Stress‐induced tRNA cleavage and tiRNA generation in rat neuronal PC12 cells

Transfer RNA (tRNA) plays a role in stress response programs involved in various pathological conditions including neurological diseases. Under cell stress conditions, intracellular tRNA is cleaved by a specific ribonuclease, angiogenin, generating tRNA‐derived fragments or tRNA‐derived stress‐induced RNA (tiRNA). Generated tiRNA contributes to the cell stress response and has potential cell protective effects. However, tiRNA generation under stress conditions in neuronal cells has not been fully elucidated. To examine angiogenin‐mediated tiRNA generation in neuronal cells, we used the rat neuronal cell line, PC12, in combination with analysis of SYBR staining and immuno‐northern blotting using anti‐1‐methyladenosine antibody, which specifically and sensitively detects tiRNA. Oxidative stress induced by arsenite and hydrogen peroxide caused tRNA cleavage and tiRNA generation in PC12 cells. We also demonstrated that oxygen‐glucose deprivation, which is an in vitro model of ischemic–reperfusion injury, induced tRNA cleavage and tiRNA generation. In these stress conditions, the amount of generated tiRNA was associated with the degree of morphological cell damage. Time course analysis indicated that generation of tiRNA was prior to severe cell damage and cell death. Angiogenin over‐expression did not influence the amount of tiRNA in normal culture conditions; however, it significantly increased tiRNA generation induced by cell stress conditions. Our findings show that angiogenin‐mediated tiRNA generation can be induced in neuronal cells by different cell stressors, including ischemia–reperfusion. Additionally, detection of tiRNA could be used as a potential cell damage marker in neuronal cells.

Cover Image for this issue: doi: 10.1111/jnc.14191 .

     

Elp3‐mediated codon‐dependent translation promotes mTORC2 activation and regulates macrophage polarization

Macrophage polarization is a process whereby macrophages acquire distinct effector states (M1 or M2) to carry out multiple and sometimes opposite functions. We show here that translational reprogramming occurs during macrophage polarization and that this relies on the Elongator complex subunit Elp3, an enzyme that modifies the wobble uridine base U34 in cytosolic tRNAs. Elp3 expression is downregulated by classical M1‐activating signals in myeloid cells, where it limits the production of pro‐inflammatory cytokines via FoxO1 phosphorylation, and attenuates experimental colitis in mice. In contrast, alternative M2‐activating signals upregulate Elp3 expression through a PI3K‐ and STAT6‐dependent signaling pathway. The metabolic reprogramming linked to M2 macrophage polarization relies on Elp3 and the translation of multiple candidates, including the mitochondrial ribosome large subunit proteins Mrpl3, Mrpl13, and Mrpl47. By promoting translation of its activator Ric8b in a codon‐dependent manner, Elp3 also regulates mTORC2 activation. Elp3 expression in myeloid cells further promotes Wnt‐driven tumor initiation in the intestine by maintaining a pool of tumor‐associated macrophages exhibiting M2 features. Collectively, our data establish a functional link between tRNA modifications, mTORC2 activation, and macrophage polarization.     

ATAD3B is a mitophagy receptor mediating clearance of oxidative stress‐induced damaged mitochondrial DNA

Mitochondrial DNA (mtDNA) encodes several key components of respiratory chain complexes that produce cellular energy through oxidative phosphorylation. mtDNA is vulnerable to damage under various physiological stresses, especially oxidative stress. mtDNA damage leads to mitochondrial dysfunction, and dysfunctional mitochondria can be removed by mitophagy, an essential process in cellular homeostasis. However, how damaged mtDNA is selectively cleared from the cell, and how damaged mtDNA triggers mitophagy, remain mostly unknown. Here, we identified a novel mitophagy receptor, ATAD3B, which is specifically expressed in primates. ATAD3B contains a LIR motif that binds to LC3 and promotes oxidative stress‐induced mitophagy in a PINK1‐independent manner, thus promoting the clearance of damaged mtDNA induced by oxidative stress. Under normal conditions, ATAD3B hetero‐oligomerizes with ATAD3A, thus promoting the targeting of the C‐terminal region of ATAD3B to the mitochondrial intermembrane space. Oxidative stress‐induced mtDNA damage or mtDNA depletion reduces ATAD3B‐ATAD3A hetero‐oligomerization and leads to exposure of the ATAD3B C‐terminus at the mitochondrial outer membrane and subsequent recruitment of LC3 for initiating mitophagy. Furthermore, ATAD3B is little expressed in m.3243A > G mutated cells and MELAS patient fibroblasts showing endogenous oxidative stress, and ATAD3B re‐expression promotes the clearance of m.3243A > G mutated mtDNA. Our findings uncover a new pathway to selectively remove damaged mtDNA and reveal that increasing ATAD3B activity is a potential therapeutic approach for mitochondrial diseases.     

Inhibition of thioredoxin reductase 1 by caveolin 1 promotes stress‐induced premature senescence

Thioredoxin reductase 1 (TrxR1) is an important antioxidant enzyme that controls cellular redox homeostasis. By using a proteomic‐based approach, here we identify TrxR1 as a caveolar membrane‐resident protein. We show that caveolin 1, the structural protein component of caveolae, is a TrxR1‐binding protein by demonstrating that the scaffolding domain of caveolin 1 (amino acids 82–101) binds directly to the caveolin‐binding motif (CBM) of TrxR1 (amino acids 454–463). We also show that overexpression of caveolin 1 inhibits TrxR activity, whereas a lack of caveolin 1 activates TrxR, both in vitro and in vivo. Expression of a peptide corresponding to the caveolin 1 scaffolding domain is sufficient to inhibit TrxR activity. A TrxR1 mutant lacking the CBM, which fails to localize to caveolae and bind to caveolin 1, is constitutively active and inhibits oxidative‐stress‐mediated activation of the p53/p21^Waf1/Cip1 pathway and induction of premature senescence. Finally, we show that caveolin 1 expression inhibits TrxR1‐mediated cell transformation. Thus, caveolin 1 links free radicals to activation of the p53/p21^Waf1/Cip1 pathway and induction of cellular senescence by acting as an endogenous inhibitor of TrxR1.     

Oxidative stress‐induced attenuation of thrombospondin‐1 expression in primary rat astrocytes

Astrocytes, the major glial population in the central nervous system (CNS), can secrete thrombospondin (TSP)‐1 that plays the role in synaptogenesis and axonal sprouting during CNS development and tissue repair. However, little is known about the regulation of TSP‐1 expression in astrocytes under oxidative stress condition. Here, a hypoxic mimetic reagent, cobalt chloride (CoCl₂), was used to initiate hypoxia‐induced oxidative stress in primary rat astrocytes. CoCl₂ at the concentration range of 0.1–0.5 mM was found to cause no significant cell death in primary rat astrocytes. However, CoCl₂ at 0.2–0.5 mM increased intracellular reactive oxygen species (ROS) levels and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) gene expression that is known as a hallmark for oxidative damage. We further found that TSP‐1 mRNA expression in astrocytes was inhibited dose‐ and time‐dependently by CoCl₂. TSP‐1 mRNA levels were increased in CoCl₂‐exposed astrocytes in the presence of the inhibitors (U0126 and PD98059) of mitogen‐activated protein kinase/extracellular signal‐regulated kinases (MAPK/ERK), when compared to that detected in the culture only exposed to CoCl₂. Moreover, the inhibition in TSP‐1 mRNA expression by CoCl₂ was blocked by the addition of the potent antioxidant, N‐acetylcysteine (NAC). Thus, we conclude that CoCl₂ inhibits TSP‐1 mRNA expression in astrocytes via a ROS mechanism possibly involving MAPK/ERK. This inhibition may occur after CNS injury and impair the supportive function of astrocytes on neurite growth in the injured CNS tissues. J. Cell. Biochem. 112: 59–70, 2011. © 2010 Wiley‐Liss, Inc.     

Oxidative stress‐induced phosphorylation of JIP4 regulates lysosomal positioning in coordination with TRPML1 and ALG2

Retrograde transport of lysosomes is recognised as a critical autophagy regulator. Here, we found that acrolein, an aldehyde that is significantly elevated in Parkinson''s disease patient serum, enhances autophagy by promoting lysosomal clustering around the microtubule organising centre via a newly identified JIP4‐TRPML1‐ALG2 pathway. Phosphorylation of JIP4 at T217 by CaMK2G in response to Ca²⁺ fluxes tightly regulated this system. Increased vulnerability of JIP4 KO cells to acrolein indicated that lysosomal clustering and subsequent autophagy activation served as defence mechanisms against cytotoxicity of acrolein itself. Furthermore, the JIP4‐TRPML1‐ALG2 pathway was also activated by H₂O₂, indicating that this system acts as a broad mechanism of the oxidative stress response. Conversely, starvation‐induced lysosomal retrograde transport involved both the TMEM55B‐JIP4 and TRPML1‐ALG2 pathways in the absence of the JIP4 phosphorylation. Therefore, the phosphorylation status of JIP4 acts as a switch that controls the signalling pathways of lysosoma l distribution depending on the type of autophagy‐inducing signal.     

Cancer‐secreted miRNAs regulate amino‐acid‐induced mTORC1 signaling and fibroblast protein synthesis

Metabolic reprogramming of non‐cancer cells residing in a tumor microenvironment, as a result of the adaptations to cancer‐derived metabolic and non‐metabolic factors, is an emerging aspect of cancer–host interaction. We show that in normal and cancer‐associated fibroblasts, breast cancer‐secreted extracellular vesicles suppress mTOR signaling upon amino acid stimulation to globally reduce mRNA translation. This is through delivery of cancer‐derived miR‐105 and miR‐204, which target RAGC, a component of Rag GTPases that regulate mTORC1 signaling. Following amino acid starvation and subsequent re‐feeding, ¹³C‐arginine labeling of de novo synthesized proteins shows selective translation of proteins that cluster to specific cellular functional pathways. The repertoire of these newly synthesized proteins is altered in fibroblasts treated with cancer‐derived extracellular vesicles, in addition to the overall suppressed protein synthesis. In human breast tumors, RAGC protein levels are inversely correlated with miR‐105 in the stroma. Our results suggest that through educating fibroblasts to reduce and re‐prioritize mRNA translation, cancer cells rewire the metabolic fluxes of amino acid pool and dynamically regulate stroma‐produced proteins during periodic nutrient fluctuations.     

Young transgenic hMTH1 mice are protected against dietary fat‐induced metabolic stress—implications for enhanced longevity

hMTH1 protects against mutation during oxidative stress. It degrades 8‐oxodGTP to exclude potentially mutagenic oxidized guanine from DNA. hMTH1 expression is linked to ageing. Its downregulation in cultured cells accelerates RAS‐induced senescence, and its overexpression in hMTH1‐Tg mice extends lifespan. In this study, we analysed the effects of a brief (5 weeks) high‐fat diet challenge (HFD) in young (2 months old) and adult (7 months old) wild‐type (WT) and hMTH1‐Tg mice. We report that at 2 months, hMTH1 overexpression ameliorated HFD‐induced weight gain, changes in liver metabolism related to mitochondrial dysfunction and oxidative stress. It prevented DNA damage as quantified by a comet assay. At 7 months old, these HFD‐induced effects were less severe and hMTH1‐Tg and WT mice responded similarly. hMTH1 overexpression conferred lifelong protection against micronucleus induction, however. Since the canonical activity of hMTH1 is mutation prevention, we conclude that hMTH1 protects young mice against HFD by reducing genome instability during the early period of rapid growth and maximal gene expression. hMTH1 protection is redundant in the largely non‐growing, differentiated tissues of adult mice. In hMTH1‐Tg mice, expression of a less heavily mutated genome throughout life provides a plausible explanation for their extended longevity.     

MEK/ERK pathway mediates cytoprotection of salvianolic acid B against oxidative stress‐induced apoptosis in rat bone marrow stem cells

To improve the survival and/or differentiation of grafted BMSCs (bone marrow stem cells) represents one of the challenges for the promising cell‐based therapy. Considerable reports have implicated Sal B (salvianolic acid B), a potent aqueous extract of Salvia miltiorrhiza, in enhancing the survival of cells under various conditions. In this study, we investigated the effect of Sal B on H₂O₂‐induced apoptosis in rat BMSCs, focusing on the survival signalling pathways. Results indicated that the MEK [MAPK (mitogen‐activated protein kinase)/ERK (extracellular‐signal‐regulated kinase) kinase] inhibitor (PD98059) and 10 μM Sal B remarkably prevented BMSCs from H₂O₂‐induced apoptosis through attenuating caspase‐3 activation, which is accompanied by the significant up‐regulation of Bcl‐2. In addition, the ROS (reactive oxygen species) accumulation was also reduced after Sal B treatment. Furthermore, Sal B inhibited the ERK1/2 phosphorylations stimulated by H₂O₂. Taken together, our results showed that H₂O₂‐induced apoptosis in BMSCs via the ROS/MEK/ERK1/2 pathway and Sal B may exert its cytoprotection through mediating the pathway.     

Tat‐antioxidant 1 protects against stress‐induced hippocampal HT‐22 cells death and attenuate ischaemic insult in animal model

Oxidative stress‐induced reactive oxygen species (ROS) are responsible for various neuronal diseases. Antioxidant 1 (Atox1) regulates copper homoeostasis and promotes cellular antioxidant defence against toxins generated by ROS. The roles of Atox1 protein in ischaemia, however, remain unclear. In this study, we generated a protein transduction domain fused Tat‐Atox1 and examined the roles of Tat‐Atox1 in oxidative stress‐induced hippocampal HT‐22 cell death and an ischaemic injury animal model. Tat‐Atox1 effectively transduced into HT‐22 cells and it protected cells against the effects of hydrogen peroxide (H₂O₂)‐induced toxicity including increasing of ROS levels and DNA fragmentation. At the same time, Tat‐Atox1 regulated cellular survival signalling such as p53, Bad/Bcl‐2, Akt and mitogen‐activate protein kinases (MAPKs). In the animal ischaemia model, transduced Tat‐Atox1 protected against neuronal cell death in the hippocampal CA1 region. In addition, Tat‐Atox1 significantly decreased the activation of astrocytes and microglia as well as lipid peroxidation in the CA1 region after ischaemic insult. Taken together, these results indicate that transduced Tat‐Atox1 protects against oxidative stress‐induced HT‐22 cell death and against neuronal damage in animal ischaemia model. Therefore, we suggest that Tat‐Atox1 has potential as a therapeutic agent for the treatment of oxidative stress‐induced ischaemic damage.     

Fumonisin B1 actuates oxidative stress‐associated colonic damage via apoptosis and autophagy activation in murine model

In the present study, we investigated the cytotoxic mechanism of Fumonisin B1 (FB1) in mice colonic region in a time course manner. Herein, after consecutive 4 days of exposure to FBI (2.5 mg/kg body weight), we observed disintegration of mice colon, as evidenced by histopathological analysis. FB1 significantly increased alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase activities in serum and plasma, decreased ceramide level, increased sphinganine level, and increased lipid peroxidase level along with the breakdown of the antioxidant system. Further, FB1‐induced ER stress caused apoptosis and autophagy activation in mice colon, evidenced by increased expression of IRE1‐α, p‐JNK, Casp3, and LC3I/II. In addition, we also noticed a reduced protein kinase C expression in mice colon exposed to FB1, suggesting its role in ER stress‐induced cell death. Taken together, study suggests both physiologically and biochemically, FB1 toxicity to mice colon induced by oxidative stress‐associated apoptosis and autophagy activation.     

Monoamine oxidase‐A is a novel driver of stress‐induced premature senescence through inhibition of parkin‐mediated mitophagy

Cellular senescence, the irreversible cell cycle arrest observed in somatic cells, is an important driver of age‐associated diseases. Mitochondria have been implicated in the process of senescence, primarily because they are both sources and targets of reactive oxygen species (ROS). In the heart, oxidative stress contributes to pathological cardiac ageing, but the mechanisms underlying ROS production are still not completely understood. The mitochondrial enzyme monoamine oxidase‐A (MAO‐A) is a relevant source of ROS in the heart through the formation of H₂O₂ derived from the degradation of its main substrates, norepinephrine (NE) and serotonin. However, the potential link between MAO‐A and senescence has not been previously investigated. Using cardiomyoblasts and primary cardiomyocytes, we demonstrate that chronic MAO‐A activation mediated by synthetic (tyramine) and physiological (NE) substrates induces ROS‐dependent DNA damage response, activation of cyclin‐dependent kinase inhibitors p21^cip, p16^ink4a, and p15^ink4b and typical features of senescence such as cell flattening and SA‐β‐gal activity. Moreover, we observe that ROS produced by MAO‐A lead to the accumulation of p53 in the cytosol where it inhibits parkin, an important regulator of mitophagy, resulting in mitochondrial dysfunction. Additionally, we show that the mTOR kinase contributes to mitophagy dysfunction by enhancing p53 cytoplasmic accumulation. Importantly, restoration of mitophagy, either by overexpression of parkin or inhibition of mTOR, prevents mitochondrial dysfunction and induction of senescence. Altogether, our data demonstrate a novel link between MAO‐A and senescence in cardiomyocytes and provides mechanistic insights into the potential role of MAO‐dependent oxidative stress in age‐related pathologies.     

LINE‐1 hypomethylation induced by reactive oxygen species is mediated via depletion of S‐adenosylmethionine

Whether long interspersed nuclear element‐1 (LINE‐1) hypomethylation induced by reactive oxygen species (ROS) was mediated through the depletion of S‐adenosylmethionine (SAM) was investigated. Bladder cancer (UM‐UC‐3 and TCCSUP) and human kidney (HK‐2) cell lines were exposed to 20 μM H₂O₂ for 72 h to induce oxidative stress. Level of LINE‐1 methylation, SAM and homocysteine (Hcy) was measured in the H₂O₂‐exposed cells. Effects of α‐tocopheryl acetate (TA), N‐acetylcysteine (NAC), methionine, SAM and folic acid on oxidative stress and LINE‐1 methylation in the H₂O₂‐treated cells were explored. Viabilities of cells treated with H₂O₂ were not significantly changed. Intracellular ROS production and protein carbonyl content were significantly increased, but LINE‐1 methylation was significantly decreased in the H₂O₂‐treated cells. LINE‐1 methylation was restored by TA, NAC, methionine, SAM and folic acid. SAM level in H₂O₂‐treated cells was significantly decreased, while total glutathione was significantly increased. SAM level in H₂O₂‐treated cells was restored by NAC, methionine, SAM and folic acid; while, total glutathione level was normalized by TA and NAC. Hcy was significantly decreased in the H₂O₂‐treated cells and subsequently restored by NAC. In conclusion, in bladder cancer and normal kidney cells exposed to H₂O₂, SAM and Hcy were decreased, but total glutathione was increased. Treatments with antioxidants (TA and NAC) and one‐carbon metabolites (SAM, methionine and folic acid) restored these changes. This pioneer finding suggests that exposure of cells to ROS activates glutathione synthesis via the transsulfuration pathway leading to deficiency of Hcy, which consequently causes SAM depletion and eventual hypomethylation of LINE‐1. Copyright © 2015 John Wiley & Sons, Ltd.