Inhibition of palmitoyltransferase ZDHHC12 sensitizes ovarian cancer cells to cisplatin through ROS-mediated mechanisms.

Platinum-based therapies have revolutionized the treatment of high-grade serous ovarian cancer (HGSOC). However, high rates of disease recurrence and progression remain a major clinical concern. Impaired mitochondrial function and dysregulated reactive oxygen species (ROS), hallmarks of cancer, hold potential as therapeutic targets for selectively sensitizing cisplatin treatment. Here, we uncover an oncogenic role of the palmitoyltransferase ZDHHC12 in regulating mitochondrial function and ROS homeostasis in HGSOC cells. Analysis of The Cancer Genome Atlas (TCGA) ovarian cancer data revealed significantly elevated ZDHHC12 expression, demonstrating the strongest positive association with ROS pathways among all ZDHHC enzymes. Transcriptomic analysis of independent ovarian cancer datasets and the SNU119 cell model corroborated this association, highlighting a strong link between ZDHHC12 expression and signature pathways involving mitochondrial oxidative metabolism and ROS regulation. Knockdown of ZDHHC12 disrupted this association, leading to increased cellular complexity, ATP levels, mitochondrial activity, and both mitochondrial and cellular ROS. This dysregulation, achieved by the siRNA knockdown of ZDHHC12 or treatment with the general palmitoylation inhibitor 2BP or the fatty acid synthase inhibitor C75, significantly enhanced cisplatin cytotoxicity in 2D and 3D spheroid models of HGSOC through ROS-mediated mechanisms. Markedly, ZDHHC12 inhibition significantly augmented the anti-tumor activity of cisplatin in an ovarian cancer xenograft tumor model, as well as in an ascites-derived organoid line of platinum-resistant ovarian cancer. Our data suggest the potential of ZDHHC12 as a promising target to improve the outcome of HGSOCs in response to platinum-based chemotherapy.

Mitochondrial GCN5L1 acts as a novel regulator for iron homeostasis to promote sorafenib sensitivity in hepatocellular carcinoma.

BACKGROUND: Sorafenib resistance is becoming increasingly common and disadvantageous for hepatocellular carcinoma (HCC) treatment. Ferroptosis is an iron dependent programmed cell death underlying the mechanism of sorafenib. Iron is crucial for synthesis of cofactors essential to mitochondrial enzymes and necessary for HCC proliferation, while mitochondrial iron overload and oxidative stress are associated with sorafenib induced ferroptosis. However, the crosstalk among iron homeostasis and sorafenib resistance is unclear. METHODS: We conducted bioinformatics analysis of sorafenib treated HCC datasets to analyze GCN5L1 and iron related gene expression with sorafenib resistance. GCN5L1 deleted HCC cell lines were generated by CRISPR technology. Sorafenib resistant HCC cell line was established to validate dataset analysis and evaluate the effect of potential target. RESULTS: We identified GCN5L1, a regulator of mitochondrial acetylation, as a modulator in sorafenib-induced ferroptosis via affecting mitochondrial iron homeostasis. GCN5L1 deficiency significantly increased sorafenib sensitivity in HCC cells by down-regulating mitochondrial iron transporters CISD1 expression to induce iron accumulation. Mitochondrial iron accumulation leads to an acceleration in cellular and lipid ROS. Sorafenib resistance is related to CISD1 overexpression to release mitochondrial iron and maintaining mitochondrial homeostasis. We combined CISD1 inhibitor NL-1 with sorafenib, which significantly enhanced sorafenib-induced ferroptosis by promoting mitochondrial iron accumulation and lipid peroxidation. The combination of NL-1 with sorafenib enhanced sorafenib efficacy in vitro and in vivo. CONCLUSIONS: Our findings demonstrate that GCN5L1/CISD1 axis is crucial for sorafenib resistance and would be a potential therapeutic strategy for sorafenib resistant HCC.

Netrin-1 attenuates cerebral ischemia/reperfusion injury by limiting mitochondrial ROS and Ca2+ levels via activation of AKT phosphorylation and mitochondrial m-AAA protease AFG3L2.

Cerebral ischemia-reperfusion (I/R) injury as the consequence of revascularization after ischemic stroke is associated with mitochondrial dysfunction, oxidative stress, and neuron loss. In this study, we used a deprivation/reoxygenation (OGD/R) model to determine whether interactions between Netrin-1, AKT, and the mitochondrial AAA protease AFG3L2 could influence mitochondrial function in neurons after I/R. We found that Netrin-1 protects primary cortical neurons from OGD/R-induced cell death and regulates mitochondrial reactive oxygen species (ROS) and Ca2+ levels. The accumulation of mitochondrial calcium uniporter (MCU) subunits was monitored in cells by immunoblot analysis. Although the regulatory subunits MICU1 and MICU2 were relatively unaffected, the accumulation of the essential MCU regulator (EMRE) subunit was impaired. In OGD/R-induced cells, the 7 kDa form of EMRE was significantly reduced. Netrin-1 inhibited the accumulation of EMRE and mitochondrial Ca2+ levels by upregulating AFG3L2 and AKT activation. Loss of AFG3L2 or inhibition of AKT increased levels of 7 kDa EMRE. Moreover, overexpression of AKT increased the expression of AFG3L2 in Netrin-1-knockdown neurons after OGD/R. Our results demonstrate that Netrin-1 enhanced AFG3L2 protein expression via activation of AKT. We also observed that overexpression of Netrin-1 significantly reduced infarction size in an I/R-induced brain injury model in rats but not when AKT was inhibited. Our data suggest that AFG3L2 is a protein substrate of AKT and indicate that Netrin-1 attenuates cerebral I/R injury by limiting mitochondrial ROS and Ca2+ levels through activating AKT phosphorylation and AFG3L2.

Macrophage MAPK7/AhR/STAT3 Signaling Mediates Mitochondrial ROS Burst and Enterohepatic Inflammatory Responses Induced by Deoxynivalenol Relevant to Low-Dose Exposure in Children.

Deoxynivalenol (DON) can induce endoplasmic reticulum (ER) stress, mitochondrial ROS burst, and macrophage polarization. Here, we investigated the mechanism linking the above three aspects with the dose range relevant to low-level exposure in children. At 0.5 µg/kg bw/day, we found remarkable liver and gut inflammatory responses after 6-week exposure in mice age comparable to humans 7-12 years old. Through antioxidant intervention, we found that ROS played a driver role in macrophage polarization and inflammatory responses induced by DON in the liver and gut. Further bioinformatics analysis uncovered that ER stress-associated protein MAPK7 (ERK5) may bind with AhR to initiate a mitochondrial ROS burst and macrophage M1 polarization. The downstream cellular events of MAPK7-AhR interaction may be mediated by the AhR/STAT3/p-STAT(Ser727) pathway. This mechanism was further supported by DON toxicity mitigation using cyanidin-3-glucoside (C-3-G), which docks to MAPK7 oligomerization region 200-400 aa and disrupts MAPK7-AhR interaction. Overall, our study provides novel evidence and mechanism for DON-induced inflammatory responses in the liver and gut system. Our findings call attention to the health risks associated with low-level DON exposure in the prepuberty children population.

Acute exposure to polystyrene nanoparticles promotes liver injury by inducing mitochondrial ROS-dependent necroptosis and augmenting macrophage-hepatocyte crosstalk.

BACKGROUND: The global use of plastic materials has undergone rapid expansion, resulting in the substantial generation of degraded and synthetic microplastics and nanoplastics (MNPs), which have the potential to impose significant environmental burdens and cause harmful effects on living organisms. Despite this, the detrimental impacts of MNPs exposure towards host cells and tissues have not been thoroughly characterized. RESULTS: In the present study, we have elucidated a previously unidentified hepatotoxic effect of 20 nm synthetic polystyrene nanoparticles (PSNPs), rather than larger PS beads, by selectively inducing necroptosis in macrophages. Mechanistically, 20 nm PSNPs were rapidly internalized by macrophages and accumulated in the mitochondria, where they disrupted mitochondrial integrity, leading to heightened production of mitochondrial reactive oxygen species (mtROS). This elevated mtROS generation essentially triggered necroptosis in macrophages, resulting in enhanced crosstalk with hepatocytes, ultimately leading to hepatocyte damage. Additionally, it was demonstrated that PSNPs induced necroptosis and promoted acute liver injury in mice. This harmful effect was significantly mitigated by the administration of a necroptosis inhibitor or systemic depletion of macrophages prior to PSNPs injection. CONCLUSION: Collectively, our study suggests a profound toxicity of environmental PSNP exposure by triggering macrophage necroptosis, which in turn induces hepatotoxicity via intercellular crosstalk between macrophages and hepatocytes in the hepatic microenvironment.

Adipose Tissue Hypoxia in Obesity: Clinical Reappraisal of Hypoxia Hypothesis.

Obese subjects exhibit lower adipose tissue oxygen consumption in accordance with the lower adipose tissue blood flow. Thereby, compared to lean subjects, obese individuals have almost half lower capillary density and more than half lower vascular endothelial growth factor (VEGF). The VEGF expression together with hypoxia-inducible transcription factor-1 alpha (HIF-1α) activity also requires phosphatidylinositol 3-kinase (PI3K) and mammalian target of rapamycin (mTOR)-mediated signaling. Especially HIF-1α is an important signaling molecule for hypoxia to induce the inflammatory responses. Hypoxia contributes to several biological functions, such as angiogenesis, cell proliferation, apoptosis, inflammation, and insulin resistance (IR). Pathogenesis of obesity-related comorbidities is attributed to intermittent hypoxia (IH), which is mostly observed in visceral obesity. Proinflammatory phenotype of the adipose tissue is a crucial link between IH and the development of IR. Inhibition of adaptive unfolded protein response (UPR) in hypoxia increases ß cell death. Moreover, deletion of HIF-1α worsens ß cell function. Oxidative stress, as well as the release of proinflammatory cytokines/adipokines in obesity, is proportional to the severity of IH. Reactive oxygen species (ROS) generation at mitochondria is responsible for propagation of the hypoxic signal; however, mitochondrial ROS production is required for hypoxic HIF-1α protein stabilization. Alterations in oxygen availability of adipose tissue directly affect the macrophage polarization and are responsible for the dysregulated adipocytokines production in obesity. Hypoxia both inhibits adipocyte differentiation from preadipocytes and macrophage migration from the hypoxic adipose tissue. Upon reaching a hypertrophic threshold beyond the adipocyte fat loading capacity, excess extracellular matrix (ECM) components are deposited, causing fibrosis. HIF-1α initiates the whole pathological process of fibrosis and inflammation in the obese adipose tissue. In addition to stressed adipocytes, hypoxia contributes to immune cell migration and activation which further aggravates adipose tissue fibrosis. Therefore, targeting HIF-1α might be an efficient way to suppress hypoxia-induced pathological changes in the ECM. The fibrosis score of adipose tissue correlates negatively with the body mass index and metabolic parameters. Inducers of browning/beiging adipocytes and adipokines, as well as modulations of matrix remodeling enzyme inhibitors, and associated gene regulators, are potential pharmacological targets for treating obesity.

The oxidoreductase CLIC4 is required to maintain mitochondrial function and resistance to exogenous oxidants in breast cancer cells.

The chloride intracellular channel-4 (CLIC4) is one of the six highly conserved proteins in the CLIC family that share high structural homology with GST-omega in the GST superfamily. While CLIC4 is a multifunctional protein that resides in multiple cellular compartments, the discovery of its enzymatic glutaredoxin-like activity in vitro suggested that it could function as an antioxidant. Here, we found that deleting CLIC4 from murine 6DT1 breast tumor cells using CRISPR enhanced the accumulation of reactive oxygen species (ROS) and sensitized cells to apoptosis in response to H2O2 as a ROS-inducing agent. In intact cells, H2O2 increased the expression of both CLIC4 mRNA and protein. In addition, increased superoxide production in 6DT1 cells lacking CLIC4 was associated with mitochondrial hyperactivity including increased mitochondrial membrane potential and mitochondrial organelle enlargement. In the absence of CLIC4, however, H2O2-induced apoptosis was associated with low expression and degradation of the antiapoptotic mitochondrial protein Bcl2 and the negative regulator of mitochondrial ROS, UCP2. Furthermore, transcriptomic profiling of H2O2-treated control and CLIC4-null cells revealed upregulation of genes associated with ROS-induced apoptosis and downregulation of genes that sustain mitochondrial functions. Accordingly, tumors that formed from transplantation of CLIC4-deficient 6DT1 cells were highly necrotic. These results highlight a critical role for CLIC4 in maintaining redox-homeostasis and mitochondrial functions in 6DT1 cells. Our findings also raise the possibility of targeting CLIC4 to increase cancer cell sensitivity to chemotherapeutic drugs that are based on elevating ROS in cancer cells.

WGX50 mitigates doxorubicin-induced cardiotoxicity through inhibition of mitochondrial ROS and ferroptosis.

BACKGROUND: Doxorubicin (DOX)-induced cardiotoxicity (DIC) is a major impediment to its clinical application. It is indispensable to explore alternative treatment molecules or drugs for mitigating DIC. WGX50, an organic extract derived from Zanthoxylum bungeanum Maxim, has anti-inflammatory and antioxidant biological activity, however, its function and mechanism in DIC remain unclear. METHODS: We established DOX-induced cardiotoxicity models both in vitro and in vivo. Echocardiography and histological analyses were used to determine the severity of cardiac injury in mice. The myocardial damage markers cTnT, CK-MB, ANP, BNP, and ferroptosis associated indicators Fe2+, MDA, and GPX4 were measured using ELISA, RT-qPCR, and western blot assays. The morphology of mitochondria was investigated with a transmission electron microscope. The levels of mitochondrial membrane potential, mitochondrial ROS, and lipid ROS were detected using JC-1, MitoSOX™, and C11-BODIPY 581/591 probes. RESULTS: Our findings demonstrate that WGX50 protects DOX-induced cardiotoxicity via restraining mitochondrial ROS and ferroptosis. In vivo, WGX50 effectively relieves doxorubicin-induced cardiac dysfunction, cardiac injury, fibrosis, mitochondrial damage, and redox imbalance. In vitro, WGX50 preserves mitochondrial function by reducing the level of mitochondrial membrane potential and increasing mitochondrial ATP production. Furthermore, WGX50 reduces iron accumulation and mitochondrial ROS, increases GPX4 expression, and regulates lipid metabolism to inhibit DOX-induced ferroptosis. CONCLUSION: Taken together, WGX50 protects DOX-induced cardiotoxicity via mitochondrial ROS and the ferroptosis pathway, which provides novel insights for WGX50 as a promising drug candidate for cardioprotection.

Targeting macrophagic PIM-1 alleviates osteoarthritis by inhibiting NLRP3 inflammasome activation via suppressing mitochondrial ROS/Cl- efflux signaling pathway.

BACKGROUND: Osteoarthritis (OA), in which macrophage-driven synovitis is considered closely related to cartilage destruction and could occur at any stage, is an inflammatory arthritis. However, there are no effective targets to cure the progression of OA. The NOD-, LRR-,and pyrin domain-containing protein 3 (NLRP3) inflammasome in synovial macrophages participates in the pathological inflammatory process and treatment strategies targeting it are considered to be an effective approach for OA. PIM-1 kinase, as a downstream effector of many cytokine signaling pathways, plays a pro-inflammatory role in inflammatory disease. METHODS: In this study, we evaluated the expression of the PIM-1 and the infiltration of synovial macrophages in the human OA synovium. The effects and mechanism of PIM-1 were investigated in mice and human macrophages stimulated by lipopolysaccharide (LPS) and different agonists such as nigericin, ATP, Monosodium urate (MSU), and Aluminum salt (Alum). The protective effects on chondrocytes were assessed by a modified co-culture system induced by macrophage condition medium (CM). The therapeutic effect in vivo was confirmed by the medial meniscus (DMM)-induced OA in mice. RESULTS: The expression of PIM-1 was increased in the human OA synovium which was accompanied by the infiltration of synovial macrophages. In vitro experiments, suppression of PIM-1 by SMI-4a, a specific inhibitor, rapidly inhibited the NLRP3 inflammasome activation in mice and human macrophages and gasdermin-D (GSDME)-mediated pyroptosis. Furthermore, PIM-1 inhibition specifically blocked the apoptosis-associated speck-like protein containing a CARD (ASC) oligomerization in the assembly stage. Mechanistically, PIM-1 inhibition alleviated the mitochondrial reactive oxygen species (ROS)/chloride intracellular channel proteins (CLICs)-dependent Cl- efflux signaling pathway, which eventually resulted in the blockade of the ASC oligomerization and NLRP3 inflammasome activation. Furthermore, PIM-1 suppression showed chondroprotective effects in the modified co-culture system. Finally, SMI-4a significantly suppressed the expression of PIM-1 in the synovium and reduced the synovitis scores and the Osteoarthritis Research Society International (OARSI) score in the DMM-induced OA model. CONCLUSIONS: Therefore, PIM-1 represented a new class of promising targets as a treatment of OA to target these mechanisms in macrophages and widened the road to therapeutic strategies for OA.

Pore formation-independent cell death induced by a ß-barrel pore-forming toxin.

Vibrio cholerae cytolysin (VCC) is a ß-barrel pore-forming toxin (ß-PFT). It exhibits potent hemolytic activity against erythrocytes that appears to be a direct outcome of its pore-forming functionality. However, VCC-mediated cell-killing mechanism is more complicated in the case of nucleated mammalian cells. It induces apoptosis in the target nucleated cells, mechanistic details of which are still unclear. Furthermore, it has never been explored whether the ability of VCC to trigger programmed cell death is stringently dependent on its pore-forming activity. Here, we show that VCC can evoke hallmark features of the caspase-dependent apoptotic cell death even in the absence of the pore-forming ability. Our study demonstrates that VCC mutants with abortive pore-forming hemolytic activity can trigger apoptotic cell death responses and cytotoxicity, similar to those elicited by the wild-type toxin. VCC as well as its pore formation-deficient mutants display prominent propensity to translocate to the target cell mitochondria and cause mitochondrial membrane damage. Therefore, our results for the first time reveal that VCC, despite being an archetypical ß-PFT, can kill target nucleated cells independent of its pore-forming functionality. These findings are intriguing for a ß-PFT, whose destination is generally expected to remain limited on the target cell membranes, and whose mode of action is commonly attributed to the membrane-damaging pore-forming ability. Taken together, our study provides critical new insights regarding distinct implications of the two important virulence functionalities of VCC for the V. cholerae pathogenesis process: hemolytic activity for iron acquisition and cytotoxicity for tissue damage by the bacteria.

MiR-206 may regulate mitochondrial ROS contribute to the progression of Myocardial infarction via TREM1.

Myocardial infarction (MI) is a leading cause of mortality. To better understand its molecular and cellular mechanisms, we used bioinformatic tools and molecular experiments to explore the pathogenesis and prognostic markers. Differential gene expression analysis was conducted using GSE60993 and GSE66360 datasets. Hub genes were identified through pathway enrichment analysis and PPI network construction, and four hub genes (AQP9, MMP9, FPR1, and TREM1) were evaluated for their predictive performance using AUC and qRT-PCR. miR-206 was identified as a potential regulator of TREM1. Finally, miR-206 was found to induce EC senescence and ER stress through upregulating mitochondrial ROS levels via TREM1. These findings may contribute to understanding the pathogenesis of MI and identifying potential prognostic markers.

Theaflavin mitigates acute gouty peritonitis and septic organ injury in mice by suppressing NLRP3 inflammasome assembly.

Activation of NLR family pyrin domain-containing 3 (NLRP3) inflammasome plays important role in defending against infections, but its aberrant activation is causally linked to many inflammatory diseases, thus being a therapeutic target for these diseases. Theaflavin, one major ingredient of black tea, exhibits potent anti-inflammatory and anti-oxidative activities. In this study, we investigated the therapeutic effects of theaflavin against NLRP3 inflammasome activation in macrophages in vitro and in animal models of related diseases. We showed that theaflavin (50, 100, 200 µM) dose-dependently inhibited NLRP3 inflammasome activation in LPS-primed macrophages stimulated with ATP, nigericin or monosodium urate crystals (MSU), evidenced by reduced release of caspase-1p10 and mature interleukin-1ß (IL-1ß). Theaflavin treatment also inhibited pyroptosis as shown by decreased generation of N-terminal fragment of gasdermin D (GSDMD-NT) and propidium iodide incorporation. Consistent with these, theaflavin treatment suppressed ASC speck formation and oligomerization in macrophages stimulated with ATP or nigericin, suggesting reduced inflammasome assembly. We revealed that theaflavin-induced inhibition on NLRP3 inflammasome assembly and pyroptosis resulted from ameliorated mitochondrial dysfunction and reduced mitochondrial ROS production, thereby suppressing interaction between NLRP3 and NEK7 downstream of ROS. Moreover, we showed that oral administration of theaflavin significantly attenuated MSU-induced mouse peritonitis and improved the survival of mice with bacterial sepsis. Consistently, theaflavin administration significantly reduced serum levels of inflammatory cytokines including IL-1ß and attenuated liver inflammation and renal injury of mice with sepsis, concomitant with reduced generation of caspase-1p10 and GSDMD-NT in the liver and kidney. Together, we demonstrate that theaflavin suppresses NLRP3 inflammasome activation and pyroptosis by protecting mitochondrial function, thus mitigating acute gouty peritonitis and bacterial sepsis in mice, highlighting a potential application in treating NLRP3 inflammasome-related diseases.

Garcinol protects SH-SY5Y cells against MPP+-induced cell death by activating DJ-1/SIRT1 and PGC-1α mediated antioxidant pathway in sequential stimulation of p-AMPK mediated autophagy.

Parkinson's disease (PD), a chronic and progressive neurodegenerative disease, can reduce the population of dopaminergic neurons in the substantia nigra. The cause of this neuronal death remains unclear. 1-Methyl-4-phenylpyridinium ion (MPP+) is a potent neurotoxin that can destroy dopaminergic (DA) neurons and promote PD. Garcinol, a polyisoprenylated benzophenone derivative, was extracted from Garcinia indica and is an important active compound it has been used as an anticancer, antioxidant, and anti-inflammatory, agent and it can suppress reactive oxygen species (ROS) mediated cell death in a PD model. Human neuroblastoma (SH-SY5Y) cells (1 × 105 cells) were treated with MPP+ (1 mM) for 24 h to induce cellular ROS production. The formation of ROS was suppressed by pretreatment with different concentrations of garcinol (0.5 and 1.0 µM) for 3 h in SH-SY5Y cells. The present study found that MPP+ treatment increased the formation of reactive oxygen species (ROS), and the increased ROS began to promote cell death in SH-SY5Y cells. However, our natural compound garcinol effectively blocked MPP+-mediated ROS formation by activating the DJ-1/SIRT1 and PGC-1α mediated antioxidant pathway. Further findings indicate that the activated SIRT1 can also regulate p-AMPK-mediated autophagy to protect the neurons from the damage it concludes that garcinol sub-sequential regulates intracellular autophagy in this model, and the productive efficacy of garcinol was confirmed by western blot analysis and MitoSOX DCFDA and MTT assays. The results showed garcinol increased protection due to the prevention of MPP+-induced ROS and the promotion of cell survival.

Acrolein increases the concentration of intracellular Zn2⁺ by producing mitochondrial reactive oxygen species in A549 cells.

Smoking or occupational exposure leads to low concentrations of acrolein on the surface of the airways. Acrolein is involved in the pathophysiological processes of various respiratory diseases. Reports showed that acrolein induced an increase in mitochondrial reactive oxygen species (mROS). Furthermore, exogenous H2O2 was found to increase intracellular Zn2⁺ concentration ([Zn2⁺]ᵢ). However, the specific impact of acrolein on changes in intracellular Zn2⁺ levels has not been fully investigated. Therefore, this study aimed to investigate the effects of acrolein on mROS and [Zn2⁺]ᵢ in A549 cells. We used Mito Tracker Red CM-H2Xros (MitoROS) and Fluozin-3 fluorescent probes to observe changes in mROS and intracellular Zn2⁺. The results revealed that acrolein increased [Zn2⁺]ᵢ in a time- and dose-dependent manner. Additionally, the production of mROS was observed in response to acrolein treatment. Subsequent experiments showed that the intracellular Zn2⁺ chelator TPEN could inhibit the acrolein-induced elevation of [Zn2⁺]ᵢ but did not affect the acrolein-induced mROS production. Conversely, the acrolein-induced elevation of mROS and [Zn2⁺]ᵢ were significantly decreased by the inhibitors of ROS formation (NaHSO3, NAC). Furthermore, external oxygen free radicals increased both [Zn2⁺]ᵢ levels and mROS production. These results demonstrated that acrolein-induced elevation of [Zn2⁺]ᵢ in A549 cells was mediated by mROS generation, rather than through a pathway where [Zn2⁺]ᵢ elevation leads to mROS production.

The Role of Intracellular Ca2+ and Mitochondrial ROS in Small Aß1-42 Oligomer-Induced Microglial Death.

Alzheimer's disease (AD) is the most common form of dementia worldwide, and it contributes up to 70% of cases. AD pathology involves abnormal amyloid beta (Aß) accumulation, and the link between the Aß1-42 structure and toxicity is of major interest. NMDA receptors (NMDAR) are thought to be essential in Aß-affected neurons, but the role of this receptor in glial impairment is still unclear. In addition, there is insufficient knowledge about the role of Aß species regarding mitochondrial redox states in neurons and glial cells, which may be critical in developing Aß-caused neurotoxicity. In this study, we investigated whether different Aß1-42 species-small oligomers, large oligomers, insoluble fibrils, and monomers-were capable of producing neurotoxic effects via microglial NMDAR activation and changes in mitochondrial redox states in primary rat brain cell cultures. Small Aß1-42 oligomers induced a concentration- and time-dependent increase in intracellular Ca2+ and necrotic microglial death. These changes were partially prevented by the NMDAR inhibitors MK801, memantine, and D-2-amino-5-phosphopentanoic acid (DAP5). Neither microglial intracellular Ca2+ nor viability was significantly affected by larger Aß1-42 species or monomers. In addition, the small Aß1-42 oligomers caused mitochondrial reactive oxygen species (mtROS)-mediated mitochondrial depolarization, glutamate release, and neuronal cell death. In microglia, the Aß1-42-induced mtROS overproduction was mediated by intracellular calcium ions and Aß-binding alcohol dehydrogenase (ABAD). The data suggest that the pharmacological targeting of microglial NMDAR and mtROS may be a promising strategy for AD therapy.

Modulation of Cellular Senescence in HEK293 and HepG2 Cells by Ultrafiltrates UPla and ULu Is Partly Mediated by Modulation of Mitochondrial Homeostasis under Oxidative Stress.

Protein probes, including ultrafiltrates from the placenta (UPla) and lung (ULu) of postnatal rabbits, were investigated in premature senescent HEK293 and HepG2 cells to explore whether they could modulate cellular senescence. Tris-Tricine-PAGE, gene ontology (GO), and LC-MS/MS analysis were applied to describe the characteristics of the ultrafiltrates. HEK293 and HepG2 cells (both under 25 passages) exposed to a sub-toxic concentration of hydrogen peroxide (H2O2, 300 µM) became senescent; UPla (10 µg/mL), ULu (10 µg/mL), as well as positive controls lipoic acid (10 µg/mL) and transferrin (10 µg/mL) were added along with H2O2 to the cells. Cell morphology; cellular proliferation; senescence-associated beta-galactosidase (SA-ß-X-gal) activity; expression of senescence biomarkers including p16 INK4A (p16), p21 Waf1/Cip1 (p21), HMGB1, MMP-3, TNF-α, IL-6, lamin B1, and phospho-histone H2A.X (γ-H2AX); senescence-related gene expression; reactive oxygen species (ROS) levels; and mitochondrial fission were examined. Tris-Tricine-PAGE revealed prominent detectable bands between 10 and 100 kDa. LC-MS/MS identified 150-180 proteins and peptides in the protein probes, and GO analysis demonstrated a distinct enrichment of proteins associated with "extracellular space" and "proteasome core complex". UPla and ULu modulated senescent cell morphology, improved cell proliferation, and decreased beta-galactosidase activity, intracellular and mitochondrial ROS production, and mitochondrial fission caused by H2O2. The results from this study demonstrated that UPla and Ulu, as well as lipoic acid and transferrin, could protect HEK293 and HepG2 cells from H2O2-induced oxidative damage via protecting mitochondrial homeostasis and thus have the potential to be explored in anti-aging therapies.

Mitochondrial calpain-1 activates NLRP3 inflammasome by cleaving ATP5A1 and inducing mitochondrial ROS in CVB3-induced myocarditis.

Treatment options for myocarditis are currently limited. Inhibition of calpains has been shown to prevent Coxsackievirus B3 (CVB3)-induced cardiac injuries, but the underlying mechanism of action of calpains has not been elucidated. We investigated whether NOD-, LRR-, and pyrin domain-containing 3 (NLRP3) inflammasome participated in CVB3-induced myocarditis, and investigated the effects of calpain-1 on CVB3-induced cardiac injury. NLRP3 inflammasome was activated in CVB3-infected hearts, evidenced by elevated protein levels of NLRP3, N-terminal domain of Gasdermin D, and cleaved caspase-1, and the increased co-localization of NLRP3 and apoptosis-associated speck-like protein. The intraperitoneal administration of MCC950, a selective inhibitor of the NLRP3 inflammasome, led to decreased levels of serum creatine kinase-MB, cardiac troponin I, lactate dehydrogenase, interleukin-18, interleukin-1ß, prevention of the infiltration of inflammatory cells, and improvement of cardiac function under CVB3 infection. Transgenic mice overexpressing the endogenous calpain inhibitor calpastatin (Tg-CAST mice) exhibited not only decreased apoptosis, inflammation, fibrosis, and enhanced cardiac function but also inhibition of NLRP3 inflammasome and pyroptosis. The selective inhibition of calpain-1 using PD151746 protected cardiomyocytes in vitro from CVB3 infection by downregulating NLRP3 inflammasome and, thus, preserved cell viability. Mechanistically, we showed that mitochondrial dysfunction preceded inflammatory response after CVB3 treatment and elimination of mitochondrial reactive oxygen species (ROS) using mitochondria-targeted antioxidants (mito-TEMPO) recapitalized the phenotype observed in Tg-CAST mice. Furthermore, the promotion or inhibition of calpain-1 activation in vitro regulated the mitochondrial respiration chain. Mito-TEMPO reversed calpain-1-mediated NLRP3 inflammation activation and cell death. We also found that mitochondrial calpain-1, which was increased after CVB3 stimulation, activated the NLRP3 inflammasome and resulted in cell death. Furthermore, ATP synthase-α (ATP5A1) was revealed to be the cleaving target of calpain-1 after CVB3 treatment. Downregulating ATP5A1 using ATP5A1-small interfering RNA impaired mitochondrial function, decreased cell viability, and induced NLRP3 inflammasome activation. In conclusion, CVB3 infection induced calpain-1 accumulation in mitochondria, and led to subsequent ATP5A1 cleavage, mitochondrial ROS overproduction, and impaired mitochondrial function, eventually causing NLRP3 inflammasome activation and inducing pyroptosis. Therefore, our findings established the role of calpain in viral myocarditis and unveiled its underlying mechanism of its action. Calpain appears as a promising target for the treatment of viral myocarditis.

TLR4 inhibitor alleviates sepsis-induced organ failure by inhibiting platelet mtROS production, autophagy, and GPIIb/IIIa expression.

Thrombocytopenia and impaired platelet function are associated with sepsis-induced organ failure. Numerous studies have shown that mitochondrial ROS (mtROS) and autophagy are related to organ injury in sepsis. However, the relationships between platelet mtROS, autophagy and sepsis organ failure remain unclear. Herein, we explored whether toll like receptor 4 (TLR4) inhibitor alleviates sepsis organ failure by inhibiting platelet mtROS production, autophagy, and GPIIb/IIIa expression.Mice were administrated with LPS, LPS + TAK242 or vehicle. The lungs and kidneys were harvested and analyzed using hematoxylin and eosin staining assay. Platelet rich plasma (PRP) was isolated from blood and platelets aggregation and TLR4 expression were analyzed using flow cytometer and western blot. PRP from healthy volunteers was treated with saline, LPS, or LPS + TAK242, and then mitoSOX and calcium were detected using flow cytometer, and NOX2 and LC3B were tested using western blot.Results showed that TAK242 effectively alleviated LPS-induced acute kidney and lung injury in mice, and decreased CD41 expression more significantly than CD62P. In vitro, by inhibiting TLR4, TAK242 suppressed Ca2+, mitoSOX fluorescence, NOX2 expression and LC3BII/LC3BI ratio in LPS treated platelets.TLR4 inhibitor TAK242 may effectively alleviate mouse lung and kidney injury by inhibition of mouse platelet GPIIb/IIIa, and reduce LPS-induced mtROS generation related to Ca2+ influx, thus reducing platelet activation.

Complement C5a induces the generation of neutrophil extracellular traps by inhibiting mitochondrial STAT3 to promote the development of arterial thrombosis.

BACKGROUND: Thrombotic events cannot be completely prevented by antithrombotics, implicating a therapeutic gap due to inflammation, a not yet sufficiently addressed mechanism. Neutrophil extracellular traps (NETs) are an essential interface between inflammation and thrombosis, but exactly how the NETotic process is initiated and maintained during arterial thrombosis remains incompletely understood. METHODS AND RESULTS: We found that the plasma concentrations of C5a were higher in patients with ST-elevation myocardial infarction (STEMI) than in patients with angina and higher in mice with left common carotid artery (LCCA) thrombosis induced by FeCl3 than in control mice. We observed that the thrombus area and weight were decreased and that NET formation in the thrombi was reduced in the group treated with the selective C5aR1 receptor inhibitor PMX53 compared with the NaCl group. In vitro, NETosis was observed when C5a was added to neutrophil cultures, and this effect was reversed by PMX53. In addition, our data showed that C5a increased the production of mitochondrial reactive oxygen species (ROS) and that the promotion of NET formation by C5a was mitochondrial ROS (Mito-ROS) dependent. Furthermore, we found that C5a induced the production of Mito-ROS by inhibiting mitochondrial STAT3 activity. CONCLUSIONS: By inhibiting mitochondrial STAT3 to elicit Mito-ROS generation, C5a triggers the generation of NETs to promote the development of arterial thrombosis. Hence, our study identifies complement C5a as a potential new target for the treatment and prevention of thrombosis.

Effects of OP2113 on Myocardial Infarct Size and No Reflow in a Rat Myocardial Ischemia/Reperfusion Model.

PURPOSE: The present study was to determine whether OP2113 could limit myocardial infarction size and the no-reflow phenomenon in a rat myocardial ischemia/reperfusion model. METHODS: Rat heart-isolated mitochondria (RHM) were used to investigate mitochondrial respiration and mitochondrial reactive oxygen species (mtROS) generation both in normal conditions and in ischemia/reperfusion-mimicking conditions (using high concentrations of succinate). Human skeletal muscle myoblasts (HSMM) in culture were used to investigate the cellular intermittent deprivation in energy substrates and oxygen as reported in ischemia/reperfusion conditions. In vivo, rats were anesthetized and subjected to 30 min of left coronary artery occlusion followed by 3 h of reperfusion. Rats were randomized to receive OP2113 as an intravenous infusion starting either 5 min prior to coronary artery occlusion (preventive), or 5 min prior to reperfusion (curative), or to receive vehicle starting 5 min prior to coronary artery occlusion. Infusions continued until the end of the study (3 h of reperfusion). RESULTS: RHM treated with OP2113 showed a concentration-dependent reduction of succinate-induced mtROS generation. In HSMM cells, OP2113 treatment (5-10 µM) during 48H prevented the reduction in the steady-state level of ATP measured just after reperfusion injuries and decreased the mitochondrial affinity to oxygen. In vivo, myocardial infarct size, expressed as the percentage of the ischemic risk zone, was significantly lower in the OP2113-treated preventive group (44.5 ± 2.9%) versus that in the vehicle group (57.0 ± 3.6%; p < 0.05), with a non-significant trend toward a smaller infarct size in the curative group (50.8 ± 3.9%). The area of no reflow as a percentage of the risk zone was significantly smaller in both the OP2113-treated preventive (28.8 ± 2.4%; p = 0.026 vs vehicle) and curative groups (30.1 ± 2.3%; p = 0.04 vs vehicle) compared with the vehicle group (38.9 ± 3.1%). OP2113 was not associated with any hemodynamic changes. CONCLUSIONS: These results suggest that OP2113 is a promising mitochondrial ROS-modulating agent to reduce no-reflow as well as to reduce myocardial infarct size, especially if it is on board early in the course of the infarction. It appears to have benefit on no-reflow even when administered relatively late in the course of ischemia.