Mitochondria-targeted Antioxidant MitoQ Research Articles

The mitochondria-targeted antioxidant MitoQ ameliorates inorganic arsenic-induced DCs/Th1/Th2/Th17/Treg differentiation partially by activating PINK1-mediated mitophagy in murine liver

Inorganic arsenic is a well-established environmental toxicant linked to acute liver injury, fibrosis, and cancer. While oxidative stress, pyroptosis, and ferroptosis are known contributors, the role of PTEN-induced kinase 1 (PINK1)-mediated mitophagy in arsenic-induced hepatic immunotoxicity remains underexplored. Our study revealed that acute arsenic exposure prompts differentiation of hepatic dendritic cells (DCs) and T helper (Th) 1, Th2, Th17, and regulatory T (Treg) cells, alongside increased transcription factors and cytokines. Inorganic arsenic triggered liver redox imbalance, leading to elevated alanine transaminase (ALT), hydrogen peroxide (H2O2), malondialdehyde (MDA), and activation of nuclear factor erythroid 2-related factor (Nrf2)/heme oxygenase-1 (HO-1) pathway. PINK1-mediated mitophagy was initiated, and its inhibition exacerbates H2O2 accumulation while promoting DCs/Th1/Th2/Treg differentiation in the liver of arsenic-exposed mice. Mitoquinone (MitoQ) pretreatment relieved arsenic-induced acute liver injury and immune imbalance by activating Nrf2/HO-1 and PINK1-mediated mitophagy. To our knowledge, this is the first report identifying PINK1-mediated mitophagy as a protective factor against inorganic arsenic-induced hepatic DCs/Th1/Th2 differentiation. This study has provided new insights on the immunotoxicity of inorganic arsenic and established a foundation for exploring preventive and therapeutic strategies targeting PINK1-mediated mitophagy in acute liver injury. Consequently, the application of mitochondrial antioxidant MitoQ may offer a promising treatment for the metalloid-induced acute liver injury.

Elevated intracellular Ca2+ functions downstream of mitodysfunction to induce Wallerian-like degeneration and necroptosis in organophosphorus-induced delayed neuropathy

Neurotoxic organophosphorus compounds can induce a type of delayed neuropathy in humans and sensitive animals, known as organophosphorus-induced delayed neuropathy (OPIDN). OPIDN is characterized by axonal degeneration akin to Wallerian-like degeneration, which is thought to be caused by increased intra-axonal Ca2+ concentrations. This study was designed to investigate that deregulated cytosolic Ca2+ may function downstream of mitodysfunction in activating Wallerian-like degeneration and necroptosis in OPIDN. Adult hens were administrated a single dosage of 750 mg/kg tri-ortho-cresyl phosphate (TOCP), and then sacrificed at 1 day, 5 day, 10 day and 21 day post-exposure, respectively. Sciatic nerves and spinal cords were examined for pathological changes and proteins expression related to Wallerian-like degeneration and necroptosis. In vitro experiments using differentiated neuro-2a (N2a) cells were conducted to investigate the relationship among mitochondrial dysfunction, Ca2+ influx, axonal degeneration, and necroptosis. The cells were co-administered with the Ca2+-chelator BAPTA-AM, the TRPA1 channel inhibitor HC030031, the RIPK1 inhibitor Necrostatin-1, and the mitochondrial-targeted antioxidant MitoQ along with TOCP. Results demonstrated an increase in cytosolic calcium concentration and key proteins associated with Wallerian degeneration and necroptosis in both in vivo and in vitro models after TOCP exposure. Moreover, co-administration with BATPA-AM or HC030031 significantly attenuated the loss of NMNAT2 and STMN2 in N2a cells, as well as the upregulation of SARM1, RIPK1 and p-MLKL. In contrast, Necrostatin-1 treatment only inhibited the TOCP-induced elevation of p-MLKL. Notably, pharmacological protection of mitochondrial function with MitoQ effectively alleviated the increase in intracellular Ca2+ following TOCP and mitigated axonal degeneration and necroptosis in N2a cells, supporting mitochondrial dysfunction as an upstream event of the intracellular Ca2+ imbalance and neuronal damage in OPIDN. These findings suggest that mitochondrial dysfunction post-TOCP intoxication leads to an elevated intracellular Ca2+ concentration, which plays a pivotal role in the initiation and development of OPIDN through inducing SARM1-mediated axonal degeneration and activating the necroptotic signaling pathway.

Abstract 4129: Mitochondria-targeted antioxidant MitoQ inhibits human breast cancer metastasis in mice

Abstract In oncology, the occurrence of distant metastases often marks the transition from curative to palliative care. Such outcome is highly predictable for triple-negative and HER2-positive breast cancer patients, even if tumors are detected early, and there is no treatment to prevent metastasis. Here, considering that mitochondria are bioenergetic sensors of the tumor microenvironment, we report in preclinical mouse models that human breast cancer relapse is not a fatality: local recurrence and metastatic dissemination can be prevented by MitoQ, a mitochondria-targeted antioxidant that already underwent Phase I safety and Phase II efficacy trials in humans for pathologies other than cancer. Metastatic prevention was confirmed in the MMTV-PyMT model of spontaneously metastatic mouse breast cancer. After oral delivery, MitoQ was effective at nanomolar concentrations at which nonmalignant cells were spared. In anticipation of future clinical trials, a robust signature of the response to MitoQ was defined, applicable to bulk primary tumor biopsies. Citation Format: Tania Capeloa, Pierre Sonveaux. Mitochondria-targeted antioxidant MitoQ inhibits human breast cancer metastasis in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4129.

In vivo mitochondria-targeted protection against uterine artery vascular dysfunction and remodelling in rodent hypoxic pregnancy.

Gestational hypoxia adversely affects uterine artery function, increasing complications. However, an effective therapy remains unidentified. Here, we show in rodent uterine arteries that hypoxic pregnancy promotes hypertrophic remodelling, increases constrictor reactivity via protein kinase C signalling, and triggers compensatory dilatation via nitric oxide-dependent mechanisms and stimulation of large conductance Ca2+ -activated K+ -channels. Maternal in vivo oral treatment with the mitochondria-targeted antioxidant MitoQ in hypoxic pregnancy normalises uterine artery reactivity and prevents vascular remodelling. From days 6-20 of gestation (term ∼22 days), female Wistar rats were randomly assigned to normoxic or hypoxic (13-14% O2 ) pregnancy±daily maternal MitoQ treatment (500 µm in drinking water). At 20 days of gestation, maternal, placental and fetal tissue was frozen to determine MitoQ uptake. The uterine arteries were harvested and, in one segment, constrictor and dilator reactivity was determined by wire myography. Another segment was fixed for unbiased stereological analysis of vessel morphology. Maternal administration of MitoQ in both normoxic and hypoxic pregnancy crossed the placenta and was present in all tissues analysed. Hypoxia increased uterine artery constrictor responses to norepinephrine, angiotensin II and the protein kinase C activator, phorbol 12,13-dibutyrate. Hypoxia enhanced dilator reactivity to sodium nitroprusside, the large conductance Ca2+ -activated K+ -channel activator NS1619 and ACh via increased nitric oxide-dependent mechanisms. Uterine arteries from hypoxic pregnancy showed increased wall thickness and MitoQ treatment in hypoxic pregnancy prevented all effects on uterine artery reactivity and remodelling. The data support mitochondria-targeted therapy against adverse changes in uterine artery structure and function in high-risk pregnancy. KEY POINTS: Dysfunction and remodelling of the uterine artery are strongly implicated in many pregnancy complications, including advanced maternal age, maternal hypertension of pregnancy, maternal obesity, gestational diabetes and pregnancy at high altitude. Such complications not only have immediate adverse effects on the growth of the fetus, but also they can also increase the risk of cardiovascular disease in the mother and offspring. Despite this, there is a significant unmet clinical need for therapeutics that treat uterine artery vascular dysfunction in adverse pregnancy. Here, we show in a rodent model of gestational hypoxia that in vivo oral treatment of the mitochondria-targeted antioxidant MitoQ protects against uterine artery vascular dysfunction and remodelling, supporting the use of mitochondria-targeted therapy against adverse changes in uterine artery structure and function in high-risk pregnancy.

Selpercatinib combination with the mitochondria-targeted antioxidant MitoQ effectively suppresses RET–mutant thyroid cancer

Genetic alternation of REarranged during Transfection (RET) that leads to constitutive RET activation is a crucial etiological factor for thyroid cancer. RET is known to regulate mitochondrial processes, although the underlying molecular mechanisms remain unclear. We previously showed that the multi-kinase inhibitors vandetanib and cabozantinib increase the mitochondrial membrane potential (Δψm) in RET-mutated thyroid tumor cells and that this effect can be exploited to increase mitochondrial enrichment of Δψm-sensitive agents in the tumor cells. In this study, we hypothesized that the RET-selective inhibitor, selpercatinib, can increase Δψm and, subsequently, tumor cell uptake of the mitochondria-targeted ubiquinone (MitoQ) to the level to break the mitochondrial homeostasis and induce lethal responses in RET-mutated thyroid tumor cells. We show that selpercatinib significantly increased Δψm, and its combination with MitoQ synergistically suppressed RET-mutated human thyroid tumor cells, which we validated using RET-targeted genetic approaches. Selpercatinib and MitoQ, in combination, also suppressed CCDC6-RET fusion cell line xenografts in mice and prolonged animal survival more effectively than single treatments of each agent. Moreover, we treated two patients with CCDC6-RET or RETM918T thyroid cancer, who could not take selpercatinib at regular doses due to adverse effects, with a dose-reduced selpercatinib and MitoQ combination. In response to this combination therapy, both patients showed tumor reduction. The quality of life of one patient significantly improved over a year until the tumor relapsed. This combination of selpercatinib with MitoQ may have therapeutic potential for patients with RET-mutated tumors and intolerant to regular selpercatinib doses.

Redox processes are major regulators of leukotriene synthesis in neutrophils exposed to bacteria Salmonella typhimurium; the way to manipulate neutrophil swarming.

Neutrophils play a primary role in protecting our body from pathogens. When confronted with invading bacteria, neutrophils begin to produce leukotriene B4, a potent chemoattractant that, in cooperation with the primary bacterial chemoattractant fMLP, stimulates the formation of swarms of neutrophils surrounding pathogens. Here we describe a complex redox regulation that either stimulates or inhibits fMLP-induced leukotriene synthesis in an experimental model of neutrophils interacting with Salmonella typhimurium. The scavenging of mitochondrial reactive oxygen species by mitochondria-targeted antioxidants MitoQ and SkQ1, as well as inhibition of their production by mitochondrial inhibitors, inhibit the synthesis of leukotrienes regardless of the cessation of oxidative phosphorylation. On the contrary, antioxidants N-acetylcysteine and sodium hydrosulfide promoting reductive shift in the reversible thiol-disulfide system stimulate the synthesis of leukotrienes. Diamide that oxidizes glutathione at high concentrations inhibits leukotriene synthesis, and the glutathione precursor S-adenosyl-L-methionine prevents this inhibition. Diamide-dependent inhibition is also prevented by diphenyleneiodonium, presumably through inhibition of NADPH oxidase and NADPH accumulation. Thus, during bacterial infection, maintaining the reduced state of glutathione in neutrophils plays a decisive role in the synthesis of leukotriene B4. Suppression of excess leukotriene synthesis is an effective strategy for treating various inflammatory pathologies. Our data suggest that the use of mitochondria-targeted antioxidants may be promising for this purpose, whereas known thiol-based antioxidants, such as N-acetylcysteine, may dangerously stimulate leukotriene synthesis by neutrophils during severe pathogenic infection.

Contribution of Mitochondrial Reactive Oxygen Species to Chronic Hypoxia-Induced Pulmonary Hypertension.

Pulmonary hypertension (PH) resulting from chronic hypoxia (CH) occurs in patients with chronic obstructive pulmonary diseases, sleep apnea, and restrictive lung diseases, as well as in residents at high altitude. Previous studies from our group and others demonstrate a detrimental role of reactive oxygen species (ROS) in the pathogenesis of CH-induced PH, although the subcellular sources of ROS are not fully understood. We hypothesized that mitochondria-derived ROS (mtROS) contribute to enhanced vasoconstrictor reactivity and PH following CH. To test the hypothesis, we exposed rats to 4 weeks of hypobaric hypoxia (PB ≈ 380 mmHg), with control rats housed in ambient air (PB ≈ 630 mmHg). Chronic oral administration of the mitochondria-targeted antioxidant MitoQ attenuated CH-induced decreases in pulmonary artery (PA) acceleration time, increases in right ventricular systolic pressure, right ventricular hypertrophy, and pulmonary arterial remodeling. In addition, endothelium-intact PAs from CH rats exhibited a significantly greater basal tone compared to those from control animals, as was eliminated via MitoQ. CH also augmented the basal tone in endothelium-disrupted PAs, a response associated with increased mtROS production in primary PA smooth muscle cells (PASMCs) from CH rats. However, we further uncovered an effect of NO synthase inhibition with Nω-nitro-L-arginine (L-NNA) to unmask a potent endothelial vasoconstrictor influence that accentuates mtROS-dependent vasoconstriction following CH. This basal tone augmentation in the presence of L-NNA disappeared following combined endothelin A and B receptor blockade with BQ123 and BQ788. The effects of using CH to augment vasoconstriction and PASMC mtROS production in exogenous endothelin 1 (ET-1) were similarly prevented by MitoQ. We conclude that mtROS participate in the development of CH-induced PH. Furthermore, mtROS signaling in PASMCs is centrally involved in enhanced pulmonary arterial constriction following CH, a response potentiated by endogenous ET-1.

Preservation of the quality and fertility potential of post-thawed rooster sperm using MitoQ

Cryopreservation of rooster spermatozoa is an efficient procedure to spread qualified semen samples for reproductive goals in commercial flocks, but the freeze-thawing process reduces the quality and fertility potential of post-thawed sperm cells. This study was aimed to investigate the effect of the mitochondria-targeted antioxidant MitoQ on rooster sperm quality and fertility potential preservation during freeze-thawing process. Semen samples were collected and diluted in the Lake medium, assigned into five equal aliquots, supplemented with 0, 1, 10, 100 and 1000 nM MitoQ, and cryopreserved in liquid nitrogen. After thawing, sperm motility, membrane functionality, abnormal morphology, mitochondria active potential, acrosome integrity, viability, apoptosis status, lipid peroxidation, DNA fragmentation, ROS concentration and fertility potential were evaluated. According to the results, freezing extender supplementation with 10 and 100 nM MitoQ presented higher (P ≤ 0.05) total motility, progressive motility, average path velocity, membrane functionality, mitochondria active potential, acrosome integrity and viability compared to the other groups. On the other hand, lipid peroxidation, apoptosis rate, DNA fragmentation and ROS concentration were lower (P ≤ 0.05) in groups received 10 and 100 nM MitoQ compared to other groups. Moreover, fertility rate was higher in groups received 10 and 100 nM MitoQ compared to control group. Therefore, MitoQ is able to preserve quality parameters and fertility potential of post-thawed spermatozoa in rooster and it could be an effective additive for supplementation of rooster's semen cryopreservation medium during reproductive programs.

(226) Mito-Q, a Mitochondrial-targeted Antioxidant, Partially Improves Relaxation of the Corpus Cavernosum in Aged Diabetic Mice

Abstract Introduction Erectile dysfunction is associated with increased contraction and decreased relaxation of the corpus cavernosum (CC). Excessive reactive oxygen species produced by mitochondria play a key role in aging- and diabetes-induced erectile dysfunction. Erectile dysfunction is associated with increased contraction and decreased relaxation of the corpus cavernosum. Mitochondrial deterioration is part of normal physiological aging, and it plays a role in the pathogenesis of age-related disorders by producing reactive oxygen species (ROS). It is also known that excessive ROS produced by mitochondria plays a key role in aging- and diabetes-induced erectile dysfunction. Objective We aimed to test the hypothesis that in vitro inhibition of mitochondrial oxidative stress improves the reactivity of the CC. Methods To test this hypothesis, the CC of young (3 to 4-month-old) and old (10 to 14-month-old) male lean and diabetic (dbdb-/-) mice were mounted in myographs. Concentration-response curves to acetylcholine (ACh 1 nM – 30 uM) and relaxation induced by electrical field stimulation (EFS – 1, 2, 4, 8 and 16 Hz) were obtained. All curves were repeated after 30 minutes of incubation with the antioxidant Mito-Q (100 nM). Results Results: Relaxation to ACh was significantly reduced in the CC of diabetic mice (Lean: 87 ± 8% vs dbdb-/-: 60 ± 6%). Similarly, aged lean mice presented impaired relaxation to ACh (Lean old: 54 ± 6%) while old diabetic mice displayed a further decrease in the relaxing response to ACh (35 ± 6%). After 30 minutes of incubation with Mito-Q, the relaxation produced by ACh was improved in the CC of the aged lean mice (62 ± 3%) and aged diabetic mice (44 ± 1%). Relaxing responses to EFS were significantly decreased in obese and aged mice compared to the lean young animals. However, no improvement in the relaxing response to EFS was seen after incubation with Mito-Q. Conclusions Impairment in the endothelium-dependent relaxation of the CC induced by aging and diabetes is associated with increased production of mitochondrial reactive oxygen species. The use of the mitochondrial-targeted antioxidant Mito-Q improved at least in part the relaxation of the CC in both aged and diabetic mice. Disclosure No

Chronic mitochondria-targeted antioxidant supplementation in older adults alters circulating factors to improve endothelial cell function

Age-related vascular endothelial dysfunction, characterized by reduced nitric oxide (NO) bioavailability, is mediated in part by excess production of mitochondrial reactive oxygen species (mtROS). Excess mtROS also oxidizes circulating lipids (i.e., low-density lipoprotein [LDL]) to promote endothelial dysfunction with aging. We recently showed that 6-weeks of treatment with the mitochondria-targeted antioxidant MitoQ (MQ) (20 mg/day) improved endothelial function in vivo in older adults. The improvements in endothelial function with MQ were mediated by reduced mtROS and associated with lower plasma levels of oxidized LDL (oxLDL). However, the complete mechanisms by which MQ improves endothelial function, and if MQ-related decreases in oxLDL are mechanistically involved, remain unknown. PURPOSE. To determine if chronic MQ supplementation-mediated changes in the circulating milieu contribute to improvements in endothelial cell (EC) function and the mechanistic role of reductions in oxLDL. METHODS. Plasma samples were obtained 24 h after the last dose from 19 older adults (mean±SEM: 67±1 yrs; 11 women) who completed our crossover design pilot trial to assess the efficacy of 6-weeks of MQ vs. placebo (PLA) treatment for improving endothelial function. Using an ex vivo model of endothelial function, acetylcholine (Ach)-stimulated NO bioavailability (DAR-4M-AM) was quantified in human aortic ECs (HAECs) following exposure to plasma collected after each phase of the study (MQ and PLA). The role of mtROS production (MitoSOX) in plasma-mediated changes in HAEC function was also assessed. To elucidate the role of lower oxLDL with MQ treatment on HAEC function, physiological levels of oxLDL were ‘added back’ to plasma collected from subjects after MQ treatment at a concentration corresponding to the average reduction after MQ (14 ng/mL). To determine if lower oxLDL with MQ treatment improves HAEC function through a reduction in binding to the oxLDL receptor LOX-1, HAECs were subjected to LOX-1 blocking antibodies prior to plasma exposure under the 3 experimental conditions (PLA; MQ; MQ + oxLDL [‘add back’]) in a subset of subjects (N=9; 67±1 yrs; 5 women). RESULTS. Ach-stimulated NO bioavailability was higher (PLA: 1.0±0.05; MQ: 1.24±0.08; P=0.0002) and mtROS was lower (PLA: 1.0±0.09; MQ: 0.76±0.08; P=0.003) in HAECs exposed to plasma collected from subjects after MQ treatment compared to PLA. Normalizing oxLDL (‘add back’) abolished the effects of MitoQ treatment. LOX-1 blockade modulated Ach-stimulated NO and mtROS production to levels comparable to (P>0.98) or lower than (P<0.047) MQ in all 3 groups, respectively. CONCLUSIONS. Chronic MQ supplementation in older adults alters circulating factors by decreasing plasma oxLDL to reduce oxLDL-LOX-1 signaling-mediated mtROS production and improve EC function. These findings extend insight into the mechanisms by which mitochondria-targeted antioxidant treatment improves endothelial function with aging. 1R21AG049451-01A1 (DRS); 5T32DK007135-46 (KOM); K01DK115524-01A1 (MJR) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

TET2 confers a mechanistic link of microRNA-210 and mtROS in hypoxia-suppressed spontaneous transient outward currents in uterine arteries of pregnant sheep.

Hypoxia during pregnancy impairs uterine vascular adaptation via microRNA-210 (miR-210)-mediated mitochondrial dysfunction and mitochondrial reactive oxygen species (mtROS) generation. TET methylcytosine dioxygenase 2 (TET2) participates in regulating inflammation and oxidative stress and its deficiency contributes to the pathogenesis of multiple cardiovascular diseases. Thus, we hypothesize a role of TET2 in hypoxia/miR-210-mediated mtROS suppressing spontaneous transient outward currents (STOCs) in uterine arteries. We found that gestational hypoxia downregulated TET2 in uterine arteries of pregnant sheep and TET2 was a target of miR-210. Knockdown of TET2 with small interfering RNAs suppressed mitochondrial respiration, increased mtROS, inhibited STOCs and elevated myogenic tone. By contrast, overexpression of TET2 negated hypoxia- and miR-210-induced mtROS. The effects of TET2 knockdown in uterine arteries on mtROS, STOCs and myogenic contractions were blocked by the mitochondria-targeted antioxidant MitoQ. In addition, the recovery effects of inhibiting endogenous miR-210 with miR-210-LNA on hypoxia-induced suppression of STOCs and augmentation of myogenic tone were reversed by TET2 knockdown in uterine arteries. Together, our study reveals a novel mechanistic link between the miR-210-TET2-mtROS pathway and inhibition of STOCs and provides new insights into the understanding of uterine vascular maladaptation in pregnancy complications associated with gestational hypoxia. KEY POINTS: Gestational hypoxia downregulates TET methylcytosine dioxygenase 2 (TET2) in uterine arteries of pregnant sheep. TET2 is a downstream target of microRNA-210 (miR-210) and miR-210mediates hypoxia-induced TET2 downregulation. Knockdown of TET2 in uterine arteries recapitulates the effect of hypoxia and miR-210 and impairs mitochondrial bioenergetics and increases mitochondrial reactive oxygen species (mtROS) . Overexpression of TET2 negates the effect of hypoxia and miR-210 on increasing mtROS. TET2 knockdown reiterates the effect of hypoxia and miR-210 and suppresses spontaneous transient outward currents (STOCs) and elevates myogenic tone, and these effects are blocked by MitoQ. Knockdown of TET2 reverses the miR-210-LNA-induced reversal of the effects of hypoxia on STOCs and myogenic tone in uterine arteries.

Mitochondria-targeted antioxidant MitoQ ameliorates ROS production and improves cell viability in cryopreserved buffalo fibroblasts

Cryopreservation commonly decreases the cellular functionality and post-thaw viability of cells. Reactive oxygen species (ROS) generated during cryopreservation degrade mitochondrial activity and promote the release of cytochrome C which activates caspases required for apoptosis. Antioxidants have the potential to improve the recovery efficiency of cells by reducing ROS production and maintaining mitochondrial membrane potential (MMP). The present study was conducted to explore the role of MitoQ, a derivative of coenzyme Q10 on cryopreserved fibroblasts derived from buffalo skin. To achieve our goal, buffalo skin fibroblasts were treated with varying concentrations of MitoQ (0, 0.1, 0.5, 1, 2, and 10 μM) for 24, 48, and 72 h. The MMP, ROS generation, cell viability was measured by flow cytometry. Furthermore, expression of genes related to mitochondrial oxidative stress (NRF2, GPX, and SOD), apoptosis (BAK and caspase 3) and cell proliferation (AKT) were also assessed. The results showed that over a period of 72 h lower concentrations of MitoQ (0.1–0.5 μM) decrease the ROS production, improves MMP and cell viability whilst the high concentration of MitoQ (2–10 μM) increased the oxidative damage to the cells. Taken together, our study provide important insights into the novel role of MitoQ in cryopreserved buffalo skin fibroblasts. In conclusion, we demonstrated the dose-dependent functional role of MitoQ on cryopreserved fibroblasts for improving post-thaw cell viability and cellular function.

Redox signalling and proteostasis in neurodegeneration: In vivo effects of the mitochondria-targeted antioxidant MitoQ in Huntington's disease mice

Redox signalling and proteostasis in neurodegeneration: In vivo effects of the mitochondria-targeted antioxidant MitoQ in Huntington's disease mice

Protective effects of different doses of MitoQ separately and combined with trehalose on oxidative stress and sperm function of cryopreserved Markhoz goat semen

The mitochondria-targeted antioxidant MitoQ has been regarded as an effective antioxidant agent against cryo-induced oxidative cellular damage. This study aimed to evaluate the use of different doses of MitoQ combined with trehalose to minimize mitochondrial impairment and oxidative stress during sperm cryopreservation of Markhoz goat. For this, semen samples (n = 50) were collected by electroejaculation every 5 days from 5 bucks in 10 replicates. On each collection day, 5 ejaculates (one ejaculate for each buck) were pooled and then diluted in eight different Tris-based extenders as follows: no additives (control), 20, 200, 2000 nM of MitoQ (MT20, MT200, MT 2000, respectively), 150 mM of trehalose (Tr), MT20+Tr, MT200+Tr, MT2000+Tr. The semen samples were frozen using a standard protocol, and sperm function and oxidative stress were evaluated after thawing. The semen extender supplemented with MT200+Tr had higher (P < 0.05) total and progressive motility, acrosome and membrane integrity, superoxide dismutase, glutathione peroxidase, total antioxidant capacity, and lower (P < 0.05) DNA fragmentation, malondialdehyde and intracellular hydrogen peroxide levels than the all other groups except MT200; meanwhile, MT200 was also improved (P < 0.05) in these parameters than in the control group. Furthermore, MT200 and MT200+Tr showed higher (P < 0.05) percentages of live cryopreserved sperm with high mitochondrial activity than other groups. However, abnormality percentage and catalase activity of frozen-thawed sperm were not affected by treatments (P > 0.05). To conclude, we have found that supplementation of 200 nM MitoQ alone or in combination with 150 mM trehalose to semen extender improved the quality of cryopreserved sperm in goats, which is associated with enhanced antioxidant enzymatic defense and mitochondrial activity and reduced DNA fragmentation.

Selective and reversible disruption of mitochondrial inner membrane protein complexes by lipophilic cations

Triphenylphosphonium (TPP) derivatives are commonly used to target chemical into mitochondria. We show that alkyl-TPP cause reversible, dose- and hydrophobicity-dependent alterations of mitochondrial morphology and function and a selective decrease of mitochondrial inner membrane proteins including subunits of the respiratory chain complexes, as well as components of the mitochondrial calcium uniporter complex. The treatment with alkyl-TPP resulted in the cleavage of the pro-fusion and cristae organisation regulator Optic atrophy-1. The structural and functional effects of alkyl-TPP were found to be reversible and not merely due to loss of membrane potential. A similar effect was observed with the mitochondria-targeted antioxidant MitoQ.

Mitochondrial Regulation of Ferroptosis in Acute Myeloid Leukemia

Background: Investigations into targeting apoptosis in acute myeloid leukemia (AML) have resulted in breakthrough therapies utilizing Bcl-2 inhibition by venetoclax; however, acquired resistance poses major clinical challenges. Non-apoptotic regulated cell death (RCD) pathways have recently drawn attention especially in solid tumors, but studies targeting these pathways including ferroptosis in leukemia are limited. While ferroptosis is well-defined by iron- and lipid peroxidation-dependency, accumulating evidence suggests that there are also context-specific mechanisms. Since leukemia cells exhibit increased oxidative stress with particular dependency of cell survival on mitochondria, we hypothesized that AML exhibits a unique vulnerability to ferroptosis involving mitochondria. We here study targeting ferroptosis pathway in AML and its mitochondrial (mito-) regulation to develop mechanism-based combination with mito-targeting agents. Results: We first analyzed DepMap datasets to determine the essentiality of ferroptosis-regulating genes in cancer cell lines. Among major anti-ferroptosis genes, GPX4 exhibits high essentiality prominently in leukemia cells, suggesting its potential as a therapeutic target. AML patient survival data from TCGA revealed that high GPX4 expression is associated with shorter survival, indicating prognostic impact. GPX4 is the most downstream molecule to block ferroptosis by reducing lipid hydroperoxide. Although GPX4 is essential for mouse embryonic development, acquired depletion is reported to have no significant effect on hematopoietic stem cells, suggesting tolerability of GPX4-targeted therapies for normal hematopoiesis. We demonstrate that GPX4 inhibition with doxycycline-inducible shRNA or the specific inhibitor ML210 induces ferroptosis in leukemia cells, evidenced by iron-dependent lipid peroxidation and blockade of cell death by the iron chelator deferoxamine. Of note, the classical ferroptosis inducers (xCT inhibitors) sulfasalazine and sorafenib did not induce ferroptosis in AML cells, suggesting a specific relevance of GPX4 in AML ferroptosis. Consistent with solid tumor cells, AML cells show early (6h post-treatment) morphological alterations in mitochondria characterized by shrinkage and increased membrane density upon GPX4 inhibition (Figure 1). Importantly, and in contrast to most solid tumor studies, mitochondria-targeted antioxidants MitoQ and MitoTEMPO completely blocked ferroptosis, suggesting that AML ferroptosis is regulated predominantly by mitochondrial rather than cytoplasmic redox metabolism. To further determine the role of mitochondria in ferroptosis, we utilized HL60 rho0 cells that exhibit abrogated mito-respiration due to lack of mito-DNA. Surprisingly, HL60 rho0 cells were nearly 10 times more sensitive to ML210 than HL60 parental cells, in clear contrast to sarcoma studies demonstrating similar or lower sensitivity to ferroptosis of mito-DNA deficient cells. Consistently, degradation of the respiratory complex with hyperactivation of mito-protease ClpP by imipridone ONC212 sensitized cells to GPX4 inhibition in HL60 parental cells but not in rho0 cells, further supporting the role of mito-respiration in the protection of cells against ferroptosis. As venetoclax (Ven) is one of the most widely used mitochondria-targeting agents in AML that also inhibits mito-respiration, we tested the combination of GPX4 inhibitor ML210 and Ven. This combinatorial treatment exerts synergistic anti-leukemia effects in AML cell lines and primary cells from AML patients including those with Ven resistance (Figure 2). Unexpectedly, and as a novel mechanism of GPX4 inhibition-mediated ferroptosis, we found that ML210 induces BAX/BAK-independent cytochrome C release from mitochondria, which is blocked by MitoQ. Combination with Ven further enhanced cytochrome C release, as one of potential mechanisms of the observed synergistic cell death. Conclusion: Our data suggest therapeutic potential of ferroptosis induction in AML by targeting GPX4 with the mechanistic involvement of mito-respiration. The combination of GPX4 and BCL-2 inhibition is synergistic and capable of overcoming Ven resistance. Studies are in progress to elucidate the molecular link between mito-respiration/redox and cytochrome C release and to investigate ferroptosis induction in vivo. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Mitochondrial-targeted antioxidant supplementation for improving age-related vascular dysfunction in humans: A study protocol.

Background: Cardiovascular disease (CVD) is the leading cause of death worldwide and aging is the primary risk factor for the development of CVD. The increased risk of CVD with aging is largely mediated by the development of vascular dysfunction. Excessive production of mitochondrial reactive oxygen species (mtROS) is a key mechanism of age-related vascular dysfunction. Therefore, establishing the efficacy of therapies to reduce mtROS to improve vascular function with aging is of high biomedical importance. Previously, in a small, randomized, crossover-design pilot clinical trial, our laboratory obtained initial evidence that chronic oral supplementation with the mitochondrial-targeted antioxidant MitoQ improves vascular function in healthy older adults. Here, we describe the protocol for an ongoing R01-funded phase IIa clinical trial to establish the efficacy of MitoQ as a therapy to improve vascular function in older adults (ClinicalTrials.gov Identifier: NCT04851288). Outcomes: The primary outcome of the study is nitric oxide (NO)-mediated endothelium-dependent dilation (EDD) as assessed by brachial artery flow-mediated dilation (FMDBA). Secondary outcomes include mtROS-mediated suppression of EDD, aortic stiffness as measured by carotid-femoral pulse wave velocity, carotid compliance and β-stiffness index, and intima media thickness. Other outcomes include the assessment of endothelial mitochondrial health and oxidative stress in endothelial cells obtained by endovascular biopsy; the effect of altered circulating factors following MitoQ treatment on endothelial cell NO bioavailability and whole cell and mitochondrial reactive oxygen species production ex vivo; and circulating markers of oxidative stress, antioxidant status, and inflammation. Methods: We are conducting a randomized, placebo-controlled, double-blind, parallel group, phase IIa clinical trial in 90 (45/group) healthy older men and women 60 years of age or older. Participants complete baseline testing and are then randomized to either 3 months of oral MitoQ (20 mg; once daily) or placebo supplementation. Outcome measures are assessed at the midpoint of treatment, i.e., 6 weeks, and again at the conclusion of treatment. Discussion: This study is designed to establish the efficacy of chronic supplementation with the mitochondrial-targeted antioxidant MitoQ for improving vascular endothelial function and reducing large elastic artery stiffness in older adults, and to investigate the mechanisms by which MitoQ supplementation improves endothelial function.

MicroRNA‐210‐mediated mtROS confer hypoxia‐induced suppression of STOCs in ovine uterine arteries

Background and PurposeHypoxia during pregnancy is associated with increased uterine vascular resistance and elevated blood pressure both in women and female sheep. A previous study demonstrated a causal role of microRNA‐210 (miR‐210) in gestational hypoxia‐induced suppression of Ca2+ sparks/spontaneous transient outward currents (STOCs) in ovine uterine arteries, but the underlying mechanisms remain undetermined. We tested the hypothesis that miR‐210 perturbs mitochondrial metabolism and increases mitochondrial reactive oxygen species (mtROS) that confer hypoxia‐induced suppression of STOCs in uterine arteries.Experimental ApproachResistance‐sized uterine arteries were isolated from near‐term pregnant sheep and were treated ex vivo in normoxia and hypoxia (10.5% O2) for 48 h.Key ResultsHypoxia increased mtROS and suppressed mitochondrial respiration in uterine arteries, which were also produced by miR‐210 mimic to normoxic arteries and blocked by antagomir miR‐210‐LNA in hypoxic arteries. Hypoxia or miR‐210 mimic inhibited Ca2+ sparks/STOCs and increased uterine arterial myogenic tone, which were inhibited by the mitochondria‐targeted antioxidant MitoQ. Hypoxia and miR‐210 down‐regulated iron–sulfur cluster scaffold protein (ISCU) in uterine arteries and knockdown of ISCU via siRNAs suppressed mitochondrial respiration, increased mtROS, and inhibited STOCs. In addition, blockade of mitochondrial electron transport chain with antimycin and rotenone inhibited large‐conductance Ca2+‐activated K+ channels, decreased STOCs and increased uterine arterial myogenic tone.Conclusion and ImplicationsThis study demonstrates a novel mechanistic role for the miR‐210‐ISCU‐mtROS axis in inhibiting Ca2+ sparks/STOCs in the maladaptation of uterine arteries and provides new insights into the understanding of mitochondrial perturbations in the pathogenesis of pregnancy complications resulted from hypoxia.

P1563: INTRACELLULAR IRON OVERLOAD IMPAIRS HSC FUNCTION BY AFFECTING MITOCHONDRIAL FITNESS IN ΒETA-THALASSEMIA

Background: β-Thalassemia (BT) is a genetic hemolytic anemia caused by reduced or absent synthesis of hemoglobin β-chains. Iron overload (IO), associated with ineffective erythropoiesis and therapeutic blood transfusions, is one of the main complications, leading to morbidities in various organs. Despite the improvement in chelation therapies over the past years, IO toxicity is an important issue for a successful therapeutic outcome. Recently, we demonstrated an impaired function and activated transcriptional responses to stress in HSCs from thalassemic th3 mice. We hypothesized that IO and the resulting oxidative stress might impair the bone marrow (BM) niche, thus interfering with the maintenance of HSCs. We also reported that IO reduces the hematopoietic supportive capacity of BM mesenchymal stromal cells in BT patients. However, there is no evidence of the direct effect of IO on HSCs in BT. Aims: To understand the key factors affecting BT HSC function it is essential to gain novel insight into BT pathophysiology. To this aim, we investigated the role of iron on HSCs. Methods: Gene expression profiling of HSCs from th3 mice was assessed by RNAseq. IO and oxidative stress were analyzed by flow cytometry. Mitochondria were evaluated by flow cytometry and transmission electron microscopy. We studied HSC metabolism by ATP analysis after in vitro inhibition of the oxidative phosphorylation (OXPHOS) using oligomycin and glucose uptake assay by flow cytometry. HSC function was evaluated following in vivo treatment with the mitochondria-targeted antioxidant MitoQ. Results: We found a positive enrichment of genes involved in iron homeostasis, such as TfR1, Steap3, Fth1 and Ftl1, in th3 HSC transcriptome suggesting iron uptake and intracellular storage. These findings are corroborated by high levels of free reactive iron (Fe2+) in th3 HSCs. Interestingly, we reported an accumulation of Fe2+ in the mitochondria, correlating with a 2-fold increase in mitochondrial reactive oxygen species (ROS) content. As a result, mitochondria in th3 HSCs are impaired, with low mass and activity, as assessed by decreased mitochondrial membrane potential. Moreover, th3 HSCs showed a reduced expression of mitochondrial biogenesis and mitophagy genes, thus suggesting an accumulation of damaged mitochondria. In line with mitochondrial dysfunction, in vitro inhibition of OXPHOS did not affect ATP levels in th3 HSCs. Interestingly, they preferentially rely on glycolysis rather than OXPHOS for their energy demands, as shown by the positive enrichment of glycolytic genes and by the 1.4-fold higher glucose uptake of th3 HSCs. Finally, in vivo reduction of mitochondrial ROS by MitoQ administration rescues mitochondrial activity and th3 HSC quiescence. Ongoing studies on iron chelation will reveal how HSCs sense iron and the molecular mechanism leading to intracellular IO, providing clues for improving BT HSC function. Summary/Conclusion: Our study unveils that IO directly affects HSCs in BT. IO promotes oxidative stress, which in turn induces mitochondrial dysfunction. Alterations in mitochondrial activity and metabolic profile, in response to IO, are expected to negatively impact HSC function. This research will add novel contribution to the field of iron regulation and HSC biology by unraveling how intracellular iron and ROS affect the metabolic and transcriptional programs underlying HSC function and fate.

MitoQ supplementation augments acute exercise-induced increases in muscle PGC1α mRNA and improves training-induced increases in peak power independent of mitochondrial content and function in untrained middle-aged men

The role of mitochondrial ROS in signalling muscle adaptations to exercise training has not been explored in detail. We investigated the effect of supplementation with the mitochondria-targeted antioxidant MitoQ on a) the skeletal muscle mitochondrial and antioxidant gene transcriptional response to acute high-intensity exercise and b) skeletal muscle mitochondrial content and function following exercise training. In a randomised, double-blind, placebo-controlled, parallel design study, 23 untrained men (age: 44 ± 7 years, VO2peak: 39.6 ± 7.9 ml/kg/min) were randomised to receive either MitoQ (20 mg/d) or a placebo for 10 days before completing a bout of high-intensity interval exercise (cycle ergometer, 10 × 60 s at VO2peak workload with 75 s rest). Blood samples and vastus lateralis muscle biopsies were collected before exercise and immediately and 3 h after exercise. Participants then completed high-intensity interval training (HIIT; 3 sessions per week for 3 weeks) and another blood sample and muscle biopsy were collected. There was no effect of acute exercise or MitoQ on systemic (plasma protein carbonyls and reduced glutathione) or skeletal muscle (mtDNA damage and 4-HNE) oxidative stress biomarkers. Acute exercise-induced increases in skeletal muscle peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1-α) mRNA expression were augmented in the MitoQ group. Despite this, training-induced increases in skeletal muscle mitochondrial content were similar between groups. HIIT-induced increases in VO2peak and 20 km time trial performance were also similar between groups while training-induced increases in peak power achieved during the VO2peak test were augmented in the MitoQ group. These data suggest that training-induced increases in peak power are enhanced following MitoQ supplementation, which may be related to the augmentation of skeletal muscle PGC1α expression following acute exercise. However, these effects do not appear to be related to an effect of MitoQ supplementation on exercise-induced oxidative stress or training-induced mitochondrial biogenesis in skeletal muscle.