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

BACKGROUND: Small 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. RESULTS: Using 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. CONCLUSION: We 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.

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

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.

Gut neurotoxin p-cresol induces brain-derived neurotrophic factor secretion and increases the expression of neurofilament subunits in PC-12 cells.

Increased p-cresol levels reportedly alter brain dopamine metabolism and exacerbate neurological disorders in experimental animals. In contrast to toxic concentrations, low doses of p-cresol may have distinct effects on neuronal metabolism. However, the role of p-cresol in synapse remodeling, neurite outgrowth, and other anabolic processes in neurons remains elusive. We propose that low doses of p-cresol affect neuronal cell structural remodeling compared with the high concentration-mediated harmful effects. Thus, the effects of p-cresol on the secretion of brain-derived neurotrophic factor (BDNF) and neurofilament subunit expression were examined using rat pheochromocytoma cells (PC-12 cells). We observed that low doses of p-cresol potentiated nerve growth factor-induced differentiation via secretion of BDNF in cultured PC-12 cells. Opioidergic compounds modulated these p-cresol effects, which were reversed by oxytocin. We propose that this effect of p-cresol has an adaptive and compensatory character and can be attributed to the induction of oxidative stress. Accordingly, we hypothesize that low doses of p-cresol induce mild oxidative stress, stimulating BDNF release by activating redox-sensitive genes. Given that the intestinal microbiome is the primary source of endogenous p-cresol, the balance between gut microbiome strains (especially Clostridium species) and opioidergic compounds may directly influence neuroplasticity.

Gut neurotoxin p-cresol induces differential expression of GLUN2B and GLUN2A subunits of the NMDA receptor in the hippocampus and nucleus accumbens in healthy and audiogenic seizure-prone rats.

Mislocalization and abnormal expression of N-methyl-D-aspartate glutamate receptor (NMDAR) subunits is observed in several brain disorders and pathological conditions. Recently, we have shown that intraperitoneal injection of the gut neurotoxin p-cresol induces autism-like behavior and accelerates seizure reactions in healthy and epilepsy-prone rats, respectively. In this study, we evaluated the expression of GLUN2B and GLUN2A NMDAR subunits, and assessed the activity of cAMP-response element binding protein (CREB) and Rac1 in the hippocampi and nucleus accumbens of healthy and epilepsy-prone rats following p-cresol administration. We have found that subchronic intraperitoneal injection of p-cresol induced differential expression of GLUN2B and GLUN2A between the two brain regions, and altered the GLUN2B/GLUN2A ratio, in rats in both groups. Moreover, p-cresol impaired CREB phosphorylation in both brain structures and stimulated Rac activity in the hippocampus. These data indicate that p-cresol differently modulates the expression of NMDAR subunits in the nucleus accumbens and hippocampi of healthy and epilepsy-prone rats. We propose that these differences are due to the specificity of interactions between dopaminergic and glutamatergic pathways in these structures.

N-methyl-D-aspartate and sigma-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 sigma1-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 sigma-ligands modulated the action of NMDA. Moreover, the effects of NMDA and sigma-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 x 10(-5)M), in contrast to low concentrations (0.3 x 10(-6)M), 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-kappaB was increased whereas the transcriptional activity of c-Fos was decreased. These findings implicate that the activities of NF-kappaB 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.

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 Aß1-42 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 Aß1-42-induced increase in ROS. σ1R ligands (agonists or antagonists) did not impact respiration but attenuated Aß1-42-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, Aß1-42 significantly decreased the activity of complexes I and IV and σ1R agonists attenuated the Aß1-42-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 (Aß) conditions.

Subcellular Distribution of S-Nitrosylated H-Ras in Differentiated and Undifferentiated PC12 Cells during Hypoxia.

OBJECTIVES: Hypoxia or exposure to excessive reactive oxygen or nitrogen species could induce S-nitrosylation of various target proteins, including GTPases of the Ras-superfamily. Under hypoxic conditions, the Ras-protein is translocated to the cytosol and interacts with the Golgi complex, endoplasmic reticulum, mitochondria. The mobility/translocation of Ras depend on the cells oxidative status. However, the importance of relocated Snitrosylated- H-Ras (NO-H-Ras) in proliferation/differentiation processes is not completely understood. We have determined the content of soluble- and membrane-bound-NO-HRas in differentiated (D) and undifferentiated (ND) rat pheochromocytoma (PC12) cells under hypoxic and normoxic conditions. MATERIALS AND METHODS: In our experimental study, we analyzed NO-H-Ras levels under hypoxic/normoxic conditions in membrane and soluble fractions of ND and D PC12 cells with/without nitric oxide donor, sodium nitroprusside (SNP) treatment. Cells were analyzed by the S-nitrosylated kit, immunoprecipitation, and Western blot. We assessed the action of NO-H-Ras on oxidative metabolism of isolated mitochondria by determining mitochondrial hydrogen peroxide generation via the scopoletin oxidation method and ATPproduction as estimated by the luminometric method. RESULTS: Hypoxia did not influence nitrosylation of soluble H-Ras in ND PC12 cells. Under hypoxic conditions, the nitrosylation of soluble-H-Ras greatly decreased in D PC12 cells. SNP didn't change the levels of nitrosylation of soluble-H-Ras, in either hypoxic or normoxic conditions. On the other hand, hypoxia, per se, did not affect the nitrosylation of membrane-bound-H-Ras in D and ND PC12 cells. SNP-dependent nitrosylation of membrane-bound-H-Ras greatly increased in D PC12 cells. Both unmodified normal and mutated H-Ras enhanced the mitochondrial synthesis of ATP, whereas the stimulatory effects on ATP synthesis were eliminated after S-nitrosylation of H-Ras. CONCLUSIONS: According to the results, it may be proposed that hypoxia can decrease S-nitrosylation of soluble-H-Ras in D PC12 cells and abolish the inhibitory effect of NO-HRas in mitochondrial oxidative metabolism.

Mitochondrial Target of Nobiletin's Action.

Nobiletin is an 0-methylated flavonoid found in citrus peels that have anticancer, antiviral, neuroprotective; anti-inflammatory activities and depending on the cell types exhibits both pro- or anti-apoptotic properties We have found that nobiletin decreases oxygen consumption by bovine brain isolated mitochondria in the presence of glutamate and malate and increases in the presence of succinate. In paralleli nobiletin increases NADH: oxidation, a-ketoghitarate dehydrogenase activities and through matrix substrate-level phosphorylation elevates the a-ketoglutarate-dependent-production-of ATP. In addition, nobiletin reduces the production of peroxides in the presence of complex I substrates and slightly enhances succinate-driven H(2)0(2) formation. Besides, nobiletin induces transient elevation of membrane potential followed by mild depolarization. Affinity purified, nobiletin binding proteins revealed one major anti-NDUFVl positive protein with 52kD and NADH: ubiquinone oxidoreductase activity. This fraction can produce peroxide that is inhibited by nobiletin. We propose that nobiletin may act as a mild "uncoupler", which through activation of a-ketoglutarate dehydrogenase (a-KGDH)-complex and acceleration of matrix substrate-level phosphorylation maintains membrane potential at an abnormal level. This switch in mitochondrial metabolism could elevate succinate-driven oxygen consumption that may underlay in both pro- and anti-apoptotic effects of nobiletin.

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.

WITHDRAWN: Role of nitrosylaton and farnesylation in the regulation of homocysteine metabolism in PC12 cells.

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L-NAME has opposite effects on the productions of S-adenosylhomocysteine and S-adenosylmethionine in V12-H-Ras and M-CR3B-Ras pheochromocytoma cells.

Homocysteine is a sulfur-containing, nonproteinogenic, neurotoxic amino acid biosynthesized during methyl cycles after demethylation of S-adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH) and subsequent hydrolysis of SAH into homocysteine and adenosine. Formed homocysteine is either catabolized into cystathionine (transsulfuration pathway) by cystathionine beta-synthase, or remethylated into methionine (remethylation pathway) by methionine synthase. To demonstrate the specificity of Ras-elicited effects on the activity of methyl cycles, wild-type pheochromocytoma PC12, mutant oncogenic rasH gene (MVR) expressing PC12 pheochromocytoma and normal c-rasH stably transfected M-CR3B cells were incubated with the N(omega)-nitro-L-arginine methyl ester (L-NAME), and manumycin, (inhibitors of nitric oxide synthase and farnesyltransferase, respectively). We have found that L-NAME significantly changes the SAM/SAH ratio in both MCR and MVR cells. Moreover, these alterations have reciprocal character; in the MCR cells, the SAM/SAH ratio was raised, whereas in the MVR cells this ratio was decreased. We conclude that depletion of endogenous NO with L-NAME increased the production of SAH only in cells with mutated oncogenic RasH, possibly through enhancement of production of reactive oxygen species (ROS). Oxidative stress can increase cystathionine beta-synthase activity that switches methyl cycles from remethylation into transsulfuration pathway to maintain the intracellular glutathione pool (essential for the redox-regulating capacity of cells) via an adaptive process.