Identification of a mechanism promoting mitochondrial sterol accumulation during myocardial ischemia-reperfusion: role of TSPO and STAR.

Hypercholesterolemia is a major risk factor for coronary artery diseases and cardiac ischemic events. Cholesterol per se could also have negative effects on the myocardium, independently from hypercholesterolemia. Previously, we reported that myocardial ischemia-reperfusion induces a deleterious build-up of mitochondrial cholesterol and oxysterols, which is potentiated by hypercholesterolemia and prevented by translocator protein (TSPO) ligands. Here, we studied the mechanism by which sterols accumulate in cardiac mitochondria and promote mitochondrial dysfunction. We performed myocardial ischemia-reperfusion in rats to evaluate mitochondrial function, TSPO, and steroidogenic acute regulatory protein (STAR) levels and the related mitochondrial concentrations of sterols. Rats were treated with the cholesterol synthesis inhibitor pravastatin or the TSPO ligand 4'-chlorodiazepam. We used Tspo deleted rats, which were phenotypically characterized. Inhibition of cholesterol synthesis reduced mitochondrial sterol accumulation and protected mitochondria during myocardial ischemia-reperfusion. We found that cardiac mitochondrial sterol accumulation is the consequence of enhanced influx of cholesterol and not of the inhibition of its mitochondrial metabolism during ischemia-reperfusion. Mitochondrial cholesterol accumulation at reperfusion was related to an increase in mitochondrial STAR but not to changes in TSPO levels. 4'-Chlorodiazepam inhibited this mechanism and prevented mitochondrial sterol accumulation and mitochondrial ischemia-reperfusion injury, underlying the close cooperation between STAR and TSPO. Conversely, Tspo deletion, which did not alter cardiac phenotype, abolished the effects of 4'-chlorodiazepam. This study reveals a novel mitochondrial interaction between TSPO and STAR to promote cholesterol and deleterious sterol mitochondrial accumulation during myocardial ischemia-reperfusion. This interaction regulates mitochondrial homeostasis and plays a key role during mitochondrial injury.

Dysregulated Phenylalanine Catabolism Plays a Key Role in the Trajectory of Cardiac Aging.

BACKGROUND: Aging myocardium undergoes progressive cardiac hypertrophy and interstitial fibrosis with diastolic and systolic dysfunction. Recent metabolomics studies shed light on amino acids in aging. The present study aimed to dissect how aging leads to elevated plasma levels of the essential amino acid phenylalanine and how it may promote age-related cardiac dysfunction. METHODS: We studied cardiac structure and function, together with phenylalanine catabolism in wild-type (WT) and p21-/- mice (male; 2-24 months), with the latter known to be protected from cellular senescence. To explore phenylalanine's effects on cellular senescence and ectopic phenylalanine catabolism, we treated cardiomyocytes (primary adult rat or human AC-16) with phenylalanine. To establish a role for phenylalanine in driving cardiac aging, WT male mice were treated twice a day with phenylalanine (200 mg/kg) for a month. We also treated aged WT mice with tetrahydrobiopterin (10 mg/kg), the essential cofactor for the phenylalanine-degrading enzyme PAH (phenylalanine hydroxylase), or restricted dietary phenylalanine intake. The impact of senescence on hepatic phenylalanine catabolism was explored in vitro in AML12 hepatocytes treated with Nutlin3a (a p53 activator), with or without p21-targeting small interfering RNA or tetrahydrobiopterin, with quantification of PAH and tyrosine levels. RESULTS: Natural aging is associated with a progressive increase in plasma phenylalanine levels concomitant with cardiac dysfunction, whereas p21 deletion delayed these changes. Phenylalanine treatment induced premature cardiac deterioration in young WT mice, strikingly akin to that occurring with aging, while triggering cellular senescence, redox, and epigenetic changes. Pharmacological restoration of phenylalanine catabolism with tetrahydrobiopterin administration or dietary phenylalanine restriction abrogated the rise in plasma phenylalanine and reversed cardiac senescent alterations in aged WT mice. Observations from aged mice and human samples implicated age-related decline in hepatic phenylalanine catabolism as a key driver of elevated plasma phenylalanine levels and showed increased myocardial PAH-mediated phenylalanine catabolism, a novel signature of cardiac aging. CONCLUSIONS: Our findings establish a pathogenic role for increased phenylalanine levels in cardiac aging, linking plasma phenylalanine levels to cardiac senescence via dysregulated phenylalanine catabolism along a hepatic-cardiac axis. They highlight phenylalanine/PAH modulation as a potential therapeutic strategy for age-associated cardiac impairment.

Chronic maternal exposure to titanium dioxide nanoparticles alters breathing in newborn offspring.

BACKGROUND: Over the last two decades, nanotechnologies and the use of nanoparticles represent one of the greatest technological advances in many fields of human activity. Particles of titanium dioxide (TiO2) are one of the nanomaterials most frequently found in everyday consumer products. But, due in particular to their extremely small size, TiO2 nanoparticles (NPs) are prone to cross biological barriers and potentially lead to adverse health effects. The presence of TiO2 NPs found in human placentae and in the infant meconium has indicated unequivocally the capacity for a materno-fetal transfer of this nanomaterial. Although chronic exposure to TiO2 NPs during pregnancy is known to induce offspring cognitive deficits associated with neurotoxicity, the impact of a gestational exposure on a vital motor function such as respiration, whose functional emergence occurs during fetal development, remains unknown. RESULTS: Using in vivo whole-body plethysmographic recordings from neonatal mice, we show that a chronic exposure to TiO2 NPs during pregnancy alters the respiratory activity of offspring, characterized by an abnormally elevated rate of breathing. Correspondingly, using ex vivo electrophysiological recordings performed on isolated brainstem-spinal cord preparations of newborn mice and medullary slice preparations containing specific nuclei controlling breathing frequency, we show that the spontaneously generated respiratory-related rhythm is significantly and abnormally accelerated in animals prenatally exposed to TiO2 NPs. Moreover, such a chronic prenatal exposure was found to impair the capacity of respiratory neural circuitry to effectively adjust breathing rates in response to excitatory environmental stimuli such as an increase in ambient temperature. CONCLUSIONS: Our findings thus demonstrate that a maternal exposure to TiO2 NPs during pregnancy affects the normal development and operation of the respiratory centers in progeny.

Cardioprotective Signaling Pathways in Obese Mice Submitted to Regular Exercise: Effect on Oxysterols.

Exercise induces cardioprotection against myocardial infarction, despite obesity, by restoring pro-survival pathways and increasing resistance of mitochondrial permeability transition pore (mPTP) opening at reperfusion. Among the mechanisms involved in the inactivation of these pathways, oxysterols appear interesting. Thus, we investigated the influence of regular exercise on the reperfusion injury salvage kinase (RISK) pathway, oxysterols, and mitochondria, in the absence of ischemia-reperfusion. We also studied 7ß-hydroxycholesterol (7ßOH) concentration (mass spectrometry) in human lean and obese subjects. Wild-type (WT) and obese (ob/ob) mice were assigned to sedentary conditions or regular treadmill exercise. Exercise significantly increased Akt phosphorylation, whereas 7ßOH concentration was reduced. Moreover, exercise induced the translocation of PKCε from the cytosol to mitochondria. However, exercise did not affect the calcium concentration required to open mPTP in the mitochondria, neither in WT nor in ob/ob animals. Finally, human plasma 7ßOH concentration was consistent with observations made in mice. In conclusion, regular exercise enhanced the RISK pathway by increasing kinase phosphorylation and PKCε translocation and decreasing 7ßOH concentration. This activation needs the combination with stress conditions, i.e., ischemia-reperfusion, in order to inhibit mPTP opening at the onset of reperfusion. The human findings suggest 7ßOH as a candidate marker for evaluating cardiovascular risk factors in obesity.

Synergistic interaction between sensory inputs and propriospinal signalling underlying quadrupedal locomotion.

KEY POINTS: Stimulation of hindlimb afferent fibres can both stabilize and increase the activity of fore- and hindlimb motoneurons during fictive locomotion. The increase in motoneuron activity is at least partially due to the production of doublets of action potentials in a subpopulation of motoneurons. These results were obtained using an in vitro brainstem/spinal cord preparation of neonatal rat. ABSTRACT: Quadrupedal locomotion relies on a dynamic coordination between central pattern generators (CPGs) located in the cervical and lumbar spinal cord, and controlling the fore- and hindlimbs, respectively. It is assumed that this CPG interaction is achieved through separate closed-loop processes involving propriospinal and sensory pathways. However, the functional consequences of a concomitant involvement of these different influences on the degree of coordination between the fore- and hindlimb CPGs is still largely unknown. Using an in vitro brainstem/spinal cord preparation of neonatal rat, we found that rhythmic, bilaterally alternating stimulation of hindlimb sensory input pathways elicited coordinated hindlimb and forelimb CPG activity. During pharmacologically induced fictive locomotion, lumbar dorsal root (DR) stimulation entrained and stabilized an ongoing cervico-lumbar locomotor-like rhythm and increased the amplitude of both lumbar and cervical ventral root bursting. The increase in cervical burst amplitudes was correlated with the occurrence of doublet action potential firing in a subpopulation of motoneurons, enabling the latter to transition between low and high frequency discharge according to the intensity of DR stimulation. Moreover, our data revealed that propriospinal and sensory pathways act synergistically to strengthen cervico-lumbar interactions. Indeed, split-bath experiments showed that fully coordinated cervico-lumbar fictive locomotion was induced by combining pharmacological stimulation of either the lumbar or cervical CPGs with lumbar DR stimulation. This study thus highlights the powerful interactions between sensory and propriospinal pathways which serve to ensure the coupling of the fore- and hindlimb CPGs for effective quadrupedal locomotion.

L-DOPA and 5-HTP modulation of air-stepping in newborn rats.

KEY POINTS: In newborn rats, L-DOPA increases the occurrence of air-stepping activity without affecting movement characteristics. L-DOPA administration increases the spinal content of dopamine in a dose-dependent manner. Injection of 5-HTP increases the spinal serotonin content but does not trigger air-stepping. 5-HTP counteracts the pro-locomotor action of L-DOPA. Less dopamine and serotonin are synthesized when L-DOPA and 5-HTP are administered as a cocktail. ABSTRACT: The catecholamine precursor, L-3,4-dihydroxyphenylalanine (L-DOPA), is a well-established pharmacological agent for promoting locomotor action in vertebrates, including triggering air-stepping activities in the neonatal rat. Serotonin is also a well-known neuromodulator of the rodent spinal locomotor networks. Here, using kinematic analysis, we compared locomotor-related activities expressed by newborn rats in response to varying doses of L-DOPA and the serotonin precursor 5-hydroxytryptophan (5-HTP) administered separately or in combination. L-DOPA alone triggered episodes of air-stepping in a dose-dependent manner (25-100 mg/kg), notably determining the duration of locomotor episodes, but without affecting step cycle frequency or amplitude. In contrast, 5-HTP (25-150 mg/kg) was ineffective in instigating air-stepping, but altered episode durations of L-DOPA-induced air-stepping, and decreased locomotor cycle frequency. High performance liquid chromatography revealed that L-DOPA, which was undetectable in control conditions, accumulated in a dose-dependent manner in the lumbar spinal cord 30 min after its administration. This was paralleled by an increase in dopamine levels, whereas the spinal content of noradrenaline and serotonin remained unaffected. In the same way, the spinal levels of serotonin increased in parallel with the dose of 5-HTP without affecting the levels of dopamine and noradrenaline. When both precursors are administrated, they counteract each other for the production of serotonin and dopamine. Our data thus indicate for the first time that both L-DOPA and 5-HTP exert opposing neuromodulatory actions on air-stepping behaviour in the developing rat, and we speculate that competition for the production of dopamine and serotonin occurs when they are administered as a cocktail.

A Phenyl-Pyrrolidine Derivative Reveals a Dual Inhibition Mechanism of Myocardial Mitochondrial Permeability Transition Pore, Which Is Limited by Its Myocardial Distribution.

Mitochondrial permeability transition pore (mPTP) opening is a key event in cell death during myocardial ischemia reperfusion. Inhibition of its modulator cyclophilin D (CypD) by cyclosporine A (CsA) reduces ischemia-reperfusion injury. The use of cyclosporine A in this indication is debated; however, targeting mPTP remains a major goal to achieve. We investigated the protective effects of a new original small-molecule cyclophilin inhibitor C31, which was specifically designed to target CypD. CypD peptidylprolyl cis-trans isomerase (PPIase) activity was assessed by the standard chemotrypsin-coupled assay. The effects of C31 on mPTP opening were investigated in isolated mouse cardiac mitochondria by measuring mitochondrial swelling and calcium retention capacity (CRC) in rat H9C2 cardiomyoblasts and in adult mouse cardiomyocytes by fluorescence microscopy in isolated perfused mouse hearts and ex vivo after drug infusion in mice. C31 potently inhibited CypD PPIase activity and mitochondrial swelling. C31 was more effective at increasing mitochondrial CRC than CsA and was still able to increase CRC in Ppif -/- (CypD-inactivated) cardiac mitochondria. C31 delayed both mPTP opening and cell death in cardiomyocytes subjected to hypoxia reoxygenation. However, high concentrations of both drugs were necessary to reduce mPTP opening in isolated perfused hearts, and neither CsA nor C31 inhibited mPTP opening in heart after in vivo infusion, underlying the importance of myocardial drug distribution for cardioprotection. C31 is an original inhibitor of mPTP opening involving both CypD-dependent and -independent mechanisms. It constitutes a promising new cytoprotective agent. Optimization of its pharmacokinetic properties is now required prior to its use against cardiac ischemia-reperfusion injury. SIGNIFICANCE STATEMENT: This study demonstrates that the new cyclophilin inhibitor C31 potently inhibits cardiac mitochondrial permeability transition pore (mPTP) opening in vitro and ex vivo. The dual mechanism of action of C31 allows the prevention of mPTP opening beyond cyclophilin D inhibition. Further development of the compound might bring promising drug candidates for cardioprotection. However, the lack of effect of both C31 and cyclosporine A after systemic administration demonstrates the difficulties of targeting myocardial mitochondria in vivo and should be taken into account in cardioprotective strategies.

Modulation of respiratory network activity by forelimb and hindlimb locomotor generators.

Early at the onset of exercise, breathing rate accelerates in order to anticipate the increasing metabolic demand resulting from the extra effort produced. Accordingly, the respiratory neural networks are the target of various input signals originating either centrally or peripherally. For example, during locomotion, the activation of muscle sensory afferents is able to entrain and thereby increase the frequency of spontaneous respiratory rhythmogenesis. Moreover, the lumbar spinal networks engaged in generating hindlimb locomotor rhythms are also capable of activating the medullary respiratory generators through an ascending excitatory command. However, in the context of quadrupedal locomotion, the influence of other spinal cord regions, such as cervical and thoracic segments, remains unknown. Using isolated brainstem-spinal cord preparations from neonatal rats and mice, we show that cervicothoracic circuitry may also contribute to locomotion-induced acceleration of respiratory cycle frequency. As previously observed for the hindlimb CPGs, the pharmacological activation of forelimb locomotor networks produces episodes of fictive locomotion that in turn increase the ongoing respiratory rhythm. Thoracic neuronal circuitry may also participate indirectly in this modulation via the activation of both cervical and lumbar CPG neurons. Furthermore, using light stimulation of CHR2-expressing glutamatergic neurons, we found that the modulation of the respiratory rate during locomotion involves lumbar glutamatergic circuitry. Our results demonstrate that during locomotion, the respiratory rhythm-generating networks receive excitatory ascending inputs from the spinal circuits responsible for generating and coordinating fore- and hindlimb movements. This constitutes a distributed central mechanism that contributes to matching breathing rate to the speed of locomotion.

Small-Molecule Inhibitors of Cyclophilins Block Opening of the Mitochondrial Permeability Transition Pore and Protect Mice From Hepatic Ischemia/Reperfusion Injury.

BACKGROUND & AIMS: Hepatic ischemia/reperfusion injury is a complication of liver surgery that involves mitochondrial dysfunction resulting from mitochondrial permeability transition pore (mPTP) opening. Cyclophilin D (PPIF or CypD) is a peptidyl-prolyl cis-trans isomerase that regulates mPTP opening in the inner mitochondrial membrane. We investigated whether and how recently created small-molecule inhibitors of CypD prevent opening of the mPTP in hepatocytes and the resulting effects in cell models and livers of mice undergoing ischemia/reperfusion injury. METHODS: We measured the activity of 9 small-molecule inhibitors of cyclophilins in an assay of CypD activity. The effects of the small-molecule CypD inhibitors or vehicle on mPTP opening were assessed by measuring mitochondrial swelling and calcium retention in isolated liver mitochondria from C57BL/6J (wild-type) and Ppif-/- (CypD knockout) mice and in primary mouse and human hepatocytes by fluorescence microscopy. We induced ischemia/reperfusion injury in livers of mice given a small-molecule CypD inhibitor or vehicle before and during reperfusion and collected samples of blood and liver for histologic analysis. RESULTS: The compounds inhibited peptidyl-prolyl isomerase activity (half maximal inhibitory concentration values, 0.2-16.2 µmol/L) and, as a result, calcium-induced mitochondrial swelling, by preventing mPTP opening (half maximal inhibitory concentration values, 1.4-132 µmol/L) in a concentration-dependent manner. The most potent inhibitor (C31) bound CypD with high affinity and inhibited swelling in mitochondria from livers of wild-type and Ppif-/- mice (indicating an additional, CypD-independent effect on mPTP opening) and in primary human and mouse hepatocytes. Administration of C31 in mice with ischemia/reperfusion injury before and during reperfusion restored hepatic calcium retention capacity and oxidative phosphorylation parameters and reduced liver damage compared with vehicle. CONCLUSIONS: Recently created small-molecule inhibitors of CypD reduced calcium-induced swelling in mitochondria from mouse and human liver tissues. Administration of these compounds to mice during ischemia/reperfusion restored hepatic calcium retention capacity and oxidative phosphorylation parameters and reduced liver damage. These compounds might be developed to protect patients from ischemia/reperfusion injury after liver surgery or for other hepatic or nonhepatic disorders related to abnormal mPTP opening.

Brainstem Steering of Locomotor Activity in the Newborn Rat.

Control of locomotion relies on motor loops conveying modulatory signals between brainstem and spinal motor circuits. We investigated the steering control of the brainstem reticular formation over the spinal locomotor networks using isolated brainstem-spinal cord preparations of male and female neonatal rats. First, we performed patch-clamp recordings of identified reticulospinal cells during episodes of fictive locomotion. This revealed that a spinal ascending phasic modulation of reticulospinal cell activity is already present at birth. Half of the cells exhibited tonic firing during locomotion, while the other half emitted phasic discharges of action potentials phase locked to ongoing activity. We next showed that mimicking the phasic activity of reticulospinal neurons by applying patterned electrical stimulation bilaterally at the ventral caudal medulla level triggered fictive locomotion efficiently. Moreover, the brainstem stimuli-induced locomotor rhythm was entrained in a one-to-one coupling over a range of cycle periods (2-6 s). Additionally, we induced turning like motor outputs by either increasing or decreasing the relative duration of the stimulation trains on one side of the brainstem compared to the other. The ability of the patterned descending command to control the locomotor output depended on the functional integrity of ventral reticulospinal pathways and the involvement of local spinal central pattern generator circuitry. Altogether, this study provides a mechanism by which brainstem reticulospinal neurons relay steering and speed commands to the spinal locomotor networks.SIGNIFICANCE STATEMENT Locomotor function allows the survival of most animal species while sustaining the expression of fundamental behaviors. Locomotor activities adapt from moment to moment to behavioral and environmental changes. We show that the brainstem can control the spinal locomotor network outputs through phasic descending commands that alternate bilaterally. Manipulating the periodicity and/or the relative durations of the left and right descending commands at the brainstem level is efficient to set the locomotor speed and sustain directional changes.

Myocardial protection by propolis during prolonged hypothermic preservation.

Oxidative stress is involved in the pathogenesis of ischemia-reperfusion during myocardial transplantation. Therefore, graft preservation solutions may be improved by supplementation with antioxidants to minimize graft dysfunction caused by cold ischemic injury. Propolis is a polyphenol-rich substance which has an important antioxidant activity. The protective effect of propolis against oxidative stress induced by prolonged cold preservation of heart was investigated. Mice were subjected to a hypothermic model of ischemia in which hearts were preserved for 24 h at 4 °C in Krebs-Hensleit (KH) solution in the absence or presence of propolis concentrations (50, 150 and 250 µg/ml). Levels of released Lactate dehydrogenase (LDH), Creatine phosphokinase (CPK) and Troponine-I (Trop I) were assessed in the preservation solution and histological assessement of heart ischemia injuries was performed. Oxidative stress biomarkers malondialdehyde (MDA) and advanced oxidation protein products (AOPP) and antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) were assessed in cardiac tissue. Mitochondria were isolated from stored hearts and production of reactive oxygen species (ROS) was tested. Propolis supplementation protected efficiently hearts during preservation by reducing significantly levels of lipids and proteins oxidation and restoring activities of antioxidant enzymes. Also, propolis preserved tissue integrity altered by hypothermic ischemia in a concentration-dependent manner. Propolis reduced significantly the rate of H2O2 produced by mitochondrial respiration, the best antioxidant effect being obtained at the highest propolis concentration (250 µg/ml). Algerian propolis is a non-temperature sensitive scavenger that protects heart from oxidative damage induced by prolonged hypothermic ischemia.

Bimodal Respiratory-Locomotor Neurons in the Neonatal Rat Spinal Cord.

Neural networks that can generate rhythmic motor output in the absence of sensory feedback, commonly called central pattern generators (CPGs), are involved in many vital functions such as locomotion or respiration. In certain circumstances, these neural networks must interact to produce coordinated motor behavior adapted to environmental constraints and to satisfy the basic needs of an organism. In this context, we recently reported the existence of an ascending excitatory influence from lumbar locomotor CPG circuitry to the medullary respiratory networks that is able to depolarize neurons of the parafacial respiratory group during fictive locomotion and to subsequently induce an increased respiratory rhythmicity (Le Gal et al., 2014b). Here, using an isolated in vitro brainstem-spinal cord preparation from neonatal rat in which the respiratory and the locomotor networks remain intact, we show that during fictive locomotion induced either pharmacologically or by sacrocaudal afferent stimulation, the activity of both thoracolumbar expiratory motoneurons and interneurons is rhythmically modulated with the locomotor activity. Completely absent in spinal inspiratory cells, this rhythmic pattern is highly correlated with the hindlimb ipsilateral flexor activities. Furthermore, silencing brainstem neural circuits by pharmacological manipulation revealed that this locomotor-related drive to expiratory motoneurons is solely dependent on propriospinal pathways. Together these data provide the first evidence in the newborn rat spinal cord for the existence of bimodal respiratory-locomotor motoneurons and interneurons onto which both central efferent expiratory and locomotor drives converge, presumably facilitating the coordination between the rhythmogenic networks responsible for two different motor functions. Significance statement: In freely moving animals, distant regions of the brain and spinal cord controlling distinct motor acts must interact to produce the best adapted behavioral response to environmental constraints. In this context, it is well established that locomotion and respiration must to be tightly coordinated to reduce muscular interferences and facilitate breathing rate acceleration during exercise. Here, using electrophysiological recordings in an isolated in vitro brainstem-spinal cord preparation from neonatal rat, we report that the locomotor-related signal produced by the lumbar central pattern generator for locomotion selectively modulates the intracellular activity of spinal respiratory neurons engaged in expiration. Our results thus contribute to our understanding of the cellular bases for coordinating the rhythmic neural circuitry responsible for different behaviors.

CORM-3, a water soluble CO-releasing molecule, uncouples mitochondrial respiration via interaction with the phosphate carrier.

Carbon monoxide is continuously produced in small quantities in tissues and is an important signaling mediator in mammalian cells. We previously demonstrated that CO delivered to isolated rat heart mitochondria using a water-soluble CO-releasing molecule (CORM-3) is able to uncouple mitochondrial respiration. The aim of this study was to explore more in depth the mechanism(s) of this uncoupling effect. We found that acceleration of mitochondrial O2 consumption and decrease in membrane potential induced by CORM-3 were associated with an increase in mitochondrial swelling. This effect was independent of the opening of the mitochondrial transition pore as cyclosporine A was unable to prevent it. Interestingly, removal of phosphate from the incubation medium suppressed the effects mediated by CORM-3. Blockade of the dicarboxylate carrier, which exchanges dicarboxylate for phosphate, decreased the effects induced by CORM-3 while direct inhibition of the phosphate carrier with N-ethylmaleimide completely abolished the effects of CORM-3. In addition, CORM-3 was able to enhance the transport of phosphate into mitochondria as evidenced by changes in mitochondrial phosphate concentration and mitochondrial swelling that evaluates the activity of the phosphate carrier in de-energized conditions. These results indicate that CORM-3 activates the phosphate carrier leading to an increase in phosphate and proton transport inside mitochondria, both of which could contribute to the non-classical uncoupling effect mediated by CORM-3. The dicarboxylate carrier amplifies this effect by increasing intra-mitochondrial phosphate concentration.

Translation of TRO40303 from myocardial infarction models to demonstration of safety and tolerance in a randomized Phase I trial.

BACKGROUND: Although reperfusion injury has been shown to be responsible for cardiomyocytes death after an acute myocardial infarction, there is currently no drug on the market that reduces this type of injury. TRO40303 is a new cardioprotective compound that was shown to inhibit the opening of the mitochondrial permeability transition pore and reduce infarct size after ischemia-reperfusion in a rat model of cardiac ischemia-reperfusion injury. METHODS: In the rat model, the therapeutic window and the dose effect relationship were investigated in order to select the proper dose and design for clinical investigations. To evaluate post-ischemic functional recovery, TRO40303 was tested in a model of isolated rat heart. Additionally, TRO40303 was investigated in a Phase I randomized, double-blind, placebo controlled study to assess the safety, tolerability and pharmacokinetics of single intravenous ascending doses of the compound (0.5 to 13 mg/kg) in 72 healthy male, post-menopausal and hysterectomized female subjects at flow rates from 0.04 to 35 mL/min (EudraCT number: 2010-021453-39). This work was supported in part by the French Agence Nationale de la Recherche. RESULTS: In the vivo model, TRO40303 reduced infarct size by 40% at 1 mg/kg and by 50% at 3 and 10 mg/kg given by intravenous bolus and was only active when administered before reperfusion. Additionally, TRO40303 provided functional recovery and reduced oxidative stress in the isolated rat heart model.These results, together with pharmacokinetic based allometry to human and non-clinical toxicology data, were used to design the Phase I trial. All the tested doses and flow rates were well tolerated clinically. There were no serious adverse events reported. No relevant changes in vital signs, electrocardiogram parameters, laboratory tests or physical examinations were observed at any time in any dose group. Pharmacokinetics was linear up to 6 mg/kg and slightly ~1.5-fold, hyper-proportional from 6 to 13 mg/kg. CONCLUSIONS: These data demonstrated that TRO40303 can be safely administered by the intravenous route in humans at doses expected to be pharmacologically active. These results allowed evaluating the expected active dose in human at 6 mg/kg, used in a Phase II proof-of-concept study currently ongoing.

Multigenerational inheritance of breathing deficits following perinatal exposure to titanium dioxide nanoparticles in the offspring of mice.

BACKGROUND: The utilization of titanium dioxide nanoparticles (TIO2NPs) has experienced a significant surge in recent decades, and these particles are now commonly found in various everyday consumer products. Due to their small size, TIO2NPs can penetrate biological barriers and elicit adverse interactions with biological tissues. Notably, exposure of pregnant females to TIO2NPs during the perinatal period has been shown to disrupt the growth of offspring. Furthermore, this exposure induces epigenetic modifications in the DNA of newborns, suggesting the possibility of multigenerational effects. Thus, perinatal exposure to TIO2NPs may induce immediate metabolic impairments in neonates, which could be transmitted to subsequent generations in the long term. RESULTS: In this study, we utilized perinatal exposure of female mice to TIO2NPs through voluntary food intake and observed impaired metabolism in newborn male and female F1 offspring. The exposed newborn mice exhibited reduced body weight gain and a slower breathing rate compared to non-exposed animals. Additionally, a higher proportion of exposed F1 newborns experienced apneas. Similar observations were made when the exposure was limited to the postnatal period, highlighting lactation as a critical period for the adverse effects of TIO2NPs on postnatal metabolism. Importantly, the breathing deficits induced by TIO2NPs were transmitted from F1 females to the subsequent F2 generation. Moreover, re-exposure of adult F1 females to TIO2NPs exacerbated the breathing deficits in newborn F2 males. CONCLUSIONS: Our findings demonstrate that perinatal exposure to TIO2NPs disrupts postnatal body weight gain and respiration in the offspring, and these deficits are transmissible to future generations.

History of Tspo deletion and induction in vivo: Phenotypic outcomes under physiological and pathological situations.

The mitochondrial translocator protein (TSPO) is an outer mitochondrial protein membrane with high affinity for cholesterol. It is expressed in most tissues but is more particularly enriched in steroidogenic tissues. TSPO is involved in various biological mechanisms and TSPO regulation has been related to several diseases. However, despite a considerable number of published studies interested in TSPO over the past forty years, the precise function of the protein remains obscure. Most of the functions attributed to TSPO have been identified using pharmacological ligands of this protein, leading to much debate about the accuracy of these findings. In addition, research on the physiological role of TSPO has been hampered by the lack of in vivo deletion models. Studies to perform genetic deletion of Tspo in animal models have long been unsuccessful, which led to the conclusions that the deletion was deleterious and the gene essential to life. During the last decades, thanks to the significant technical advances allowing genome modification, several models of animal genetically modified for TSPO have been developed. These models have modified our view regarding TSPO and profoundly improved our fundamental knowledge on this protein. However, to date, they did not allow to elucidate the precise molecular function of TSPO and numerous questions persist concerning the physiological role of TSPO and its future as a therapeutic target. This article chronologically reviews the development of deletion and induction models of TSPO.

Effect of chronic prenatal exposure to the food additive titanium dioxide E171 on respiratory activity in newborn mice.

Nanoparticles (NPs) possess unique properties that make their use valuable in all industries. Titanium dioxide (TiO2) NPs are extensively used as a white pigment in food (labeled under the European number E171) and personal care products, which creates a significant potential for chronic consumer exposure. Concerns about the potential toxic effects of TiO2 NPs have arisen, particularly in vulnerable populations, including pregnant women and infants. Recently, human materno-fetal transfer of E171 was demonstrated, and simultaneously, we reported that chronic prenatal exposure to reference P25 TiO2 NPs was found to alter the developing respiratory neural networks. In this study, using whole body plethysmography from postnatal day (P) 0 to P7, we assessed the respiratory function of newborn mice born to mothers fed with E171 during pregnancy. We also evaluated the potential alterations to respiratory centers by using brainstem-spinal cord electrophysiological recordings from P0 to P6. Our study reveals that E171-prenatally exposed animals displayed an abnormally elevated breathing rate from P3 onwards. From P5 to P6, the respiratory-related burst frequency generated by the isolated brainstem-spinal cord preparations was significantly higher in E171-exposed animals than in non-exposed animals. These findings demonstrate prenatal toxicity of E171 to the developing respiratory function and may contribute to policy-making regarding the use of TiO2 NPs.

Elucidating the pivotal role of TSPO in porphyrin-related cellular processes, in Bacillus cereus.

A structural homolog of the mammalian TSPO has been identified in the human pathogen Bacillus cereus. BcTSPO, in its recombinant form, has previously been shown to bind and degrade porphyrins. In this study, we generated a ΔtspO mutant strain in B. cereus ATCC 14579 and assessed the impact of the absence of BcTSPO on cellular proteomics and physiological characteristics. The proteomic analysis revealed correlations between the lack of BcTSPO and the observed growth defects, increased oxygen consumption, ATP deficiency, heightened tryptophan catabolism, reduced motility, and impaired biofilm formation in the ΔtspO mutant strain. Our results also suggested that BcTSPO plays a crucial role in regulating intracellular levels of metabolites from the coproporphyrin-dependent branch of the heme biosynthetic pathway. This regulation potentially underlies alterations in the metabolic landscape, emphasizing the pivotal role of BcTSPO in B. cereus aerobic metabolism. Notably, our study unveils, for the first time, the involvement of TSPO in tryptophan metabolism. These findings underscore the multifaceted role of TSPO, not only in metabolic pathways but also potentially in the microorganism's virulence mechanisms.

TSPO in pancreatic beta cells and its possible involvement in type 2 diabetes.

Amyloidosis forms a large family of pathologies associated with amyloid deposit generated by the formation of amyloid fibrils or plaques. The amyloidogenic proteins and peptides involved in these processes are targeted against almost all organs. In brain they are associated with neurodegenerative disease, and the Translocator Protein (TSPO), overexpressed in these inflammatory conditions, is one of the target for the diagnostic. Moreover, TSPO ligands have been described as promising therapeutic drugs for neurodegenerative diseases. Type 2 diabetes, another amyloidosis, is due to a beta cell mass decrease that has been linked to hIAPP (human islet amyloid polypeptide) fibril formation, leading to the reduction of insulin production. In the present study, in a first approach, we link overexpression of TSPO and inflammation in potentially prediabetic patients. In a second approach, we observed that TSPO deficient rats have higher level of insulin secretion in basal conditions and more IAPP fibrils formation compared with wild type animals. In a third approach, we show that diabetogenic conditions also increase TSPO overexpression and IAPP fibril formation in rat beta pancreatic cell line (INS-1E). These data open the way for further studies in the field of type 2 diabetes treatment or prevention.

Cervicolumbar coordination in mammalian quadrupedal locomotion: role of spinal thoracic circuitry and limb sensory inputs.

Effective quadrupedal locomotion requires a close coordination between the spatially distant central pattern generators (CPGs) controlling forelimb and hindlimb movements. Using isolated preparations of the neonatal rat spinal cord, we explore the role of intervening thoracic circuitry in cervicolumbar CPG coordination and the contribution to this remote coupling of limb somatosensory inputs. In preparations activated with bath-applied N-methyl-D,L-aspartate, serotonin, and dopamine, the coordination between locomotor-related bursts recorded in cervical and lumbar ventral roots was substantially weakened, although not abolished, when the thoracic segments were selectively withheld from neurochemical stimulation or were exposed to a low Ca(2+) solution to block synaptic transmission. Moreover, cervicolumbar CPG coordination was reduced after a thoracic midsagittal section, suggesting that cross-cord projections participate in the anteroposterior coupling. In quiescent preparations, either cyclic or tonic electrical stimulation of low-threshold afferent pathways in C8 or L2 dorsal roots (DRs) could elicit coordinated ventral root bursting at both cervical and lumbar levels via an activation of the underlying CPG networks. When lumbar rhythmogenesis was prevented by local synaptic transmission blockade, L2 DR stimulation could still drive left-right alternating cervical bursting in preparations otherwise exposed to normal bathing medium. In contrast, when the cervical generators were selectively blocked, C8 DR stimulation was unable to activate the lumbar CPGs. Thus, in the newborn rat, anteroposterior limb coordination relies on active burst generation within midcord thoracic circuitry that additionally conveys ascending and weaker descending coupling influences of distant limb proprioceptive inputs to the cervical and lumbar generators, respectively.