Folic Acid Protects Against Glutamate-Induced Excitotoxicity in Hippocampal Slices Through a Mechanism that Implicates Inhibition of GSK-3ß and iNOS.

Folic acid (folate) is a vitamin of the B-complex group crucial for neurological function. Considering that excitotoxicity and cell death induced by glutamate are involved in many disorders, the potential protective effect of folic acid on glutamate-induced cell damage in rat hippocampal slices and the possible intracellular signaling pathway involved in such effect were investigated. The treatment of hippocampal slices with folic acid (100 µM) significantly abrogated glutamate (1 mM)-induced reduction of cell viability measured by MTT reduction assay and inhibited glutamate-induced D-[3H]-aspartate release. To investigate the putative intracellular signaling pathways implicated in the protective effect of folic acid, we used a PI3K inhibitor, LY294002, which abolished the protective effects of folic acid against glutamate-induced cell damage and D-[3H] aspartate release. Moreover, hippocampal slices incubated with folic acid alone for 30 min presented increased phosphorylation of GSK-3ß at Ser9, indicating an inhibition of the activity of this enzyme. Furthermore, folic acid in the presence of glutamate insult in hippocampal slices maintained for an additional period of 6 h in fresh culture medium without glutamate and/or folic acid induced phosphorylation of GSK-3ß and ß-catenin expression. In addition, glutamate-treated hippocampal slices showed increased iNOS expression that was reversed by folic acid. In conclusion, the results of this study show that the protective effect of folic acid against glutamate-induced excitotoxicity may involve the modulation of PI3K/GSK-3ß/ß-catenin pathway and iNOS inhibition.

Guanosine controls inflammatory pathways to afford neuroprotection of hippocampal slices under oxygen and glucose deprivation conditions.

Guanosine (GUO) is an endogenous modulator of glutamatergic excitotoxicity and has been shown to promote neuroprotection in in vivo and in vitro models of neurotoxicity. This study was designed to understand the neuroprotective mechanism of GUO against oxidative damage promoted by oxygen/glucose deprivation and reoxygenation (OGD). GUO (100 µM) reduced reactive oxygen species production and prevented mitochondrial membrane depolarization induced by OGD. GUO also exhibited anti-inflammatory actions as inhibition of nuclear factor kappa B activation and reduction of inducible nitric oxide synthase induction induced by OGD. These GUO neuroprotective effects were mediated by adenosine A1 receptor, phosphatidylinositol-3 kinase and MAPK/ERK. Furthermore, GUO recovered the impairment of glutamate uptake caused by OGD, an effect that occurred via a Pertussis toxin-sensitive G-protein-coupled signaling, blockade of adenosine A2A receptors (A2A R), but not via A1 receptor. The modulation of glutamate uptake by GUO also involved MAPK/ERK activation. In conclusion, GUO, by modulating adenosine receptor function and activating MAPK/ERK, affords neuroprotection of hippocampal slices subjected to OGD by a mechanism that implicates the following: (i) prevention of mitochondrial membrane depolarization, (ii) reduction of oxidative stress, (iii) regulation of inflammation by inhibition of nuclear factor kappa B and inducible nitric oxide synthase, and (iv) promoting glutamate uptake.

Lectin from Canavalia brasiliensis (ConBr) protects hippocampal slices against glutamate neurotoxicity in a manner dependent of PI3K/Akt pathway.

The excitotoxicity induced by excessive activation of the glutamatergic neurotransmission pathway is involved in several neuropathologies. In this sense, molecules that prevent the release of glutamate or the excessive activation of its receptors can be useful in preventing the neuronal cell death observed in these diseases. Lectins are proteins capable of reversible binding to the carbohydrates in glycoconjugates, and some have been used in the study and purification of glutamate receptors. ConBr is a mannose/glucose-binding lectin purified from Canavalia brasiliensis seeds. In the present study, we aimed to evaluate the neuroprotective activity of ConBr against glutamate-induced excitotoxicity. Hippocampal slices were isolated from adult male mice and incubated for 6h in Krebs saline/DMEM buffer alone (control), in the presence of glutamate or glutamate plus ConBr. The phosphorylation of Akt and mitogen activated protein kinases (MAPKs) such as ERK1/2, p38(MAPK) and JNK1/2/3 was evaluated with western blotting. The results indicate that glutamate provoked a reduction in the hippocampal slice viability (-25%), diminished the phosphorylation of Akt and augmented p38(MAPK) and ERK1 phosphorylation. No changes were observed in the phosphorylation of JNK1/2/3 or ERK2. Notably, ConBr, through a mechanism dependent on carbohydrate interaction, prevented the reduction of cell viability and Akt phosphorylation induced by glutamate. Furthermore, in the presence of the PI3K inhibitor LY294002, ConBr was unable to reverse glutamate neurotoxicity. Taken together, our data suggest that the neuroprotective effect of ConBr against glutamate neurotoxicity requires oligosaccharide interaction and is dependent on the PI3K/Akt pathway.

Guanosine protects human neuroblastoma SH-SY5Y cells against mitochondrial oxidative stress by inducing heme oxigenase-1 via PI3K/Akt/GSK-3ß pathway.

Mitochondrial perturbation and oxidative stress are key factors in neuronal vulnerability in several neurodegenerative diseases or during brain ischemia. Here we have investigated the protective mechanism of action of guanosine, the guanine nucleoside, in a human neuroblastoma cell line, SH-SY5Y, subjected to mitochondrial oxidative stress. Blockade of mitochondrial complexes I and V with rotenone plus oligomycin (Rot/oligo) caused a significant decrease in cell viability and an increase in ROS production. Guanosine that the protective effect of guanosine incubated concomitantly with Rot/oligo abolished Rot/oligo-induced cell death and ROS production in a concentration dependent manner; maximum protection was achieved at the concentration of 1mM. The cytoprotective effect afforded by guanosine was abolished by adenosine A(1) or A(2A) receptor antagonists (DPCPX or ZM241385, respectively), or by a large (big) conductance Ca(2+)-activated K(+) channel (BK) blocker (charybdotoxin). Evaluation of signaling pathways showed that the protective effect of guanosine was not abolished by a MEK inhibitor (PD98059), by a p38(MAPK) inhibitor (SB203580), or by a PKC inhibitor (cheleritrine). However, when blocking the PI3K/Akt pathway with LY294002, the neuroprotective effect of guanosine was abolished. Guanosine increased Akt and p-Ser-9-GSK-3ß phosphorylation confirming this pathway plays a key role in guanosine's neuroprotective effect. Guanosine induced the antioxidant enzyme heme oxygenase-1 (HO-1) expression. The protective effects of guanosine were prevented by heme oxygenase-1 inhibitor, SnPP. Moreover, bilirubin, an antioxidant and physiologic product of HO-1, is protective against mitochondrial oxidative stress. In conclusion, our results show that guanosine can afford protection against mitochondrial oxidative stress by a signaling pathway that implicates PI3K/Akt/GSK-3ß proteins and induction of the antioxidant enzyme HO-1.

The antinociceptive effects of AR-A014418, a selective inhibitor of glycogen synthase kinase-3 beta, in mice.

UNLABELLED: We investigated the antinociceptive effects of AR-A014418, a selective inhibitor of glycogen synthase kinase-3ß (GSK-3ß) in mice. A 30-minute pretreatment with AR-A014418 (.1 and 1 mg/kg, intraperitoneal [ip]) inhibited nociception induced by an ip injection of acetic acid. AR-A014418 pretreatment (.1 and .3 mg/kg, ip) also decreased the late (inflammatory) phase of formalin-induced licking, without affecting responses of the first (neurogenic) phase. In a different set of experiments, AR-A014418 (.1-10 µg/site) coinjected intraplantarly (ipl) with formalin inhibited the late phase of formalin-induced nociception. Furthermore, AR-A014418 administration (1 and 10 ng/site, intrathecal [it]) inhibited both phases of formalin-induced licking. In addition, AR-A014418 coinjection (10 ng/site, it) inhibited nociception induced by glutamate, N-methyl-D-aspartate (NMDA), (±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD), tumor necrosis factor-alpha (TNF-α), and interleukin-1beta (IL-1ß) by 47 ± 12%, 48 ± 11%, 31 ± 8%, 46 ± 13%, and 44 ± 11%, respectively. In addition, a 30-minute pretreatment with NP031115 (3 and 10 mg/kg, ip), a different GSK-3 ß inhibitor, also attenuated the late phase of formalin-induced nociception. Collectively, these results provide convincing evidence that AR-A014418, given by local, systemic, and central routes, produces antinociception in several mouse models of nociception. The AR-A014418-dependent antinociceptive effects were induced by modulation of the glutamatergic system through metabotropic and ionotropic (NMDA) receptors and the inhibition of the cytokine (TNF-α and IL-1ß) signaling. PERSPECTIVE: These results suggest that GSK-3ß may be a novel pharmacological target for the treatment of pain.