Coenzyme Q0 Inhibits NLRP3 Inflammasome Activation through Mitophagy Induction in LPS/ATP-Stimulated Macrophages

Coenzyme Q (CoQ) analogs with a variable number of isoprenoid units have exhibited as anti-inflammatory as well as antioxidant molecules. Using novel quinone derivative CoQ0 (2,3-dimethoxy-5-methyl-1,4-benzoquinone, zero side chain isoprenoid), we studied its molecular activities against LPS/ATP-induced inflammation and redox imbalance in murine RAW264.7 macrophages. CoQ0’s non- or subcytotoxic concentration suppressed the NLRP3 inflammasome and procaspase-1 activation, followed by downregulation of IL1β expression in LPS/ATP-stimulated RAW264.7 macrophages. Similarly, treatment of CoQ0 led to LC3-I/II accumulation and p62/SQSTM1 activation. An increase in the Beclin-1/Bcl-2 ratio and a decrease in the expression of phosphorylated PI3K/AKT, p70 S6 kinase, and mTOR showed that autophagy was activated. Besides, CoQ0 increased Parkin protein to recruit damaged mitochondria and induced mitophagy in LPS/ATP-stimulated RAW264.7 macrophages. CoQ0 inhibited LPS/ATP-stimulated ROS generation in RAW264.7 macrophages. Notably, when LPS/ATP-stimulated RAW264.7 macrophages were treated with CoQ0, Mito-TEMPO (a mitochondrial ROS inhibitor), or N-acetylcysteine (NAC, a ROS inhibitor), there was a significant reduction of LPS/ATP-stimulated NLRP3 inflammasome activation and IL1β expression. Interestingly, treatment with CoQ0 or Mito-TEMPO, but not NAC, significantly increased LPS/ATP-induced LC3-II accumulation indicating that mitophagy plays a key role in the regulation of CoQ0-inhibited NLRP3 inflammasome activation. Nrf2 knockdown significantly decreased IL1β expression in LPS/ATP-stimulated RAW264.7 macrophages suggesting that CoQ0 inhibited ROS-mediated NLRP3 inflammasome activation and IL1β expression was suppressed due to the Nrf2 activation. Hence, this study showed that CoQ0 might be a promising candidate for the therapeutics of inflammatory disorders due to its effective anti-inflammatory as well as antioxidant properties.

Targeting prooxidant MnSOD effect inhibits triple-negative breast cancer (TNBC) progression and M2 macrophage functions under the oncogenic stress

Triple-negative breast cancer (TNBC) has been shown with high mitochondrial oxidative phosphorylation and production of reactive oxygen species (ROS). MnSOD (SOD2) is a mitochondrial antioxidant defense that has been implicated in inhibition of human malignancies. However, the impact of MnSOD on immunosuppressive macrophage functions and TNBC aggressiveness has never been explored. We found here that SOD2high is primarily observed in the aggressive subtypes of HER2(+) breast cancers and TNBCs patients. Further analyses demonstrated that the oncoprotein multiple copies in T-cell malignancy-1 (MCT-1 or MCTS1) induces mitochondrial superoxide dismutase (MnSOD) in TNBC cells by stabilizing the transcription factor Nrf2. SOD2high/MCTS1high expression correlates with a poor prognosis in breast cancer patients. MnSOD in TNBC cells functions as a prooxidant peroxidase that increases mitochondrial ROS (mROS) and adaptation to oxidative stress under the oncogenic effect. Interleukin-6 (IL-6) in the MCT-1 pathway elevates Nrf2/MnSOD and mROS levels. Knockdown of MnSOD inhibits TNBC cell invasion, breast cancer stem cells (BCSCs), mROS, and IL-6 excretion promoted by MCT-1. TNBC cells deficient in MnSOD prevent the polarization and chemotaxis of M2 macrophages but improve the ability of M1 macrophages to engulf cancer cells. Quenching mROS with MitoQ, a mitochondria-targeted non-metal-based antioxidant MnSOD mimics, effectively suppresses BCSCs and M2 macrophage invasion exacerbated by MnSOD and MCT-1. Consistently, silencing MnSOD impedes TNBC progression and intratumoral M2 macrophage infiltration. We revealed a novel stratagem for TNBC management involving targeting the MCT-1 oncogene-induced mitochondrial prooxidant MnSOD pathway, which prevents the development of an immunosuppressive tumor microenvironment.

Short OGA is targeted to the mitochondria and regulates mitochondrial reactive oxygen species level

O-GlcNAcylation is a reversible post-translational modification involved the regulation of cytosolic, nuclear and mitochondrial proteins. Only two enzymes, OGT and OGA, control attachment and removal of O-GlcNAc on proteins, respectively. Whereas a variant OGT (mOGT) has been proposed as the main isoform that O-GlcNAcylates proteins in mitochondria, identification of a mitochondrial OGA has not been performed yet. Two splice variants of OGA (short and long isoforms) have been described previously. In this work, using cell fractionation experiments, we show that short-OGA is preferentially recovered in mitochondria-enriched fractions from HEK-293T cells as well as mouse embryonic fibroblasts. Moreover, fluorescent microscopy imaging confirmed that GFP-tagged short-OGA is addressed to mitochondria. In addition, using a BRET-based mitochondrial O-GlcNAcylation biosensor, we show that co-transfection of short-OGA markedly reduced O-GlcNAcylation of the biosensor, whereas long-OGA had no significant effect. Finally, using genetically encoded or chemical fluorescent mitochondrial probes, we showed that short-OGA overexpression increases mitochondrial ROS levels, whereas long-OGA had no significant effect. Together, our work reveals that the short-OGA isoform is targeted to the mitochondria where it regulates ROS homoeostasis.

Akt Inhibition as Preconditioning Treatment to Protect Kidney Cells against Anoxia

Lesions issued from the ischemia/reperfusion (I/R) stress are a major challenge in human pathophysiology. Of human organs, the kidney is highly sensitive to I/R because of its high oxygen demand and poor regenerative capacity. Previous studies have shown that targeting the hypusination pathway of eIF5A through GC7 greatly improves ischemic tolerance and can be applied successfully to kidney transplants. The protection process correlates with a metabolic shift from oxidative phosphorylation to glycolysis. Because the protein kinase B Akt is involved in ischemic protective mechanisms and glucose metabolism, we looked for a link between the effects of GC7 and Akt in proximal kidney cells exposed to anoxia or the mitotoxic myxothiazol. We found that GC7 treatment resulted in impaired Akt phosphorylation at the Ser473 and Thr308 sites, so the effects of direct Akt inhibition as a preconditioning protocol on ischemic tolerance were investigated. We evidenced that Akt inhibitors provide huge protection for kidney cells against ischemia and myxothiazol. The pro-survival effect of Akt inhibitors, which is reversible, implied a decrease in mitochondrial ROS production but was not related to metabolic changes or an antioxidant defense increase. Therefore, the inhibition of Akt can be considered as a preconditioning treatment against ischemia.

Mitophagy in Yeast: Molecular Mechanism and Regulation

Mitophagy is a type of autophagy that selectively degrades mitochondria. Mitochondria, known as the “powerhouse of the cell”, supply the majority of the energy required by cells. During energy production, mitochondria produce reactive oxygen species (ROS) as byproducts. The ROS damages mitochondria, and the damaged mitochondria further produce mitochondrial ROS. The increased mitochondrial ROS damages cellular components, including mitochondria themselves, and leads to diverse pathologies. Accordingly, it is crucial to eliminate excessive or damaged mitochondria to maintain mitochondrial homeostasis, in which mitophagy is believed to play a major role. Recently, the molecular mechanism and physiological role of mitophagy have been vigorously studied in yeast and mammalian cells. In yeast, Atg32 and Atg43, mitochondrial outer membrane proteins, were identified as mitophagy receptors in budding yeast and fission yeast, respectively. Here we summarize the molecular mechanisms of mitophagy in yeast, as revealed by the analysis of Atg32 and Atg43, and review recent progress in our understanding of mitophagy induction and regulation in yeast.

TLR4 Inhibitor Alleviates Sepsis-Induced Organ Failure by Inhibiting Platelet Mtros Production, Autophagy, and Gpiib/Iiia Expression

Thrombocytopenia and impaired platelet aggregation are associated with sepsis-induced organ failure. Many studies have shown that mitochondrial ROS, autophagy is related to organ injury in sepsis. However, the relationship between them is unknown. Here, we explored whether Toll Like Receptor 4 inhibitor alleviates sepsis organ failure by inhibiting platelet mitochondrial ROS production, autophagy, and GPIIb/IIIa expression. We found Toll Like Receptor 4 inhibitor TAK242 alleviated LPS-induced acute kidney and lung injury and decrease platelet activation in a mouse model of sepsis mice. In vitro study, inhibiting Toll Like Receptor 4 reduced inward flow of Ca2+ and decreased endogenous mitochondrial ROS production in platelets treated with LPS, and TAK242 inhibited autophagy and NOX expression in LPS treated platelets. Thus, we supposed that Toll Like Receptor 4 inhibitor effectively alleviate lung and kidney injury in a mouse model of sepsis induced by LPS and its effects are related to the inhibition of mouse platelets GPIIb/IIIa, and also can reduce LPS-induced mitochondrial ROS generation related to Ca2+ influx, thus reducing platelet activation. LPS can induce platelet autophagy by generating mitochondrial ROS, which may be the pathophysiological mechanism of organ injury in sepsis.

Short senolytic or senostatic interventions rescue progression of radiation-induced frailty and premature ageing in mice

Cancer survivors suffer from progressive frailty, multimorbidity and premature morbidity. We hypothesize that therapy-induced senescence and senescence progression via bystander effects is a significant cause of this premature ageing phenotype. Accordingly, the study addresses the question whether a short anti-senescence intervention is able to block progression of radiation-induced frailty and disability in a pre-clinical setting. Male mice were sub-lethally irradiated at 5 months of age and treated (or not) with either a senolytic drug (Navitoclax or dasatinib + quercetin) for 10 days or with the senostatic metformin for 10 weeks. Follow up was for one year. Treatments commencing within a month after irradiation effectively reduced frailty progression (p<0.05) and improved muscle (p<0.01) and liver (p<0.05) function as well as short-term memory (p<0.05) until advanced age with no need for repeated interventions. Senolytic interventions that started late, after radiation-induced premature frailty was manifest, still had beneficial effects on frailty (p<0.05) and short-term memory (p<0.05). Metformin was similarly effective as senolytics. At therapeutically achievable concentrations metformin acted as a senostatic neither via inhibition of mitochondrial complex I, nor via improvement of mitophagy or mitochondrial function, but by reducing non-mitochondrial ROS production via NOX4 inhibition in senescent cells. Our study suggests that the progression of adverse long-term health and quality-of-life effects of radiation exposure, as experienced by cancer survivors, might be rescued by short-term adjuvant antisenescence interventions.

Ginsenoside Rg3 Attenuates TNF-α-Induced Damage in Chondrocytes through Regulating SIRT1-Mediated Anti-Apoptotic and Anti-Inflammatory Mechanisms

The upregulation of tumor necrosis factor-alpha (TNF-α) is a common event in arthritis, and the subsequent signaling cascade that leads to tissue damage has become the research focus. To explore a potential therapeutic strategy to prevent cartilage degradation, we tested the effect of ginsenoside Rg3, a bioactive component of Panax ginseng, on TNF-α-stimulated chondrocytes.TC28a2 Human Chondrocytes were treated with TNF-α to induce damage of chondrocytes. SIRT1 and PGC-1a expression levels were investigated by Western blotting assay. Mitochondrial SIRT3 and acetylated Cyclophilin D (CypD) were investigated using mitochondrial isolation. The mitochondrial mass number and mitochondrial DNA copy were studied for mitochondrial biogenesis. MitoSOX and JC-1 were used for the investigation of mitochondrial ROS and membrane potential. Apoptotic markers, pro-inflammatory events were also tested to prove the protective effects of Rg3. We showed Rg3 reversed the TNF-α-inhibited SIRT1 expression. Moreover, the activation of the SIRT1/PGC-1α/SIRT3 pathway by Rg3 suppressed the TNF-α-induced acetylation of CypD, resulting in less mitochondrial dysfunction and accumulation of reactive oxygen species (ROS). Additionally, we demonstrated that the reduction of ROS ameliorated the TNF-α-elicited apoptosis. Furthermore, the Rg3-reverted SIRT1/PGC-1α/SIRT3 activation mediated the repression of p38 MAPK, which downregulated the NF-κB translocation in the TNF-α-treated cells. Our results revealed that administration of Rg3 diminished the production of interleukin 8 (IL-8) and matrix metallopeptidase 9 (MMP-9) in chondrocytes via SIRT1/PGC-1α/SIRT3/p38 MAPK/NF-κB signaling in response to TNF-α stimulation. Taken together, we showed that Rg3 may serve as an adjunct therapy for patients with arthritis.

Mito-TIPTP Increases Mitochondrial Function by Repressing the Rubicon-p22phox Interaction in Colitis-Induced Mice

The run/cysteine-rich-domain-containing Beclin1-interacting autophagy protein (Rubicon) is essential for the regulation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase by interacting with p22phox to trigger the production of reactive oxygen species (ROS) in immune cells. In a previous study, we demonstrated that the interaction of Rubicon with p22phox increases cellular ROS levels. The correlation between Rubicon and mitochondrial ROS (mtROS) is poorly understood. Here, we report that Rubicon interacts with p22phox in the outer mitochondrial membrane in macrophages and patients with human ulcerative colitis. Upon lipopolysaccharide (LPS) activation, the binding of Rubicon to p22phox was elevated, and increased not only cellular ROS levels but also mtROS, with an impairment of mitochondrial complex III and mitochondrial biogenesis in macrophages. Furthermore, increased Rubicon decreases mitochondrial metabolic flux in macrophages. Mito-TIPTP, which is a p22phox inhibitor containing a mitochondrial translocation signal, enhances mitochondrial function by inhibiting the association between Rubicon and p22phox in LPS-primed bone-marrow-derived macrophages (BMDMs) treated with adenosine triphosphate (ATP) or dextran sulfate sodium (DSS). Remarkably, Mito-TIPTP exhibited a therapeutic effect by decreasing mtROS in DSS-induced acute or chronic colitis mouse models. Thus, our findings suggest that Mito-TIPTP is a potential therapeutic agent for colitis by inhibiting the interaction between Rubicon and p22phox to recover mitochondrial function.