Search

When ROS production exceeds the cellular antioxidant capacity, the cell needs to eliminate the defective mitochondria responsible for excessive ROS production. It has been proposed that the removal of these defective mitochondria involves mitophagy, but the mechanism of this regulation remains unclear. Here, we demonstrate that moderate mitochondrial superoxide and hydrogen peroxide production oxidates KEAP1, thus breaking the interaction between this protein and PGAM5, leading to the inhibition of its proteasomal degradation. Accumulated PGAM5 interferes with the processing of the PINK1 in the mitochondria leading to the accumulation of PINK1 on the outer mitochondrial membrane. In turn, PINK1 promotes Parkin recruitment to mitochondria and sensitizes mitochondria for autophagic removal. We also demonstrate that inhibitors of the KEAP1-PGAM5 protein-protein interaction (including CPUY192018) mimic the effect of mitochondrial ROS and sensitize mitophagy machinery, suggesting that these inhibitors could be used as pharmacological regulators of mitophagy. Together, our results show that KEAP1/PGAM5 complex senses mitochondrially generated superoxide/hydrogen peroxide to induce mitophagy.

Summary: Purpose: Progressive myoclonus epilepsy of the Unverricht–Lundborg type (EPM1) is a rare neurologic disorder, associated with mutations in the Cystatin B (Cstb) gene. Mice lacking Cstb, a cysteine protease inhibitor of the cathepsine family of proteases, provide a mammalian model for EPM1 by displaying similarly progressive ataxia, myoclonic seizures, and neurodegeneration. However, the linkage of Cstb deficit on the molecular level to pathologic features like myoclonic jerks or tonic–clonic seizures has remained unclear. We examined the tryptophan (TRP) metabolism, along the serotonin (5HT) and kynurenine (KYN) pathway in the brain of Cstb-deficient mice, in relation to their possible involvement in the seizure phenotype. Methods: TRP and its metabolites, along the 5HT and KYN pathways, were assayed in brain tissue by high-pressure liquid chromatography (HPLC) with electrochemical detection. The inverted wire grid and mild handling tests were used for evaluation of ataxia and myoclonic activity. Results: The Cstb-deficient mice had constitutively increased TRP, 5HT, and 5-hydroxyindole acetic acid (5HIAA) levels in the cerebral cortex and cerebellum and increased levels of KYN in the cerebellum. These neurochemical changes were accompanied with ataxia and an apparent myoclonic phenotype among the Cstb-deficient mice. Conclusions: Our findings suggest that secondary processes (i.e., overstimulation of serotoninergic transmission) on the cellular level, initiated by Cstb deficiency in specific brain regions, may be responsible for the myoclonic/seizure phenotype in EPM1.

A novel and simple method has been developed for the simultaneous quantification of tryptophan, kynurenine and indole derivatives as well as four catecholamines, including dopamine, noradrenaline, homovanillic acid and 3,4-dihydroxyphenylacetic acid. The method utilises isocratic reversed-phase high-performance liquid chromatography with electrochemical coulometric array detection. The influence of various parameters on chromatographic performance, such as the composition and the pH of the mobile phase and the detection potentials, was investigated. Separation of 13 compounds was achieved by a mobile phase consisting of 10% methanol in 50 mM sodium phosphate-acetate buffer, pH 4.10, containing 0.42 mM octanesulphonic acid. The calibration curve was linear over the range 12 pg to 300 ng on-column. The detection limits (SIN 3) depended on the working potential and were found to be between 10 and 100 pg injected. The method was reproducible with intra-day RSDs of 0.3 to 1.5% and inter-day RSDs of 0.5 to 4%.

(1) The study was aimed to investigate the effects of the minimal effective doses of acute citalopram (5 mg/kg), (+/-)-8-hydroxydipropylaminotetralin HBr (8-OH-DPAT; 0.1 mg/kg), and their combined treatment on the rat open field and forced swimming behaviour and post-mortem monoamine content. (2) The animals were prospectively divided into the vehicle- and para-chlorophenylalanine (p-CPA)-pretreated (350 mg/kg) groups. (3) Acute citalopram (5 mg/kg), 8-OH-DPAT (0.1 mg/kg), or their combined treatment had no major effect on the rat open field and forced swimming behaviour. (4) The post-mortem catecholamine content in four brain regions studied was unchanged in all treatment groups. (5) The combined 8-OH-DPAT (0.1 mg/kg) and citalopram (5 mg/kg) treatment partially reversed the p-CPA-induced decrease of serotonin (5-HT) and 5-hydroxy-indolacetic acid (5-HIAA) content. (6) The present experiments demonstrate that the 5-HT1A receptors mediate some of the selective serotonin reuptake inhibitor (SSRI)-induced biochemical phenomena.

Pseudo-total analysis (PTA) for metals in siliceous soil by atomic absorption spectrometry was carried out using digestion of siliceous soil with HNO3/H2O2 and aqua regia. The dissolved silicate was found to interfere with the absorbance of Ca, Mg, Mn, Zn and Fe. No interference was found with Cu and Pb. The effect of silicate can be decreased by dilution of solutions and/or using lanthanum as the releasing element. The rate of release of various elements was studied.

Parkinson disease is characterized by the accumulation of aggregated α-synuclein as the major component of the Lewy bodies. α-Synuclein accumulation in turn leads to compensatory effects that may include the up-regulation of autophagy. Another common feature of Parkinson disease (PD) is mitochondrial dysfunction. Here, we provide evidence that the overactivation of autophagy may be a link that connects the intracellular accumulation of α-synuclein with mitochondrial dysfunction. We found that the activation of macroautophagy in primary cortical neurons that overexpress mutant A53T α-synuclein leads to massive mitochondrial destruction and loss, which is associated with a bioenergetic deficit and neuronal degeneration. No mitochondrial removal or net loss was observed when we suppressed the targeting of mitochondria to autophagosomes by silencing Parkin, overexpressing wild-type Mitofusin 2 and dominant negative Dynamin-related protein 1 or blocking autophagy by silencing autophagy-related genes. The inhibition of targeting mitochondria to autophagosomes or autophagy was also partially protective against mutant A53T α-synuclein-induced neuronal cell death. These data suggest that overactivated mitochondrial removal could be one of the contributing factors that leads to the mitochondrial loss observed in PD models.

Dopamine is a neurotransmitter that plays a major role in a variety of brain functions, as well as in disorders such as Parkinson disease and schizophrenia. In cultured astrocytes, we have found that dopamine induces sporadic cytoplasmic calcium ([Ca(2+)](c)) signals. Importantly, we show that the dopamine-induced calcium signaling is receptor-independent in midbrain, cortical, and hippocampal astrocytes. We demonstrate that the calcium signal is initiated by the metabolism of dopamine by monoamine oxidase, which produces reactive oxygen species and induces lipid peroxidation. This stimulates the activation of phospholipase C and subsequent release of calcium from the endoplasmic reticulum via the inositol 1,4,5-trisphosphate receptor mechanism. These findings have major implications on the function of astrocytes that are exposed to dopamine and may contribute to understanding the physiological role of dopamine.

Dopamine is one of the key neurotransmitters in the central nervous system and plays an important role in physiological processes, as well as in the development of many diseases. Here we report a receptor-independent signalling pathway induced by dopamine in CNS neurons. In cultured neurons from midbrain, cortex and hippocampus, dopamine uptake via dopamine or monoamine transporters induces plasmalemmal membrane depolarization, leading to opening of voltage gated calcium channels and a cytosolic calcium signal. This dopamine-induced calcium signal is unaffected by inhibition of the known dopamine receptors. In anaesthetized rats, application of dopamine in the presence of dopamine receptor antagonists to brainstem structures controlling cardiovascular activity results in an increase in heart rate, arterial blood pressure and sympathetic nerve activity. These data identify a novel dopamine-induced signalling pathway in CNS neurons which may have an important functional role in the central mechanisms controlling complex behaviours.

International audience; Creatine kinase (CK) is a phosphotransfer kinase that catalyzes the reversible transfer of a phosphate moiety between ADP and creatine and that is highly expressed in skeletal muscle. In fast glycolytic skeletal muscle, deletion of the cytosolic M isoform of CK in mice (M-CK-/-) leads to a massive increase in the oxidative capacity and of mitochondrial volume. This study was aimed at investigating the transcriptional pathways leading to mitochondrial biogenesis in response to CK deficiency. Wild type and M-CK-/- mice of eleven months of age were used for this study. Gastrocnemius muscles of M-CK-/- mice exhibited a dramatic increase in citrate synthase (+120%) and cytochrome oxidase (COX, +250%) activity, and in mitochondrial DNA (+60%), showing a clear activation of mitochondrial biogenesis. Similarly, mRNA expression of the COXI (mitochondria-encoded) and COXIV (nuclear-encoded) subunits were increased by +103 and +94 % respectively. This was accompanied by an increase in the expression of the nuclear respiratory factor (NRF2Α and the mitochondrial transcription factor (mtTFA). Expression of the co-activator PGC-1Α, a master gene in mitochondrial biogenesis was not significantly increased while that of PGC-1Β and PRC, two members of the same family, was moderately increased (+45% and +55% respectively). While the expression of the modulatory calcineurin-interacting protein 1 (MCIP1) was dramatically decreased (minus 68%) suggesting inactivation of the calcineurin pathway, the metabolic sensor AMPK was activated (+86%) in M-CK-/- mice. These results evidence that mitochondrial biogenesis in response to a metabolic challenge exhibits a unique pattern of regulation, involving activation of the AMPK pathway.

Summary: Purpose: To evaluate the levels of tryptophan and its metabolites along serotonin (5-HT) and kynurenine (KYN) pathways in serum of progressive myoclonus epilepsy (EPM1) patients and cystatin B (CSTB)-deficient mice, a model system for EPM1. Methods: Tryptophan and its metabolites along serotonin (5HT) and KYN pathways were determined in serum of EPM1 patients and CSTB-deficient mice by reverse-phase high-pressure liquid chromatography (HPLC) with electrochemical detection. Results: Reduced levels of 5-HT and KYN intermediate metabolite 3-hydroxyanthranilic acid were found in serum of CSTB-deficient mice. A similar trend was found in EPM1 patients. Although tryptophan concentration was reduced in serum of EPM1 patients, no such decrease was observed in CSTBdeficient mice. Conclusions: The present study demonstrates that tryptophan metabolism along 5-HT and KYN pathways are disrupted in EPM1. Further studies are needed to elucidate the role of KYN pathway in pathogenesis of EPM1. Key Words: Progressive myoclonus epilepsy—Cystatin B-deficient mice—Kynurenines—Serotonin—Tryptophan. Progressive myoclonus epilepsy of the UnverrichtLundborg type (EPM1) is a recessively inherited neurodegenerative disease caused by loss of function mutations in the gene encoding cystatin B (CSTB), a cysteine protease inhibitor of the cathepsin family of proteases (1). The disease has an onset of 6‐15 years of age and is characterized by stimulus-sensitive myoclonus, clonic‐tonic seizures, ataxia, and slow decline in the cognitive functions. Previous clinical investigations in patients with EPM1 have demonstrated reduced 5-hydroxyindoleacetic acid (5-HIAA), suggesting a disrupted serotonin metabolism (2). Furthermore, the concentrations of 5HIAA seemed to correlate to the severity of the EPM1 (3). Also, it was found that administration of L-tryptophan, a precursor of serotonin, improved ambulation, myoclonic jerks, and the general condition in six of seven EPM1 patients. The quantitative EEG revealed

Cocaine- and amphetamine-regulated transcript (CART) is a novel neuropeptide with neurotransmitter-like effects. In the present study we examined the influence of CART peptide fragment, CART(62-76), on the levels of catecholamines (dopamine and norepinephrine), serotonin and their metabolites in five regions of the rat brain. CART(62-76) was administered at 0.5 or 5.0 microg dose intracerebroventricularly. A high-pressure liquid chromatograph coupled to electrochemical array detector was used to analyse the tissue homogenates. In the frontal cortex, CART(62-76) increased the levels of 5-hydroxyindoleacetic acid (5-HIAA), a metabolite of serotonin (5-HT). In the striatum, CART(62-76) decreased the levels of noradrenaline and 5-HT. In the hypothalamus, CART(62-76) increased the levels of 5-HIAA. CART(62-76) had no significant effect in the hippocampus and cerebellum. Our data suggest that CART(62-76) peptide has no major effect on dopaminergic pathways, but it modulates the activity of striatal noradrenergic and corticostriatal and hypothalamic serotoninergic system in the rat brain. These regionally selective neurochemical changes may explain the effects of CART peptides on appetitive, emotional and locomotor behaviour.

The aim of this study was to investigate the monoamine content in post-mortem brain samples of control, apomorphine-aggressive, and apomorphine-non-aggressive adult male Wistar rats. The repeated apomorphine (1.0 mg/kg, (s.c.) once daily during 2 weeks) gradually induced aggressive behaviour in 18 animals out of 24. No unidirectional changes in the brain monoamine contents in four regions (frontal cortex, striatum, hippocampus, and hypothalamus) were detected as measured by high pressure liquid chromatography-electrochemical detection. In conclusion, our present experiment demonstrates that the development and intensity of apomorphine-induced aggressive behaviour do not correlate with the brain post-mortem monoamine content.

Glutamate excitotoxicity is responsible for neuronal death in acute neurological disorders including stroke, trauma and neurodegenerative disease. Loss of calcium homeostasis is a key mediator of glutamate-induced cell death. The neurotransmitter dopamine (DA) is known to modulate calcium signalling, and here we show that it can do so in response to physiological concentrations of glutamate. Furthermore, DA is able to protect neurons from glutamate-induced cell death at pathological concentrations of glutamate. We demonstrate that DA has a novel role in preventing delayed calcium deregulation in cortical, hippocampal and midbrain neurons. The effect of DA in abolishing glutamate excitotoxicity can be induced by DA receptor agonists, and is abolished by DA receptor antagonists. Our data indicate that the modulation of glutamate excitotoxicity by DA is receptor-mediated. We postulate that DA has a major physiological function as a safety catch to restrict the glutamate-induced calcium signal, and thereby prevent glutamate-induced cell death in the brain.

Potentiometric titration with ISE is adopted for the analysis of phosphate ions in natural waters. The method is based on the low solubility of lead phosphate. To increase the sensitivity of the determination, coprecipitation of phosphate ions with magnesium hydroxide was used.