandro Santagata.

Proliferation is a fundamental trait of cancer cells, but its properties and spatial organization in tumours are poorly characterized. Here we use highly multiplexed tissue imaging to perform single-cell quantification of cell cycle regulators and then develop robust, multivariate, proliferation metrics. Across diverse cancers, proliferative architecture is organized at two spatial scales: large domains, and smaller niches enriched for specific immune lineages. Some tumour cells express cell cycle regulators in the (canonical) patterns expected of freely growing cells, a phenomenon we refer to as 'cell cycle coherence'. By contrast, the cell cycles of other tumour cell populations are skewed towards specific phases or exhibit non-canonical (incoherent) marker combinations. Coherence varies across space, with changes in oncogene activity and therapeutic intervention, and is associated with aggressive tumour behaviour. Thus, multivariate measures from high-plex tissue images capture clinically significant features of cancer proliferation, a fundamental step in enabling more precise use of anti-cancer therapies.

DOI: https://doi.org/10.1038/s41556-022-00860-9