Mitochondrial complex I activity in microglia sustains neuroinflammation.

Sustained smouldering, or low-grade activation, of myeloid cells is a common hallmark of several chronic neurological diseases, including multiple sclerosis1. Distinct metabolic and mitochondrial features guide the activation and the diverse functional states of myeloid cells2. However, how these metabolic features act to perpetuate inflammation of the central nervous system is unclear. Here, using a multiomics approach, we identify a molecular signature that sustains the activation of microglia through mitochondrial complex I activity driving reverse electron transport and the production of reactive oxygen species. Mechanistically, blocking complex I in pro-inflammatory microglia protects the central nervous system against neurotoxic damage and improves functional outcomes in an animal disease model in vivo. Complex I activity in microglia is a potential therapeutic target to foster neuroprotection in chronic inflammatory disorders of the central nervous system3.

Mitochondrial reverse electron transport in myeloid cells perpetuates neuroinflammation.

Sustained smouldering, or low grade, activation of myeloid cells is a common hallmark of several chronic neurological diseases, including multiple sclerosis (MS) 1 . Distinct metabolic and mitochondrial features guide the activation and the diverse functional states of myeloid cells 2 . However, how these metabolic features act to perpetuate neuroinflammation is currently unknown. Using a multiomics approach, we identified a new molecular signature that perpetuates the activation of myeloid cells through mitochondrial complex II (CII) and I (CI) activity driving reverse electron transport (RET) and the production of reactive oxygen species (ROS). Blocking RET in pro-inflammatory myeloid cells protected the central nervous system (CNS) against neurotoxic damage and improved functional outcomes in animal disease models in vivo . Our data show that RET in myeloid cells is a potential new therapeutic target to foster neuroprotection in smouldering inflammatory CNS disorders 3 .

Chronic Fragmentation of the Daily Sleep-Wake Rhythm Increases Amyloid-beta Levels and Neuroinflammation in the 3xTg-AD Mouse Model of Alzheimer's Disease.

Fragmentation of the daily sleep-wake rhythm with increased nighttime awakenings and more daytime naps is correlated with the risk of development of Alzheimer's disease (AD). To explore whether a causal relationship underlies this correlation, the present study tested the hypothesis that chronic fragmentation of the daily sleep-wake rhythm stimulates brain amyloid-beta (Aß) levels and neuroinflammation in the 3xTg-AD mouse model of AD. Female 3xTg-AD mice were allowed to sleep undisturbed or were subjected to chronic sleep fragmentation consisting of four daily sessions of enforced wakefulness (one hour each) evenly distributed during the light phase, five days a week for four weeks. Piezoelectric sleep recording revealed that sleep fragmentation altered the daily sleep-wake rhythm to resemble the pattern observed in AD. Levels of amyloid-beta (Aß40 and Aß42) determined by ELISA were higher in hippocampal tissue collected from sleep-fragmented mice than from undisturbed controls. In contrast, hippocampal levels of tau and phospho-tau differed minimally between sleep fragmented and undisturbed control mice. Sleep fragmentation also stimulated neuroinflammation as shown by increased expression of markers of microglial activation and proinflammatory cytokines measured by q-RT-PCR analysis of hippocampal samples. No significant effects of sleep fragmentation on Aß, tau, or neuroinflammation were observed in the cerebral cortex. These studies support the concept that improving sleep consolidation in individuals at risk for AD may be beneficial for slowing the onset or progression of this devastating neurodegenerative disease.

Mitochondria-targeted antioxidant MitoQ ameliorates ischaemia-reperfusion injury in kidney transplantation models.

BACKGROUND: Ischaemia-reperfusion (IR) injury makes a major contribution to graft damage during kidney transplantation. Oxidative damage to mitochondria is an early event in IR injury. Therefore, the uptake, safety, and efficacy of the mitochondria-targeted antioxidant MitoQ were investigated in models of transplant IR injury. METHODS: MitoQ uptake by warm and cooled pairs of pig and declined human kidneys was measured when preserved in cold static storage or by hypothermic machine perfusion. Pairs of pigs' kidneys were exposed to defined periods of warm and cold ischaemia, flushed and stored at 4°C with or without MitoQ (50 nmol/l to 250 µmol/l), followed by reperfusion with oxygenated autologous blood in an ex vivo normothermic perfusion (EVNP). Pairs of declined human kidneys were flushed and stored with or without MitoQ (5-100 µmol/l) at 4°C for 6 h and underwent EVNP with ABO group-matched blood. RESULTS: Stable and concentration-dependent uptake of MitoQ was demonstrated for up to 24 h in pig and human kidneys. Total blood flow and urine output were significantly greater in pig kidneys treated with 50 µmol/l MitoQ compared with controls (P = 0.006 and P = 0.007 respectively). In proof-of-concept experiments, blood flow after 1 h of EVNP was significantly greater in human kidneys treated with 50 µmol/l MitoQ than in controls (P ≤ 0.001). Total urine output was numerically higher in the 50-µmol/l MitoQ group compared with the control, but the difference did not reach statistical significance (P = 0.054). CONCLUSION: Mitochondria-targeted antioxidant MitoQ can be administered to ischaemic kidneys simply and effectively during cold storage, and may improve outcomes after transplantation.

Ex vivo normothermic perfusion of isolated segmental porcine bowel: a novel functional model of the small intestine.

BACKGROUND: There is an unmet need for suitable ex vivo large animal models in experimental gastroenterology and intestinal transplantation. This study details a reliable and effective technique for ex vivo normothermic perfusion (EVNP) of segmental porcine small intestine. METHODS: Segments of small intestine, 1.5-3.0 m in length, were retrieved from terminally anaesthetized pigs. After a period of cold ischaemia, EVNP was performed for 2 h at 37°C with a mean pressure of 80 mmHg using oxygenated autologous blood diluted with Ringer's solution. The duration of EVNP was extended to 4 h for a second set of experiments in which two segments of proximal to mid-ileum (1.5-3.0 m) were retrieved from each animal and reperfused with whole blood (control) or leucocyte-depleted blood to examine the impact of leucocyte depletion on reperfusion injury. RESULTS: After a mean cold ischaemia time of 5 h and 20 min, EVNP was performed in an initial group of four pigs. In the second set of experiments, five pigs were used in each group. In all experiments bowel segments were well perfused and exhibited peristalsis during EVNP. Venous glucose levels significantly increased following luminal glucose stimulation (mean(s.e.m.) basal level 1.8(0.6) mmol/l versus peak 15.5(5.8) mmol/l; P < 0.001) and glucagon-like peptide 1 (GLP-1) levels increased in all experiments, demonstrating intact absorptive and secretory intestinal functions. There were no significant differences between control and leucocyte-depleted animals regarding blood flow, venous glucose, GLP-1 levels or histopathology at the end of 4 h of EVNP. CONCLUSIONS: This novel model is suitable for the investigation of gastrointestinal physiology, pathology and ischaemia reperfusion injury, along with evaluation of potential therapeutic interventions.

Translatable mitochondria-targeted protection against programmed cardiovascular dysfunction.

The prenatal origins of heart disease in offspring have been established. However, research in species with developmental milestones comparable to humans is lacking, preventing translation of this knowledge to clinical contexts. Using sheep and chickens, two species with similar cardiovascular developmental milestones to humans, we combined in vivo experiments with in vitro studies at organ, cellular, mitochondrial, and molecular levels. We tested mitochondria-targeted antioxidant intervention with MitoQ against cardiovascular dysfunction programmed by developmental hypoxia, a common complication in human pregnancy. Experiments in sheep determined in vivo fetal and adult cardiovascular function through surgical techniques not possible in humans, while those in chicken embryos isolated effects independent of maternal or placental influences. We show that hypoxia generates mitochondria-derived oxidative stress during cardiovascular development, programming endothelial dysfunction and hypertension in adult offspring. MitoQ treatment during hypoxic development protects against this cardiovascular risk via enhanced nitric oxide signaling, offering a plausible intervention strategy.

Correction to: Protection against cardiac ischemia-reperfusion injury by hypothermia and by inhibition of succinate accumulation and oxidation is additive.

The original version of this article unfortunately contained a mistake.

Protection against cardiac ischemia-reperfusion injury by hypothermia and by inhibition of succinate accumulation and oxidation is additive.

Hypothermia induced at the onset of ischemia is a potent experimental cardioprotective strategy for myocardial infarction. The aim of our study was to determine whether the beneficial effects of hypothermia may be due to decreasing mitochondria-mediated mechanisms of damage that contribute to the pathophysiology of ischemia/reperfusion injury. New Zealand male rabbits were submitted to 30 min of myocardial ischemia with hypothermia (32 °C) induced by total liquid ventilation (TLV). Hypothermia was applied during ischemia alone (TLV group), during ischemia and reperfusion (TLV-IR group) and normothermia (Control group). In all the cases, ischemia was performed by surgical ligation of the left anterior descending coronary artery and was followed by 3 h of reperfusion before assessment of infarct size. In a parallel study, male C57BL6/J mice underwent 30 min myocardial ischemia followed by reperfusion under either normothermia (37 °C) or conventionally induced hypothermia (32 °C). In both the models, the levels of the citric acid cycle intermediate succinate, mitochondrial complex I activity were assessed at various times. The benefit of hypothermia during ischemia on infarct size was compared to inhibition of succinate accumulation and oxidation by the complex II inhibitor malonate, applied as the pro-drug dimethyl malonate under either normothermic or hypothermic conditions. Hypothermia during ischemia was cardioprotective, even when followed by normothermic reperfusion. Hypothermia during ischemia only, or during both, ischemia and reperfusion, significantly reduced infarct size (2.8 ± 0.6%, 24.2 ± 3.0% and 49.6 ± 2.6% of the area at risk, for TLV-IR, TLV and Control groups, respectively). The significant reduction of infarct size by hypothermia was neither associated with a decrease in ischemic myocardial succinate accumulation, nor with a change in its rate of oxidation at reperfusion. Similarly, dimethyl malonate infusion and hypothermia during ischemia additively reduced infarct size (4.8 ± 2.2% of risk zone) as compared to either strategy alone. Hypothermic cardioprotection is neither dependent on the inhibition of succinate accumulation during ischemia, nor of its rapid oxidation at reperfusion. The additive effect of hypothermia and dimethyl malonate on infarct size shows that they are protective by distinct mechanisms and also suggests that combining these different therapeutic approaches could further protect against ischemia/reperfusion injury during acute myocardial infarction.

Age-related endothelial dysfunction in human skeletal muscle feed arteries: the role of free radicals derived from mitochondria in the vasculature.

AIM: This study sought to determine the role of free radicals derived from mitochondria in the vasculature in the recognized age-related endothelial dysfunction of human skeletal muscle feed arteries (SMFAs). METHODS: A total of 44 SMFAs were studied with and without acute exposure to the mitochondria-targeted antioxidant MitoQ and nitric oxide synthase (NOS) blockade. The relative abundance of proteins from the electron transport chain, phosphorylated (p-) to endothelial (e) NOS ratio, manganese superoxide dismutase (MnSOD) and the mitochondria-derived superoxide (O2-) levels were assessed in SMFA. Endothelium-dependent and endothelium-independent SMFA vasodilation was assessed in response to flow-induced shear stress, acetylcholine (ACh) and sodium nitroprusside (SNP). RESULTS: MitoQ restored endothelium-dependent vasodilation in the old to that of the young when stimulated by both flow (young: 68 ± 5; old: 25 ± 7; old + MitoQ 65 ± 9%) and ACh (young: 97 ± 4; old: 59 ± 10; old + MitoQ: 98 ± 5%), but did not alter the initially uncompromised, endothelium-independent vasodilation (SNP). Compared to the young, MitoQ in the old diminished the initially elevated mitochondria-derived O2- levels and appeared to attenuate the breakdown of MnSOD. Furthermore, MitoQ increased the ratio of p-eNOS to NOS and the restoration of endothelium-dependent vasodilation in the old by MitoQ was ablated by NOS blockade. CONCLUSION: This study demonstrated that MitoQ reverses age-related vascular dysfunction by what appears to be an NO-dependent mechanism in human SMFAs. These findings suggest that mitochondria-targeted antioxidants may have utility in terms of counteracting the attenuated blood flow and vascular dysfunction associated with advancing age.

In vivo evidence of mitochondrial dysfunction and altered redox homeostasis in a genetic mouse model of propionic acidemia: Implications for the pathophysiology of this disorder.

Accumulation of toxic metabolites has been described to inhibit mitochondrial enzymes, thereby inducing oxidative stress in propionic acidemia (PA), an autosomal recessive metabolic disorder caused by the deficiency of mitochondrial propionyl-CoA carboxylase. PA patients exhibit neurological deficits and multiorgan complications including cardiomyopathy. To investigate the role of mitochondrial dysfunction in the development of these alterations we have used a hypomorphic mouse model of PA that mimics the biochemical and clinical hallmarks of the disease. We have studied the tissue-specific bioenergetic signature by Reverse Phase Protein Microarrays and analysed OXPHOS complex activities, mtDNA copy number, oxidative damage, superoxide anion and hydrogen peroxide levels. The results show decreased levels and/or activity of several OXPHOS complexes in different tissues of PA mice. An increase in mitochondrial mass and OXPHOS complexes was observed in brain, possibly reflecting a compensatory mechanism including metabolic reprogramming. mtDNA depletion was present in most tissues analysed. Antioxidant enzymes were also found altered. Lipid peroxidation was present along with an increase in hydrogen peroxide and superoxide anion production. These data support the hypothesis that oxidative damage may contribute to the pathophysiology of PA, opening new avenues in the identification of therapeutic targets and paving the way for in vivo evaluation of compounds targeting mitochondrial biogenesis or reactive oxygen species production.

Obesity, diabetes, and leptin resistance promote tau pathology in a mouse model of disease.

Obesity and type 2 diabetes mellitus (T2DM) convey an increased risk for developing dementia. The microtubule-associated protein tau is implicated in neurodegenerative disease by undergoing hyperphosphorylation and aggregation, leading to cytotoxicity and neurodegeneration. Enzymes involved in the regulation of tau phosphorylation, such as GSK3ß, are tightly associated with pathways found to be dysregulated in T2DM. We have shown previously that leptin-resistant mice, which develop obesity and a diabetic phenotype, display elevated levels of tau phosphorylation. Here we show cells cultured with leptin, an adipokine shown to have neuroprotective effects, reduces tau phosphorylation. To explore how this mechanism works in vivo we transduced an existing diabetic mouse line (Lepr(db/db)) with a tau mutant (tau(P301L)) via adeno-associated virus (AAV). The resulting phenotype included a striking increase in tau phosphorylation and the number of neurofibrillary tangles (NFTs) found within the hippocampus. We conclude that leptin resistance-induced obesity and diabetes accelerates the development of tau pathology. This model of metabolic dysfunction and tauopathy provides a new system in which to explore the mechanisms underlying the ways in which leptin resistance and diabetes influence development of tau pathology, and may ultimately be related to the development of NFTs.

Increased fragmentation of sleep-wake cycles in the 5XFAD mouse model of Alzheimer's disease.

Sleep perturbations including fragmented sleep with frequent night-time awakenings and daytime naps are common in patients with Alzheimer's disease (AD), and these daily disruptions are a major factor for institutionalization. The objective of this study was to investigate if sleep-wake patterns are altered in 5XFAD mice, a well-characterized double transgenic mouse model of AD which exhibits an early onset of robust AD pathology and memory deficits. These mice have five distinct human mutations in two genes, the amyloid precursor protein (APP) and Presenilin1 (PS1) engineered into two transgenes driven by a neuron-specific promoter (Thy1), and thus develop severe amyloid deposition by 4 months of age. Age-matched (4-6.5 months old) male and female 5XFAD mice were monitored and compared to wild-type littermate controls for multiple sleep traits using a non-invasive, high throughput, automated piezoelectric system which detects breathing and gross body movements to characterize sleep and wake. Sleep-wake patterns were recorded continuously under baseline conditions (undisturbed) for 3 days and after sleep deprivation of 4h, which in mice produces a significant sleep debt and challenge to sleep homeostasis. Under baseline conditions, 5XFAD mice exhibited shorter bout lengths (14% lower values for males and 26% for females) as compared to controls (p<0.001). In females, the 5XFAD mice also showed 12% less total sleep than WT (p<0.01). Bout length reductions were greater during the night (the active phase for mice) than during the day, which does not model the human condition of disrupted sleep at night (the inactive period). However, the overall decrease in bout length suggests increased fragmentation and disruption in sleep consolidation that may be relevant to human sleep. The 5XFAD mice may serve as a useful model for testing therapeutic strategies to improve sleep consolidation in AD patients.

The LRRK2 inhibitor GSK2578215A induces protective autophagy in SH-SY5Y cells: involvement of Drp-1-mediated mitochondrial fission and mitochondrial-derived ROS signaling.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene have been associated with Parkinson's disease, and its inhibition opens potential new therapeutic options. Among the drug inhibitors of both wild-type and mutant LRRK2 forms is the 2-arylmethyloxy-5-subtitutent-N-arylbenzamide GSK257815A. Using the well-established dopaminergic cell culture model SH-SY5Y, we have investigated the effects of GSK2578215A on crucial neurodegenerative features such as mitochondrial dynamics and autophagy. GSK2578215A induces mitochondrial fragmentation of an early step preceding autophagy. This increase in autophagosome results from inhibition of fusion rather than increases in synthesis. The observed effects were shared with LRRK2-IN-1, a well-described, structurally distinct kinase inhibitor compound or when knocking down LRRK2 expression using siRNA. Studies using the drug mitochondrial division inhibitor 1 indicated that translocation of the dynamin-related protein-1 has a relevant role in this process. In addition, autophagic inhibitors revealed the participation of autophagy as a cytoprotective response by removing damaged mitochondria. GSK2578215A induced oxidative stress as evidenced by the accumulation of 4-hydroxy-2-nonenal in SH-SY5Y cells. The mitochondrial-targeted reactive oxygen species scavenger MitoQ positioned these species as second messengers between mitochondrial morphologic alterations and autophagy. Altogether, our results demonstrated the relevance of LRRK2 in mitochondrial-activated pathways mediating in autophagy and cell fate, crucial features in neurodegenerative diseases.

Radiosynthesis of 11-[(18) F]fluoroundecyltriphenylphosphonium (MitoF) as a potential mitochondria-specific positron emission tomography radiotracer.

Changes in the magnitude of the mitochondrial membrane potential occur in a range of important pathologies. To assess changes in membrane potential in patients, we set out to develop an improved mitochondria-targeted positron emission tomography probe comprising a lipophilic triphenylphosphonium cation attached to a fluorine-18 radionuclide via an 11-carbon alkyl chain, which is well-established to effectively transport to and localise within mitochondria. Here, we describe the radiosynthesis of this probe, 11-[(18) F]fluoroundecyl-triphenylphosphonium (MitoF), from no-carrier-added [(18) F]fluoride and a fully automated synthetic protocol to prepare it in good radiochemical yields (2-3 GBq at end-of-synthesis) and radiochemical purity (97-99%).

Antioxidants that protect mitochondria reduce interleukin-6 and oxidative stress, improve mitochondrial function, and reduce biochemical markers of organ dysfunction in a rat model of acute sepsis.

BACKGROUND: Sepsis-induced organ failure is the major cause of death in critical care units, and is characterized by a massive dysregulated inflammatory response and oxidative stress. We investigated the effects of treatment with antioxidants that protect mitochondria (MitoQ, MitoE, or melatonin) in a rat model of lipopolysaccharide (LPS) plus peptidoglycan (PepG)-induced acute sepsis, characterized by inflammation, mitochondrial dysfunction and early organ damage. METHODS: Anaesthetized and ventilated rats received an i.v. bolus of LPS and PepG followed by an i.v. infusion of MitoQ, MitoE, melatonin, or saline for 5 h. Organs and blood were then removed for determination of mitochondrial and organ function, oxidative stress, and key cytokines. RESULTS: MitoQ, MitoE, or melatonin had broadly similar protective effects with improved mitochondrial respiration (P<0.002), reduced oxidative stress (P<0.02), and decreased interleukin-6 levels (P=0.0001). Compared with control rats, antioxidant-treated rats had lower levels of biochemical markers of organ dysfunction, including plasma alanine amino-transferase activity (P=0.02) and creatinine concentrations (P<0.0001). CONCLUSIONS: Antioxidants that act preferentially in mitochondria reduce mitochondrial damage and organ dysfunction and decrease inflammatory responses in a rat model of acute sepsis.

Circulating endothelial progenitor cells and erectile dysfunction: possibility of nutritional intervention?

To provide an overview of molecular and cellular processes involved in erectile dysfunction (ED) with emphasis on circulating endothelial progenitor cells (EPC) and discuss possible nutraceutical means of intervention. A review of literature on Pubmed related to EPC and ED was conducted. Patients with ED appear to possess a reduced number of circulating EPC, which is associated with poor endothelial function possibly as a result of underlying low-grade inflammation. Several studies support the possibility of improving erectile function by inhibition of inflammation as well as administration of various stem cell types. One particularly interesting approach is nutraceutical supplementation to increase circulating EPC, as demonstrated in the product Stem-Kine. Interventions aimed at increasing circulating EPC may have potential in treatment of vascular ED.

MitoQ administration prevents endotoxin-induced cardiac dysfunction.

Sepsis elicits severe alterations in cardiac function, impairing cardiac mitochondrial and pressure-generating capacity. Currently, there are no therapies to prevent sepsis-induced cardiac dysfunction. We tested the hypothesis that administration of a mitochondrially targeted antioxidant, 10-(6'-ubiquinonyl)-decyltriphenylphosphonium (MitoQ), would prevent endotoxin-induced reductions in cardiac mitochondrial and contractile function. Studies were performed on adult rodents (n = 52) given either saline, endotoxin (8 mg x kg(-1) x day(-1)), saline + MitoQ (500 microM), or both endotoxin and MitoQ. At 48 h animals were killed and hearts were removed for determination of either cardiac mitochondrial function (using polarography) or cardiac pressure generation (using the Langendorf technique). We found that endotoxin induced reductions in mitochondrial state 3 respiration rates, the respiratory control ratio, and ATP generation. Moreover, MitoQ administration prevented each of these endotoxin-induced abnormalities, P < 0.001. We also found that endotoxin produced reductions in cardiac pressure-generating capacity, reducing the systolic pressure-diastolic relationship. MitoQ also prevented endotoxin-induced reductions in cardiac pressure generation, P < 0.01. One potential link between mitochondrial and contractile dysfunction is caspase activation; we found that endotoxin increased cardiac levels of active caspases 9 and 3 (P < 0.001), while MitoQ prevented this increase (P < 0.01). These data demonstrate that MitoQ is a potent inhibitor of endotoxin-induced mitochondrial and cardiac abnormalities. We speculate that this agent may prove a novel therapy for sepsis-induced cardiac dysfunction.

Hydrogen peroxide produced inside mitochondria takes part in cell-to-cell transmission of apoptotic signal.

In monolayer of HeLa cells treated with tumor necrosis factor (TNF), apoptotic cells formed clusters indicating possible transmission of apoptotic signal via the culture media. To investigate this phenomenon, a simple method of enabling two cell cultures to interact has been employed. Two coverslips were placed side by side in a Petri dish, one coverslip covered with apoptogen-treated cells (the inducer) and another with non-treated cells (the recipient). TNF, staurosporine, or H2O2 treatment of the inducer cells is shown to initiate apoptosis on the recipient coverslip. This effect is increased by a catalase inhibitor aminotriazole and is arrested by addition of catalase or by pre-treatment of either the inducer or the recipient cells with nanomolar concentrations of mitochondria-targeted cationic antioxidant MitoQ (10-(6 -ubiquinolyl)decyltriphenylphosphonium), which specifically arrests H2O2-induced apoptosis. The action of MitoQ is abolished by an uncoupler preventing accumulation of MitoQ in mitochondria. It is concluded that reactive oxygen species (ROS) produced by mitochondria in the apoptotic cells initiate the release of H2O2 from these cells. The H2O2 released is employed as a long-distance cell suicide messenger. In processing of such a signal by the recipient cells, mitochondrial ROS production is also involved. It is suggested that the described phenomenon may be involved in expansion of the apoptotic region around a damaged part of the tissue during heart attack or stroke as well as in "organoptosis", i.e. disappearance of organs during ontogenesis.

Disulphide formation on mitochondrial protein thiols.

A large number of proteins contain free thiols that can be modified by the formation of internal disulphides or by mixed disulphides with low-molecular-mass thiols. The majority of these latter modifications result from the interaction of protein thiols with the endogenous glutathione pool. Protein glutathionylation and disulphide formation are of significance both for defence against oxidative damage and in redox signalling. As mitochondria are central to both oxidative damage and redox signalling within the cell, these modifications of mitochondrial proteins are of particular importance. In the present study, we review the mechanisms and physiological significance of these processes.