Elene Zhuravliova

Haloperidol induces neurotoxicity by the NMDA receptor downstream signaling pathway, alternative from glutamate excitotoxicity

The NMDA receptor is believed to be important in a wide range of nervous system functions including neuronal migration, synapse formation, learning and memory. In addition, it is involved in excitotoxic neuronal cell death that occurs in a variety of acute and chronic neurological disorders. Besides of agonist/coagonist sites, other modulator sites, including butyrophenone site may regulate the N-methyl-D-aspartate receptor. It has been shown that haloperidol, an antipsychotic neuroleptic drug, interacts with the NR2B subunit of NMDA receptor and inhibits NMDA response in neuronal cells. We found that NMDA receptor was co-immunoprecipitated by anti-Ras antibody and this complex, beside NR2 subunit of NMDA receptor contained haloperidol-binding proteins, nNOS and Ras-GRF. Furthermore, we have shown that haloperidol induces neurotoxicity of neuronal cells via NMDA receptor complex, accompanied by dissociation of Ras-GRF from membranes and activation of c-Jun-kinase. Inclusion of insulin prevented relocalization of Ras-GRF and subsequent neuronal death. Haloperidol-induced dissociation of Ras-GRF leads to inhibition of membrane-bound form of Ras protein and changes downstream regulators activity that results in the initiation of the apoptotic processes via the mitochondrial way. Our results suggest that haloperidol induces neuronal cell death by the interaction with NMDA receptor, but through the alternative from glutamate excitotoxicity signaling pathway.

Social isolation in rats inhibits oxidative metabolism, decreases the content of mitochondrial K-Ras and activates mitochondrial hexokinase.

Recent observations have suggested that Ras signaling includes combinations of extracellular-signal-regulated Ras activation at the plasma membrane and endomembranes, and translocation of Ras from the plasma membrane to intracellular compartments. In this study we have shown that social isolation of rat decreases the content of Bcl-2-associated K-Ras in hippocampal mitochondria, whereas the amount of H-Ras is increased in the microsomal fraction. Furthermore, we have found that galectin 1, a binding partner of activated Ras, was increased in the soluble fractions. The redistribution of Ras isoforms was accompanied by acceleration in mitochondrial hexokinase and inhibition of mitochondrial aconitase, succinate dehydrogenase, and creatine kinase, whereas the activity of aldolase, as well as cytoplasmic creatine kinase was not changed. Our data suggest that inhibition of mitochondrial oxidative metabolism by reactive oxygen species (ROS) and compensatory elevation of glycolysis in hippocampus occurs during social isolation of rats and Ras trafficking could play an important role in switching of impaired oxidative phosphorylation to anaerobic glycolysis.

Sigma-1 receptor directly interacts with Rac1-GTPase in the brain mitochondria

BackgroundSmall Rho-GTPases are critical mediators of neuronal plasticity and are involved in the pathogenesis of several psychiatric and neurological disorders. Rac-GTPase forms a multiprotein complex with upstream and downstream regulators that are essential for the spatiotemporal transmission of Rac signaling. The sigma-1 receptor (Sig1R) is a ligand-regulated membrane protein chaperone, and multiprotein complex assembly is essential to sigma-receptor function.ResultsUsing immunoprecipitation techniques, we have shown that in mitochondrial membranes Sig1R could directly interact with Rac1. Besides Rac1, the Sig1R forms complexes with inositol 1,4,5-trisphosphate receptor and Bcl2, suggesting that mitochondrial associated membranes (MAM) are involved in this macromolecular complex formation. Assembly of this complex is ligand-specific and depends on the presence of sigma agonist/antagonist, as well as on the presence of GTP/GDP. Treatment of mitochondrial membranes with (+)-pentazocine leads to the (+)-pentazocine-sensitive phosphorylation of Bad and the pentazocine-sensitive NADPH-dependent production of ROS.ConclusionWe suggest that Sig1R through Rac1 signaling induces mild oxidative stress that possibly is involved in the regulation of neuroplasticity, as well as in the prevention of apoptosis and autophagy.

N-Methyl-D-Aspartate and σ-Ligands Change the Production of Interleukins 8 and 10 in Lymphocytes through Modulation of the NMDA Glutamate Receptor

Human T lymphocytes express both ionotropic and metabotropic glutamate receptors that control immune responses, cell activation, maturation and death. In this study, we examined the effect of N-methyl-D-aspartate (NMDA) and σ1-receptor ligands on the secretion of the proinflammatory chemokine interleukin 8 (IL-8) and the anti-inflammatory cytokine interleukin 10 (IL-10) in human leukemia Jurkat cells and peripheral blood lymphocytes (PBLs). We have shown that NMDA increased IL-8 and decreased IL-10 secretion and that σ-ligands modulated the action of NMDA. Moreover, the effects of NMDA and σ-ligands were interrelated with the nitric oxide (NO) content, suggesting that the intracellular concentration of NO could play a major role in the synthesis of cytokines. Western blots against the NR2A and NR2B subunits of the NMDA glutamate receptor revealed that long-term (48 h) treatment of PBLs with glutamate at concentrations within normal plasma levels (1 × 10–5M), in contrast to low concentrations (0.3 × 10–6M), downregulates the NR2A subunit, probably by internalization. Furthermore, we found that PBLs with noninternalized NR2A secreted less IL-10 than lymphocytes with downregulated NR2A; under these conditions, the transcriptional activity of NF-κB was increased whereas the transcriptional activity of c-Fos was decreased. These findings implicate that the activities of NF-κB and c-Fos control the expression of the IL8 and IL10 genes, depending on the subunit composition of the NMDA receptor. In conclusion, we suggest that lymphocytes express an active NMDA receptor only in a low-glutamate milieu.

Inhibition of nitric oxide synthase and farnesyltransferase change the activities of several transcription factors.

Several types of cellular proteins can be modified by farnesylation and nitrosylation, of which the most significant is Ras. We used manumycin, a farnesyltransferase inhibitor, and L-NAME (Nω-nitro-l-arginine methyl ester), a nitric oxide synthase (NOS) inhibitor, for characterization of Ras-dependent downstream targets activities. Our results suggest that change of the steady-state levels of nitric oxide and inhibition of farnesylation modified the activities of several transcription factors. We have found that the inhibition of farnesylation by manumycin decreased the DNA-binding activity of nuclear factor (NF)-κB, did not change the DNA-binding activities of STAT, Sp1, ATF-2, and CREB, and increased the activities of c-Fos, JunD, and c-Jun. Under such conditions, phosphorylation of Akt was decreased, whereas phosphorylation of extracellular signal-regulated kinase (ERK) was increased and phosphorylation of JNK did not change. Furthermore, our results show that reduction of intracellular concentration of nitric oxides by L-NAME increases the activities of c-Fos, ATF-2 and JunD and decreases the activities of CREB, STAT, Sp1, and c-Jun. The activities of all of these transcription factors are restored to normal levels in the presence of manumycin, suggesting that simultaneous modifications of proteins by farnesylation and nitrosylation change the direction of Ras-controlled downstream pathways. Our results provide further evidence of the significance of posttranslational modifications of Ras for the specificity of transducing cascade networks and physiological outcome.

Hypoinsulinemia Alleviates the Grf1/Ras/Akt Anti-Apoptotic Pathway and Induces Alterations of Mitochondrial Ras Trafficking in Neuronal Cells

Recent observations have established that interruption of insulin production causes deficits in learning and memory formation. We have studied the mechanism of insulin’s neuroprotective effect on primary neuronal cells and in streptozotocin (STZ)-induced diabetic rat brain. We have found that in hippocampal neuronal cells insulin increases the content of farnesylated Ras and phosphorylated form of Akt. Besides, the treatment of cells by insulin leads to the activation of mitochondrial cytochrome oxidase, which is inhibited by manumycin, a farnesyltransferase inhibitor. During experimental diabetes, the content of membrane-bound GRF1 was decreased in rat hippocampus that was correlated with the reduction in mitochondrial Ras and phosphorylated forms of Akt. This redistribution in Ras-GRF system was accompanied by the alteration in the activities of CREB, NF-kB (p65) and c-Rel transcription factors. We have proposed that hypoinsulinemia induces the inhibition of Ras signalling in the neuronal cells additionally by abnormality of Ras trafficking into mitochondria.

S-Nitrosylation Decreases the Adsorption of H-Ras in Lipid Bilayer and Changes Intrinsic Catalytic Activity

Structural, chemical, and mutational studies have shown that C-terminal cysteine residues on H-Ras could potentially be oxidized by nitrosylation. For investigating the effect of nitrosylation of Ras molecule on the adsorption of farnesylated H-Ras into lipid layer, experiments with optical waveguide lightmode spectroscopy were used. The analysis of association/dissociation kinetics to planar phospholipids under controlled hydrodynamic conditions has shown that preliminary treatment of protein by S-nitroso-cysteine decreased the adsorption of farnesylated H-Ras. The authors have found that compared with nitrosylated forms, farnesylated H-Ras has more compact configuration, because of the smaller area occupied by protein upon absorption at the membrane. The association rate coefficient for unmodified H-Ras was lower than similar parameter for farnesylated and nitrosylated forms. However, the desorbability, i.e., parameter, which reflects the rate of dissociation of protein from lipids is higher for farnesylated H-Ras. In addition, it was have found that farnesylation of cytoplasmic H-Ras, in contrast to membrane-derived forms, inhibits intrinsic GTPase activity of protein, and preliminary treatment of H-Ras by S-nitroso-cysteine restores the activity to the control level. These data suggest that nitrosylation of H-Ras rearranges the adsorptive potential and intrinsic GTPase activity of H-Ras through modification of C-terminal cysteines of molecule.

mGluR1 interacts with cystic fibrosis transmembrane conductance regulator and modulates the secretion of IL-10 in cystic fibrosis peripheral lymphocytes

Cystic fibrosis (CF) is caused by the mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. CFTR dysfunction in T cells could lead directly to aberrant immune responses. The action of glutamate on the secretion of IL-8 and IL-10 by lymphocytes derived from healthy subjects and cystic CF patients, as well as the expression of metabotropic glutamate receptor subtype 1 (mGluR1) in the membrane fractions of lymphocytes was investigated. Our results have shown that CF-derived T-cells in the presence of IL-2 produce more IL-8 and IL-10, than T-cell from healthy control. However, only in normal lymphocytes a significant increase (144%) in the IL-10 secretion during exposure to high concentration of glutamate (10(-4)M) was detected. Glutamate-dependent secretion of IL-10 was not inhibited either by NMDA-receptor (NMDAR), or by AMPA-receptor (AMPAR) antagonist. Only mGluR1 antagonist, LY367385, strongly decreases the production of IL-10. Furthermore, the content of mGluR1, as well as cystic fibrosis transmembrane conductance regulator-associated ligand (CAL), Na(+)/H(+) exchanger regulatory factor 1 (NHERF-1), was analyzed in plasma membrane of lymphocytes after immunoprecipitation of CFTR. We have found that normal, non-mutated CFTR, as well as mutated forms of CFTR were associated with metabotropic mGluR1, but the level of surface exposed mGluR1 in CF-lymphocytes was much lower than in normal cells. Besides, our results have shown that normal, non-mutated CFTR, as well as mutated forms of CFTR were associated with NHERF-1 and CAL; however in lymphocytes with CFTR mutation the amount of cell-surface expressed CFTR-CAL complex was greatly decreased. We have concluded that CFTR and mGluR1 could compete for binding to CAL, which in turn downregulates the post-synthetic trafficking of mGluR1 and decreases the synthesis of IL-10.

Sigma-1 Receptor Agonists Induce Oxidative Stress in Mitochondria and Enhance Complex I Activity in Physiological Condition but Protect Against Pathological Oxidative Stress

The sigma1 receptor (σ1R) is a chaperone protein residing at mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs), where it modulates Ca2+ exchange between the ER and mitochondria by interacting with inositol-1,4,5 trisphosphate receptors (IP3Rs). The σ1R is highly expressed in the central nervous system and its activation stimulates neuromodulation and neuroprotection, for instance in Alzheimer’s disease (AD) models in vitro and in vivo. σ1R effects on mitochondria pathophysiology and the downstream signaling are still not fully understood. We here evaluated the impacts of σ1R ligands in mouse mitochondria preparations on reactive oxygen species (ROS) production, mitochondrial respiration, and complex activities, in physiological condition and after direct application of amyloid Aβ1–42 peptide. σ1R agonists (2-(4-morpholinethyl)-1-phenylcyclohexanecarboxylate hydrochloride (PRE-084), tetrahydro-N,N-dimethyl-5,5-diphenyl-3-furanmethanamine (ANAVEX1-41, AN1-41), (S)-1-(2,8-dimethyl-1-thia-3,8-diazaspiro[4.5]dec-3-yl)-3-(1H-indol-3-yl)propan-1-one (ANAVEX3-71, AN3-71), dehydroepiandrosterone-3 sulfate (DHEA), donepezil) increased mitochondrial ROS in a σ1R antagonist-sensitive manner but decreased Aβ1–42-induced increase in ROS. σ1R ligands (agonists or antagonists) did not impact respiration but attenuated Aβ1–42-induced alteration. σ1R agonists (PRE-084, AN1-41, tetrahydro-N,N-dimethyl-2,2-diphenyl-3-furanmethanamine hydrochloride (ANAVEX2-73, AN2-73), AN3-71) increased complex I activity, in a Ca2+-dependent and σ1R antagonist-sensitive manner. σ1R ligands failed to affect complex II, III, and IV activities. The increase in complex I activity explain the σ1R-induced increase in ROS since ligands failed to affect other sources of ROS accumulation in mitochondria and homogenates, namely NADPH oxidase (NOX) and superoxide dismutase (SOD) activities. Furthermore, Aβ1–42 significantly decreased the activity of complexes I and IV and σ1R agonists attenuated the Aβ1–42-induced complex I and IV dysfunctions. σ1R activity in mitochondria therefore results in a Ying-Yang effect, by triggering moderate ROS increase acting as a physiological signal and promoting a marked anti-oxidant effect in pathological (Aβ) conditions.

Nobiletin restores impaired hippocampal mitochondrial bioenergetics in hypothyroidism through activation of matrix substrate-level phosphorylation

Abstract Objective Evaluation of the effect of citrus flavonoid – nobiletin on the bioenergetics of synaptic and non-synaptic mitochondria in the hippocampus of hypothyroid rats. Methods Male Wistar rats were divided into hypothyroid (methimazole-treated), nobiletin supplemented hypothyroid, thyroxine-treated hypothyroid, and euthyroid (control) groups. Synaptic and non-synaptic (cell) mitochondria were isolated from hippocampus. Oligomycin-sensitive, oligomycin-insensitive, α-ketoglutarate dehydrogenase-dependent synthesis of adenosine triphosphate (ATP), succinate dehydrogenase, and hexokinase activities were determined luminometrically and spectrophotometrically, respectively. Results Decreased synthesis of oligomycin-sensitive and oligomycin-insensitive ATP in hypothyroid rat hippocampus was observed in synaptic and non-synaptic mitochondria. Supplementation of hypothyroid rats with nobiletin increases oligomycin-insensitive and α-ketoglutarate-dependent production of ATP in both types of mitochondria. The activity of succinate dehydrogenase in non-synaptic mitochondria and the activities of hexokinase in both types of mitochondria were normalized in nobiletin-treated hypothyroid rats. Discussion Nobiletin restores reduced mitochondrial metabolism in hypothyroid rat hippocampus through acceleration of matrix substrate-level phosphorylation that may be important for the prevention of hypometabolic complications in neurological disorders.