Mitochondrial Superoxide Scavenger Research Articles

Exposure to polystyrene nanoplastics promotes premature cellular senescence through mitochondrial ROS production and dysfunction in pre-differentiated skeletal myoblasts

Nanoplastics (NPs) are emerging environmental contaminants present in atmospheric, freshwater, and aquatic environments. NPs can rapidly permeate cell membranes and build up in human tissues and organs, causing a potential threat to human health. As the skeletal muscle undergoes aging, myogenesis gradually deteriorates, leading to loss of muscle mass. While previous studies have demonstrated the adverse and toxic effects of polystyrene (PS)-NPs, gaps remain in understanding aging effects and specific mechanisms by PS-NPs in pre-differentiated myoblasts. In this study, we investigated the cellular internalization, aggregation, and senescent effects of PS-NPs using an in vitro model of pre-differentiated C2C12 myoblasts. Pre-differentiated C2C12 myoblasts were exposed to increasing concentrations of PS-NPs and internalization was observed in myoblasts using flow cytometry and transmission electron microscopy (TEM). We further investigated whether internalization of these PS-NPs at sublethal cytotoxic concentrations led to an increase in senescence hallmarks, such as increased β-galactosidase activity, increased expression of p16, p21 and senescence-related secretory phenotypes, and cell cycle arrest. In addition, PS-NP treatment caused notable mitochondrial superoxide production and damage, including mitochondrial membrane depolarization, content loss, fragmentation, and decreased ATP production. Rotenone, a mitochondrial function inhibitor, and exacerbated PS-NP-induced cell proliferation inhibition, whereas Mito-TEMPO, a mitochondrial superoxide scavenger, restored the cell proliferation rate and rescued cellular senescence. Therefore, our findings indicate the senescent effects of PS-NPs through mitochondrial superoxide production and dysfunction in pre-differentiated myoblasts.

HMGCR Inhibitor Restores Mitochondrial Dynamics by Regulating Signaling Cascades in a Rodent Alzheimer's Disease Model.

Atorvastatin an HMGCR inhibitor may play a role in enhancing spatial and long-term memory and combating anxious behavior deficits induced by Aβ1-42. Behavioral deficit studies, immunoblotting for the antioxidant/apoptotic protein expression, flow cytometry (FACS) for mitochondrial ROS, membrane potential (▲ψm), and histopathological alterations were performed against Aβ1-42 toxicity. Aβ1-42 was infused directly into the brain through i.c.v for the establishment of the AD model. Atorvastatin (ATOR) was administered orally and was used to treat AD in adult male Wistar rats aged between 200 and 250g. We confirmed that ATOR administration significantly attenuates the Aβ1-42-induced cognitive decline targeted mitochondrial-mediated age-dependent disease progression. Nrf2 stabilizes to interact SOD2 antioxidant enzyme, allowing transcriptional activity by the steep increase in ▲ψm and a reduction in ROS by activating mitochondrial superoxide scavenger and Nrf2-dependent pathway. These findings confirmed that ATOR has the potential efficacy to modulate the interference in cognitive decline induced by Aβ1-42.

Methamphetamine Enhancement of HIV-1 gp120-Mediated NLRP3 Inflammasome Activation and Resultant Proinflammatory Responses in Rat Microglial Cultures.

Human Immunodeficiency Virus type 1 (HIV-1)-associated neurocognitive disorders (HANDs) remain prevalent in HIV-1-infected individuals despite the evident success of combined antiretroviral therapy (cART). The mechanisms underlying HAND prevalence in the cART era remain perplexing. Ample evidence indicates that HIV-1 envelope glycoprotein protein 120 (gp120), a potent neurotoxin, plays a pivotal role in HAND pathogenesis. Methamphetamine (Meth) abuse exacerbates HANDs, but how this occurs is not fully understood. We hypothesize that Meth exacerbates HANDs by enhancing gp120-mediated neuroinflammation. To test this hypothesis, we studied the effect of Meth on gp120-induced microglial activation and the resultant production of proinflammatory cytokines in primary rat microglial cultures. Our results show that Meth enhanced gp120-induced microglial activation, as revealed by immunostaining and Iba-1 expression, and potentiated gp120-mediated NLRP3 expression and IL-1β processing and release, as assayed by immunoblotting and ELISA. Meth also augmented the co-localization of NLRP3 and caspase-1, increased the numbers of NLRP3 puncta and ROS production, increased the levels of iNOS expression and NO production, and increased the levels of cleaved gasderminD (GSDMD-N; an executor of pyroptosis) in gp120-primed microglia. The Meth-associated effects were attenuated or blocked by MCC950, an NLRP3 inhibitor, or Mito-TEMPO, a mitochondrial superoxide scavenger. These results suggest that Meth enhances gp120-associated microglial NLRP3 activation and the resultant proinflammatory responses via mitochondria-dependent signaling.

Mito-TEMPO protects preimplantation porcine embryos against mitochondrial fission-driven apoptosis through DRP1/PINK1-mediated mitophagy

AimsMdivi-1 (Md-1) is a well-known inhibitor of mitochondrial fission and mitophagy. The mitochondrial superoxide scavenger Mito-TEMPO (MT) exerts positive effects on the developmental competence of pig embryos. This study aimed to explore the adverse effects of Md-1 on developmental capacity in porcine embryos and the protective effects of MT against Md-1-induced injury. Main methodsWe exposed porcine embryos to Md-1 (10 and 50 μM) for 2 days after in vitro fertilization (IVF). MT (0.1 μM) treatment was applied for 4 days after exposing embryos to Md-1. We assessed blastocyst development, DNA damage, mitochondrial superoxide production, and mitochondrial distribution using TUNEL assay, Mito-SOX, and Mito-tracker, respectively. Subsequently, the expression of PINK1, DRP1, and p-DRP1Ser616 was evaluated via immunofluorescence staining and Western blot analysis. Key findingsMd-1 compromised the developmental competence of blastocysts. Apoptosis and mitochondrial superoxide production were significantly upregulated in 50 μM Md-1-treated embryos, accompanied by a downregulation of p-DRP1Ser616, PINK1, and LC3B levels and lower mitophagy activity at the blastocyst stage. We confirmed the protective effects of MT against the detrimental effect of Md-1 on blastocyst developmental competence, mitochondrial fission, and DRP1/PINK1-mediated mitophagy activation. Eventually, MT recovered DRP1/PINK1-mediated mitophagy and mitochondrial fission by inhibiting superoxide production in Md-1-treated embryos. SignificanceMT protects against detrimental effects of Md-1 on porcine embryos by suppressing superoxide production. These findings expand available scientific knowledge on improving outcomes of IVF.

Cigarette smoke induces pulmonary arterial dysfunction through an imbalance in the redox status of the soluble guanylyl cyclase.

Chronic obstructive pulmonary disease (COPD), whose main risk factor is cigarette smoking (CS), is one of the most common diseases globally. Some COPD patients also develop pulmonary hypertension (PH), a severe complication that leads to premature death. Evidence suggests reactive oxygen species (ROS) involvement in COPD and PH, especially regarding pulmonary artery smooth muscle cells (PASMC) dysfunction. However, the effects of CS-driven oxidative stress on the pulmonary vasculature are not completely understood. Herein we provide evidence on the effects of CS extract (CSE) exposure on PASMC regarding ROS production, antioxidant response and its consequences on vascular tone dysregulation. Our results indicate that CSE exposure promotes mitochondrial fission, mitochondrial membrane depolarization and increased mitochondrial superoxide levels. However, this superoxide increase did not parallel a counterbalancing antioxidant response in human pulmonary artery (PA) cells. Interestingly, the mitochondrial superoxide scavenger mitoTEMPO reduced mitochondrial fission and membrane potential depolarization caused by CSE. As we have previously shown, CSE reduces PA vasoconstriction and vasodilation. In this respect, mitoTEMPO prevented the impaired nitric oxide-mediated vasodilation, while vasoconstriction remained reduced. Finally, we observed a CSE-driven downregulation of the Cyb5R3 enzyme, which prevents soluble guanylyl cyclase oxidation in PASMC. This might explain the CSE-mediated decrease in PA vasodilation. These results provide evidence that there might be a connection between mitochondrial ROS and altered vasodilation responses in PH secondary to COPD, and strongly support the potential of antioxidant strategies specifically targeting mitochondria as a new therapy for these diseases.

Neonatal Hyperoxia Activates Activating Transcription Factor 4 to Stimulate Folate Metabolism and Alveolar Epithelial Type 2 Cell Proliferation.

Oxygen supplementation in preterm infants disrupts alveolar epithelial type 2 (AT2) cell proliferation through poorly understood mechanisms. Here, newborn mice are used to understand how hyperoxia stimulates an early aberrant wave of AT2 cell proliferation that occurs between Postnatal Days (PNDs) 0 and 4. RNA-sequencing analysis of AT2 cells isolated from PND4 mice revealed hyperoxia stimulates expression of mitochondrial-specific methylenetetrahydrofolate dehydrogenase 2 and other genes involved in mitochondrial one-carbon coupled folate metabolism and serine synthesis. The same genes are induced when AT2 cells normally proliferate on PND7 and when they proliferate in response to the mitogen fibroblast growth factor 7. However, hyperoxia selectively stimulated their expression via the stress-responsive activating transcription factor 4 (ATF4). Administration of the mitochondrial superoxide scavenger mitoTEMPO during hyperoxia suppressed ATF4 and thus early AT2 cell proliferation, but it had no effect on normative AT2 cell proliferation seen on PND7. Because ATF4 and methylenetetrahydrofolate dehydrogenase are detected in hyperplastic AT2 cells of preterm infant humans and baboons with bronchopulmonary dysplasia, dampening mitochondrial oxidative stress and ATF4 activation may provide new opportunities for controlling excess AT2 cell proliferation in neonatal lung disease.

Abstract 119: Mitochondrial Redox Mechanisms Leading To Sustained Radiation-induced Endothelial Injury

Rationale: Radiation therapy strongly increases the risk of developing atherosclerotic vascular disease, such as coronary and carotid artery disease. Recently, we found that in vitro inhibition of the mitochondrial Ca 2+ uniporter (MCU) protects from endothelial barrier breakdown following irradiation. Objective: To determine the mechanisms by which irradiation initiates vascular wall injury and test potential mitigators. Methods and Results: At 24 and 240 h After head and neck irradiation with 12 Gy x-ray, decreased endothelium-dependent vasodilation was seen in carotid arteries of C57Bl/6 mice. This was prevented by pre-infusion of the mitochondrial superoxide scavenger mitoTEMPO. Altered dilation correlated with increased mitochondrial ROS, loss of NO production and mitochondrial, but not nuclear DNA damage in human coronary endothelial cells in vitro. Enhancing mitochondrial in contrast to nuclear base excision repair by overexpression of subcellular targeted 8-Oxoguanine glycosylase blocked mitochondrial ROS and maintained NO production at all time points. Similarly, treatment with pravastatin, knockdown of MCU or its pharmacologic inhibition blocked irradiation-induced mitochondrial DNA damage and excess oxidative stress while maintaining NO production. These findings were recapitulated in vivo in a transgenic model of endothelial MCU knockout and in mice pretreated with pravastatin at 24 and 240 h after irradiation. Irradiation hyperpolarized the mitochondrial membrane potential (αψ mito ) and increased baseline matrix Ca 2+ levels ([Ca 2+ ] m ) as well as Ca 2+ transients. MCU knockdown decreased αψ mito , [Ca 2+ ] m and Ca 2+ transients as did mitoTEMPO, whereas pravastatin pretreatment hyperpolarized αψ mito despite lowering [Ca 2+ ] m . Conclusion: These findings demonstrate that mitochondrial DNA damage after irradiation drives a feed-forward circuit with ROS production and is sufficient to maintain endothelial dysfunction. Irradiation hyperpolarizes αψ mito and blocking this or its sequela, increased [Ca 2+ ] m , prevents irradiation-induced endothelial injury. These findings also suggest additional mechanisms of statins in mitochondria and MCU as a new approach to mitigate irradiation-induced vascular disease.

Biomimetic Design of Mitochondria-Targeted Hybrid Nanozymes as Superoxide Scavengers.

Development of enzyme mimics for the scavenging of excessive mitochondrial superoxide (O2 •- ) can serve as an effective strategy in the treatment of many diseases. Here, protein reconstruction technology and nanotechnology is taken advantage of to biomimetically create an artificial hybrid nanozyme. These nanozymes consist of ferritin-heavy-chain-based protein as the enzyme scaffold and a metal nanoparticle core as the enzyme active center. This artificial cascade nanozyme possesses superoxide dismutase- and catalase-like activities and also targets mitochondria by overcoming multiple biological barriers. Using cardiac ischemia-reperfusion animal models, the protective advantages of the hybrid nanozymes are demonstrated in vivo during mitochondrial oxidative injury and in the recovery of heart functionality following infarction via systemic delivery and localized release from adhesive hydrogels (i.e., cardiac patch), respectively. This study illustrates a de novo design strategy in the development of enzyme mimics and provides a promising therapeutic option for alleviating oxidative damage in regenerative medicine.

Neonatal hyperoxia enhances age-dependent expression of SARS-CoV-2 receptors in mice

The severity of COVID-19 lung disease is higher in the elderly and people with pre-existing co-morbidities. People who were born preterm may be at greater risk for COVID-19 because their early exposure to oxygen (hyperoxia) at birth increases the severity of respiratory viral infections. Hyperoxia at birth increases the severity of influenza A virus infections in adult mice by reducing the number of alveolar epithelial type 2 (AT2) cells. Since AT2 cells express the SARS-CoV-2 receptors angiotensin converting enzyme (ACE2) and transmembrane protease/serine subfamily member 2 (TMPRSS2), their expression should decline as AT2 cells are depleted by hyperoxia. Instead, ACE2 was detected in airway Club cells and endothelial cells at birth, and then AT2 cells at one year of age. Neonatal hyperoxia stimulated expression of ACE2 in Club cells and in AT2 cells by 2 months of age. It also stimulated expression of TMPRSS2 in the lung. Increased expression of SARS-CoV-2 receptors was blocked by mitoTEMPO, a mitochondrial superoxide scavenger that reduced oxidative stress and DNA damage seen in oxygen-exposed mice. Our finding that hyperoxia enhances the age-dependent expression of SARS-CoV-2 receptors in mice helps explain why COVID-19 lung disease is greater in the elderly and people with pre-existing co-morbidities.

Mitochondrial dysfunction triggers a catabolic response in chondrocytes via ROS-mediated activation of the JNK/AP1 pathway.

Mitochondrial function is impaired in osteoarthritis (OA) but its impact on cartilage catabolism is not fully understood. Here, we investigated the molecular mechanism of mitochondrial dysfunction-induced activation of the catabolic response in chondrocytes. Using cartilage slices from normal and OA cartilage, we showed that mitochondrial membrane potential was lower in OA cartilage, and that this was associated with increased production of mitochondrial superoxide and catabolic genes [interleukin 6 (IL-6), COX-2 (also known as PTGS2), MMP-3, -9, -13 and ADAMTS5]. Pharmacological induction of mitochondrial dysfunction in chondrocytes and cartilage explants using carbonyl cyanide 3-chlorophenylhydrazone increased mitochondrial superoxide production and the expression of IL-6, COX-2, MMP-3, -9, -13 and ADAMTS5, and cartilage matrix degradation. Mitochondrial dysfunction-induced expression of catabolic genes was dependent on the JNK (herein referring to the JNK family)/activator protein 1 (AP1) pathway but not the NFκB pathway. Scavenging of mitochondrial superoxide with MitoTEMPO, or pharmacological inhibition of JNK or cFos and cJun, blocked the mitochondrial dysfunction-induced expression of the catabolic genes in chondrocytes. We demonstrate here that mitochondrial dysfunction contributes to OA pathogenesis via JNK/AP1-mediated expression of catabolic genes. Our data shows that AP1 could be used as a therapeutic target for OA management.This article has an associated First Person interview with the first author of the paper.

Cytoplasmic Radiation Induced Radio-Adaptive Response in Human Lung Fibroblast WI-38 Cells.

It has been reported that in cells exposed to low-dose radiation, radio-adaptive responses can be induced which make irradiated cells refractory to subsequent high-dose irradiation. However, whether adaptive responses are possible when only the cytoplasm, not the nucleus, of the cell is exposed to radiation is still unclear. In this study, using the proton microbeam facility at the National Institute of Radiological Sciences (Japan), we found that cytoplasmic irradiation activates radio-adaptive responses in normal human lung fibroblast WI-38 cells. Our results showed that when cells received cytoplasmic irradiation with 500 protons prior to 2 Gy or 6 Gy X-ray broad-beam irradiation, the DNA double-strand break levels were significantly reduced. In contrast, at cytoplasmic irradiation with less than 100 protons, the radio-adaptive response was not detected. Moreover, the time interval between cytoplasmic irradiation and whole-cell X-ray irradiation should be longer than 6 h for the induction of adaptive responses. In addition, cytoplasmic irradiation elevated the level of cellular mitochondrial superoxide, which enhanced the phosphorylation of extracellular signal-regulated kinases 1/2 (ERK 1/2) and its mediated nuclear accumulation of nuclear factor (erythroid-derived 2)-like 2 (NRF2). This signaling pathway contributed to cytoplasmic irradiation-induced adaptive response as supported by the observations that treatment with the mitochondrial superoxide scavenger mito-tempol, ERK 1/2 inhibitor U0126 or NRF2 inhibitor ML385 could repress the adaptive response. Overall, we showed that cytoplasmic irradiation induces radio-adaptive responses and that mitochondrial superoxide/ERK 1/2/NRF2 signaling is a mechanism. Our results provide new information on the biological effects induced by cytoplasm-targeted irradiation.

SVCT2-Dependent plasma and mitochondrial membrane transport of ascorbic acid in differentiating myoblasts

The Na+-dependent Vitamin C transporter 2 (SVCT2) is expressed in the plasma and mitochondrial membranes of various cell types. This notion was also established in proliferating C2C12 myoblasts (Mb), in which the transporter was characterised by a high and low affinity in the plasma and mitochondrial membranes, respectively. In addition, the mitochondrial expression of SVCT2 appeared particularly elevated and, consistently, a brief pre-exposure to low concentrations of Ascorbic Acid (AA) abolished mitochondrial superoxide formation selectively induced by the cocktail arsenite/ATP. Early myotubes (Mt) derived from these cells after 4 days of differentiation presented evidence of slightly increased SVCT2 expression, and were characterised by kinetic parameters for plasma membrane transport of AA in line with those detected in Mb. Confocal microscopy studies indicated that the mitochondrial expression of SVCT2 is well preserved in Mt with one or two nuclei, but progressively reduced in Mt with three or more nuclei. Cellular and mitochondrial expression of SVCT2 was found reduced in day 7 Mt. While the uptake studies were compromised by the poor purity of the mitochondrial preparations obtained from day 4 Mt, we nevertheless obtained evidence of poor transport of the vitamin using the same functional studies successfully employed with Mb. Indeed, even greater concentrations of/longer pre-exposure to AA failed to induce scavenging of mitochondrial superoxide in Mt. These results are therefore indicative of a severely reduced mitochondrial uptake of the vitamin in early Mt, attributable to decreased expression as well as impaired activity of mitochondrial SVCT2.

Manganese porphyrin, MnTE-2-PyP, treatment protects the prostate from radiation-induced fibrosis (RIF) by activating the NRF2 signaling pathway and enhancing SOD2 and sirtuin activity

Radiation therapy is a frequently used treatment for prostate cancer patients. Manganese (III) meso-tetrakis (N-ethylpyridinium-2-yl) porphyrin (MnTE-2-PyP or T2E or BMX-010) and other similar manganese porphyrin compounds that scavenge superoxide molecules have been demonstrated to be effective radioprotectors and prevent the development of radiation-induced fibrosis (RIF). However, understanding the molecular pathway changes associated with these compounds remains limited for radioprotection. Recent RNA-sequencing data from our laboratory revealed that MnTE-2-PyP treatment activated the nuclear factor erythroid 2-related factor 2 (NRF2) signaling pathway. Therefore, we hypothesize that MnTE-2-PyP protects the prostate from RIF by activating the NRF2 signaling pathway. We identified that MnTE-2-PyP is a post-translational activator of NRF2 signaling in prostate fibroblast cells, which plays a major role in fibroblast activation and myofibroblast differentiation. The mechanism of NRF2 activation involves an increase in hydrogen peroxide and a corresponding decrease in kelch-like ECH-associated protein 1 (KEAP1) levels. Activation of NRF2 signaling leads to an increase in expression of NAD(P)H dehydrogenase [quinone] 1 (NQO1), nicotinamide adenine dinucleotide (NAD+) levels, sirtuin activity (nuclear and mitochondrial), and superoxide dismutase 2 (SOD2) expression/activity. Increase in mitochondrial sirtuin activity correlates with a decrease in SOD2 (K122) acetylation. This decrease in SOD2 K122 acetylation correlates with an increase in SOD2 activity and mitochondrial superoxide scavenging capacity. Further, in human primary prostate fibroblast cells, the NRF2 pathway plays a major role in the fibroblast to myofibroblast transformation, which is responsible for the fibrotic phenotype. In the context of radiation protection, MnTE-2-PyP fails to prevent fibroblast to myofibroblast transformation in the absence of NRF2 signaling. Collectively, our results indicate that the activation of the NRF2 signaling pathway by MnTE-2-PyP is at least a partial mechanism of radioprotection in prostate fibroblast cells.

The impact of bilirubin ditaurate on platelet quality during storage

Bilirubin ditaurate (BRT), a conjugated bilirubin analogue, has demonstrated anti-platelet characteristics following acute ex vivo exposure. Scavenging of mitochondrial superoxide and attenuation of granule exocytosis suggested a potential benefit for including BRT for storage. With no reports of cytotoxicity following acute exposure, the impact of 35µM BRT on platelet function was investigated, in clinically suppled units, for up to seven days. Exposure to 35µM BRT significantly reduced mitochondrial membrane potential and increased glucose consumption until exhaustion after 72 hours. Platelet aggregation and activation was significantly impaired by BRT. Mitochondrial superoxide production and phosphatidylserine expression were significantly elevated following glucose exhaustion, with decreased viability observed from day five onwards. Lactate accumulation and loss of bicarbonate, support a metabolic disturbance, leading to a decline of quality following BRT inclusion. Although acute ex vivo BRT exposure reported potentially beneficial effects, translation from acute to chronic exposure failed to combat declining platelet function during storage. BRT exposure resulted in perturbations of platelet quality, with the utility of BRT during storage therefore limited. However, these are the first data of prolonged platelet exposure to analogues of conjugated bilirubin and may improve our understanding of platelet function in the context of conjugated hyperbilirubinemia.

Abstract GMM-029: MITOCHONDRIAL STRESS RESPONSE ADAPTATIONS ARE REQUIRED FOR OVARIAN CANCER ANCHORAGE-INDEPENDENT SURVIVAL AND METASTASIS

Abstract Late stage ovarian cancer is marked by poor patient survival and significant metastatic spread throughout the peritoneal cavity. During transcoelomic spread cells must adapt to survive anchorage-independence and to cope with stress associated with matrix detachment and the hostile environment of the ascites. Our findings demonstrate that an important ovarian cancer cell adaptation during anchorage-independence is the up-regulation of the mitochondrial stress response proteins, the mitochondrial antioxidant enzyme superoxide dismutase 2 (Sod2), and it's regulator, the nutrient and redox sensing deacetylase SIRT3. We show that these proteins are necessary for anchorage-independent cell survival and transcoelomic metastasis in vivo. In addition to Sod2's role as a mitochondrial superoxide scavenger, mechanistic data show that Sod2 shifts the redox landscape of cancer cells to a higher hydrogen peroxide (H2O2) steady-state. This novel non-canonical function of Sod2 results in H2O2-dependent oxidation and inactivation of phosphatases, including PTEN. Sod2/H2O2-dependent redox signaling may hence be a novel driver of anchorage-independent survival and an important contributor to the high frequency of Akt activation observed in metastatic ovarian cancers. While these sub-lethal increases in cellular H2O2 lead to pro-tumorigenic signaling, this also places cells closer to the cytotoxic threshold of H2O2, making cells with high Sod2 expression exquisitely sensitive to exogenous H2O2 application, and H2O2 generating agents, including high dose ascorbate. These findings provide an important clue to the vulnerability of Sod2 expressing cancer cells. Our findings also demonstrate that SIRT3 is an important regulator of Sod2 activity during anchorage-independence, which challenges previous studies demonstrating that SIRT3 is a tumor suppressor, and points to a novel pro-survival role for SIRT3 during ovarian cancer spread. Given that SIRT3 is sensitive to nutrient stress, this regulation has further consequences on the ability of ovarian cancer cells to survive potentially nutrient deprived tumor environments. Our study has identified two important mitochondrial stress response genes that are specifically increased in response to matrix detachment and required for anchorage independent survival and metastatic spread of ovarian cancer. Our ongoing studies are investigating their role in the context of redox and nutrient stress of ascites and targeting these stress adaptations for therapeutic intervention. Citation Format: Piyushi Gupta-Vallur, Yeon Soo Kim, Dong-Hui Shin, LP Madhubhani Hemachandra, Rebecca Phaeton, Karthikeyan Mythreye, and Nadine Hempel. MITOCHONDRIAL STRESS RESPONSE ADAPTATIONS ARE REQUIRED FOR OVARIAN CANCER ANCHORAGE-INDEPENDENT SURVIVAL AND METASTASIS [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-029.

Ryanodine Receptor to Mitochondrial Reactive Oxygen Species Pathway Plays an Important Role in Chronic Human Immunodeficiency Virus gp120MN-Induced Neuropathic Pain in Rats.

Chronic pain is one of the most common complaints in patients with human immunodeficiency virus (HIV)-associated sensory neuropathy. Ryanodine receptor (RyR) and mitochondrial oxidative stress are involved in neuropathic pain induced by nerve injury. Here, we investigated the role of RyR and mitochondrial superoxide in neuropathic pain induced by repeated intrathecal HIV glycoprotein 120 (gp120) injection. Recombinant HIV glycoprotein gp120MN was intrathecally administered to induce neuropathic pain. Mechanical threshold was tested using von Frey filaments. Peripheral nerve fiber was assessed by the quantification of the intraepidermal nerve fiber density in the skin of the hindpaw. The expression of spinal RyR was examined using Western blots. Colocalization of RyR with neuronal nuclei (NeuN; neuron marker), glial fibrillary acidic protein (GFAP; astrocyte marker), or ionizing calcium-binding adaptor molecule 1 (Iba1; microglia marker) in the spinal cord was examined using immunohistochemistry. MitoSox-positive profiles (a mitochondrial-targeted fluorescent superoxide indicator) were examined. The antiallodynic effects of intrathecal administration of RyR antagonist, dantrolene (a clinical drug for malignant hyperthermia management), or selective mitochondrial superoxide scavenger, Mito-Tempol, were evaluated in the model. We found that repeated but not single intrathecal injection of recombinant protein gp120 induced persistent mechanical allodynia. Intraepidermal nerve fibers in repeated gp120 group was lower than that in sham at 2 weeks, and the difference in means (95% confidence interval) was 8.495 (4.79-12.20), P = .0014. Repeated gp120 increased expression of RyR, and the difference in means (95% confidence interval) was 1.50 (0.504-2.495), P = .007. Repeated gp120 also increased mitochondrial superoxide cell number in the spinal cord, and the difference in means (95% confidence interval) was 6.99 (5.99-8.00), P < .0001. Inhibition of spinal RyR or selective mitochondrial superoxide scavenger dose dependently reduced mechanical allodynia induced by repeated gp120 injection. RyR and mitochondrial superoxide were colocalized in the neuron, but not glia. Intrathecal injection of RyR inhibitor lowered mitochondrial superoxide in the spinal cord dorsal horn in the gp120 neuropathic pain model. These data suggest that repeated intrathecal HIV gp120 injection induced an acute to chronic pain translation in rats, and that neuronal RyR and mitochondrial superoxide in the spinal cord dorsal horn played an important role in the HIV neuropathic pain model. The current results provide evidence for a novel approach to understanding the molecular mechanisms of HIV chronic pain and treating chronic pain in patients with HIV.

Voltage gated proton channels modulate mitochondrial reactive oxygen species production by complex I in renal medullary thick ascending limb

Hv1 is a voltage-gated proton channel highly expressed in immune cells where, it acts to maintain NAD(P)H oxidase activity during the respiratory burst. We have recently reported that Hv1 is expressed in cells of the medullary thick ascending limb (mTAL) of the kidney and is critical to augment reactive oxygen species (ROS) production by this segment. While Hv1 is associated with NOX2 mediated ROS production in immune cells, the source of the Hv1 dependent ROS in mTAL remains unknown. In the current study, the rate of ROS formation was quantified in freshly isolated mTAL using dihydroethidium and ethidium fluorescence. Hv1 dependent ROS production was stimulated by increasing bath osmolality and ammonium chloride (NH4Cl) loading. Loss of either p67phox or NOX4 did not abolish the formation of ROS in mTAL. Hv1 was localized to mitochondria within mTAL, and the mitochondrial superoxide scavenger mitoTEMPOL reduced ROS formation. Rotenone significantly increased ROS formation and decreased mitochondrial membrane potential in mTAL from wild-type rats, while treatment with this inhibitor decreased ROS formation and increased mitochondrial membrane potential in mTAL from Hv1−/− mutant rats. These data indicate that NADPH oxidase is not the primary source of Hv1 dependent ROS production in mTAL. Rather Hv1 localizes to the mitochondria in mTAL and modulates the formation of ROS by complex I. These data provide a potential explanation for the effects of Hv1 on ROS production in cells independent of its contribution to maintenance of cell membrane potential and intracellular pH.

Treadmill Exercise Ameliorates Depression-Like Behavior in the Rats With Prenatal Dexamethasone Exposure: The Role of Hippocampal Mitochondria.

Prenatal exposure to synthetic glucocorticoids (sGCs) can increase the risk of affective disorders, such as depression, in adulthood. Given that exercise training can ameliorate depression and improve mitochondrial function, we sought to investigate whether exercise can ameliorate depression-like behavior induced by prenatal sGC exposure and mitochondria function contributes to that behavior. At first, we confirmed that prenatal dexamethasone (Dex) administration in late pregnancy resulted in depression-like behavior and elevated level of circulatory corticosterone in adult offspring. We then found that mRNA and protein expression of a number of mitochondrial genes was changed in the hippocampus of Dex offspring. Mitochondria in the hippocampus showed abnormal morphology, oxidative stress and dysfunction in Dex offspring. Intracerebroventricular (ICV) injection of the mitochondrial superoxide scavenger mitoTEMPO significantly alleviated depression-like behavior but did not significantly affect circulatory corticosterone level in Dex offspring. The adult Dex offspring treated with treadmill exercise starting at four-weeks of age showed ameliorated depressive-like behavior, improved mitochondrial morphology and function and reduced circulatory corticosterone level. Our data suggest mitochondria dysfunction contributes to depression-like behavior caused by prenatal sGC exposure. Intervention with exercise training in early life can reverse depression caused by prenatal Dex exposure, which is associated with improvement of mitochondrial function in the hippocampus.

Elevated peritoneal fluid ceramides in human endometriosis-associated infertility and their effects on mouse oocyte maturation

To characterize the peritoneal fluid (PF) sphingolipid profile in endometriosis-associated infertility (EAI), and to assess the plausible functional role(s) of ceramides in oocyte maturation potential. Retrospective case-control study and invitro mouse oocyte study. University-affiliated hospital and university laboratory. Twenty-seven infertile patients diagnosed with endometriosis and 20 infertile patients who did not have endometriosis; BALB/c female mice. None. PF sphingolipid concentrations. Number of metaphase II (MII) mouse oocytes. Liquid chromatography-tandem mass spectrometry revealed 11 significantly elevated PF sphingolipids in infertile women with severe endometriosis compared with infertile women without endometriosis (change >50%, false discovery rate ≤10%). Logistic regression analysis identified three very-long-chain ceramides potentially associated with EAI. Functional studies revealed that very-long-chain ceramides may compromise or induce murine MII oocyte maturation. The oocyte maturation effects induced by the very long-chain ceramides were triggered by alterations in mitochondrial superoxide production in a concentration-dependent manner. Scavenging of mitochondrial superoxide reversed the maturation effects of C24:0 ceramide. EAI is associated with accumulation of PF very-long-chain ceramides. Mouse studies demonstrated how ceramides affect MII oocyte maturation, mediating through mitochondrial superoxide. These results provide an opportunity for direct functional readout of pathophysiology in EAI, and future therapies targeted at this sphingolipid metabolism may be harnessed for improved oocyte maturation.

PO-173 Critical requirement of the Sos1 and Sos2 RasGEFs for maintenance of mitochondrial homeostasis

IntroductionThe homologous, ubiquitously expressed, members (Sos1 and Sos2) of the Sos family of RasGEFs participate in multiple signalling pathways but their specific cellular functions are not clearly defined yet.Material and methodsUsing a tamoxifen (4OHT)-inducible, conditional Sos1 null mutation, here we generated and analysed wild type (WT), single Sos1-KO, constitutive Sos2-KO and double Sos1/2-DKO primary mouse embryonic fibroblasts (MEFs) in an effort to ascertain the functional specificity or redundancy of Sos1 and Sos2 at the cellular level.Results and discussionsSos1-KO and Sos1/2-DKO MEFs exhibited distinct flat morphology, enlarged cell perimeter and altered cytoskeletal organisation that were not observed in WT and Sos2 KO counterparts. Sos1-KO and Sos1/2-DKO MEFs also displayed significant accumulation of cytoplasmic bodies identified as autophagosomes containing degraded mitochondria by means of electron microscopy and specific markers. Consistent with a mitophagic phenotype, in vivolabelling using specific fluorophores revealed increased levels of mitochondrial oxidative stress in the Sos1-KO and the Sos1/2-DKO cells as compared to Sos2-KO or WT MEFs. Treatment of the MEF cultures with antioxidants such as GSH and NAC corrected the altered perimeter size and proliferative rate of Sos1-KO and Sos1/2-DKO MEFs to levels similar to those of WT and Sos2-KO, but not recover oxidative stress. Furthermore, treatment with the specific mitochondrial superoxide scavenger mitoTEMPO recovered endogenous redox-homeostasis in Sos1-KO and Sos1/2-DKO to normal levels. Analysis of MEFs concomitantly loaded with MitoTracker Green (Δψ-independent) and MitoTracker Red (Δψ-dependent) showed a significantly increased of dysfunctional mitochondria in the Sos1/2-DKO MEFs in comparison to all other genotypes. Finally, Seahorse-based measurements of mitochondrial parameters showed that basal and maximal mitochondrial respiration, spare respiratory capacity, and ATP production, were also significantly decreased in Sos1/2-DKO cells as compared to MEFs of all the other genotypes.ConclusionOur data uncover a direct mechanistic link between Sos RasGEF proteins and the control of mitochondrial oxidative stress and respiration. Dysfunctional mitochondria are a key element in a variety of serious diseases, including cancer, and thus a promising therapeutic target.This work was supported by grants PI16/02137 (FIS) and CIBERONC (ONCG19//2017) from ISCIII; SA043U16 from FEDER-JCyL; FS/35–2017 from FSB and AECC, Spain.