The impact of purine nucleosides on neuroplasticity in the adult brain.

Neuroplasticity refers to the nervous system's ability to adapt and reorganize its cell structures and neuronal networks in response to internal and external stimuli. In adults, this process involves neurogenesis, synaptogenesis, and synaptic and neurochemical plasticity. Several studies have reported the significant impact of the purinergic system on neuroplasticity modulation. And, there is considerable evidence supporting the role of purine nucleosides, such as adenosine, inosine, and guanosine, in this process. This review presents extensive research on how these nucleosides enhance the neuroplasticity of the adult central nervous system, particularly in response to damage. The mechanisms through which these nucleosides exert their effects involve complex interactions with various receptors and signaling pathways. Adenosine's influence on neurogenesis involves interactions with adenosine receptors, specifically A1R and A2AR. A1R activation appears to inhibit neuronal differentiation and promote astrogliogenesis, while A2AR activation supports neurogenesis, neuritogenesis, and synaptic plasticity. Inosine and guanosine positively impact cell proliferation, neurogenesis, and neuritogenesis. Inosine seems to modulate extracellular adenosine levels, and guanosine might act through interactions between purinergic and glutamatergic systems. Additionally, the review discusses the potential therapeutic implications of purinergic signaling in neurodegenerative and neuropsychiatric diseases, emphasizing the importance of these nucleosides in the neuroplasticity of brain function and recovery.

Guanosine increases global SUMO1-ylation in the hippocampus of young and aged mice and improves the short-term memory of young mice.

The nucleoside guanosine is an endogenous neuromodulator associated with neuroprotection. The roles of guanosine during aging are still not fully elucidated. Guanosine modulates SUMOylation in neurons and astrocytes in vitro, but it is not known whether guanosine can modulate SUMOylation in vivo and improve cognitive functions during aging. SUMOylation is a post-translational protein modification with potential neuroprotective roles. In this follow-up study, we investigated whether guanosine could modulate SUMOylation in vivo and behavior in young and aged mice. Young (3-month-old) and aged (24-month-old) C57BL/6 mice were treated with guanosine (8 mg/kg intraperitoneal) daily for 14 days. Starting on day 8 of treatment, the following behavioral tests were performed: open field, novel object location, Y-maze, sucrose splash test, and tail suspension test. Treatment with guanosine did not change the locomotor activity of young or aged mice in the open-field test. Treatment with guanosine improved short-term memory only for young mice but did not change the working memory of either young or aged mice, as evaluated using object recognition and the Y-maze tests, respectively. Depressive-like behaviors, such as impaired grooming evaluated through the splash test, did not change in either young or aged mice. However, young mice treated with guanosine increased their immobility time in the tail suspension test, suggesting an effect on behavioral coping strategies. Global SUMO1-ylation was significantly increased in the hippocampus of young and aged mice after 14 days of treatment with guanosine, whereas no changes were detected in the cerebral cortex of either young or aged mice. Our findings demonstrate that guanosine also targets hippocampal SUMOylation in vivo, thereby contributing to a deeper understanding of its mechanisms of action. This highlights the involvement of SUMOylation in guanosine's modulatory and neuroprotective effects.

The lectin DrfL inhibits cell migration, adhesion and triggers autophagy-dependent cell death in glioma cells.

Glioblastoma multiforme (GBM) is the most aggressive type of glioma, displaying atypical glycosylation pattern that may modulate signaling pathways involved in tumorigenesis. Lectins are glycan binding proteins with antitumor properties. The present study was designed to evaluate the antitumor capacity of the Dioclea reflexa lectin (DrfL) on glioma cell cultures. Our results demonstrated that DrfL induced morphological changes and cytotoxic effects in glioma cell cultures of C6, U-87MG and GBM1 cell lines. The action of DrfL was dependent upon interaction with glycans, and required a carbohydrate recognition domain (CRD), and the cytotoxic effect was apparently selective for tumor cells, not altering viability and morphology of primary astrocytes. DrfL inhibited tumor cell migration, adhesion, proliferation and survival, and these effects were accompanied by activation of p38MAPK and JNK (p46/54), along with inhibition of Akt and ERK1/2. DrfL also upregulated pro-apoptotic (BNIP3 and PUMA) and autophagic proteins (Atg5 and LC3 cleavage) in GBM cells. Noteworthy, inhibition of autophagy and caspase-8 were both able to attenuate cell death in GBM cells treated with DrfL. Our results indicate that DrfL cytotoxicity against GBM involves modulation of cell pathways, including MAPKs and Akt, which are associated with autophagy and caspase-8 dependent cell death.

Functional interplay between adenosine A2A receptor and NMDA preconditioning in fear memory and glutamate uptake in the mice hippocampus.

N-methyl D-aspartate (NMDA) administered at subtoxic dose plays a protective role against neuronal excitotoxicity, a mechanism described as preconditioning. Since the activation of adenosinergic receptors influences the achievement of NMDA preconditioning in the hippocampus, we evaluated the potential functional interplay between adenosine A1 and A2A receptors (A1R and A2AR) activities and NMDA preconditioning. Adult male Swiss mice received saline (NaCl 0.9 g%, i.p.) or a nonconvulsant dose of NMDA (75 mg/kg, i.p.) and 24 h later they were treated with the one of the ligands: A1R agonist (CCPA, 0.2 mg/kg, i.p.) or antagonist (DPCPX, 3 mg/kg, i.p.), A2AR agonist (CGS21680, 0.05 mg/kg, i.p.) or antagonist (ZM241385, 0.1 mg/kg, i.p.) and subjected to contextual fear conditioning task. Binding properties and content of A2AR and glutamate uptake were assessed in the hippocampus of mice subjected to NMDA preconditioning. Treatment with CGS21680 increased the time of freezing during the exposure of animals to the new environment. NMDA preconditioning did not affect the freezing time of mice per se, but it prevented the response observed after the activation of A2AR. Furthermore, the activation of A2AR by CGS21680 after the preconditioning blocked the increase of glutamate uptake induced by NMDA preconditioning. The immunodetection of A2AR in total hippocampal homogenates showed no significant differences evoked by NMDA preconditioning and did not alter A2AR maximum binding for the selective ligand [3H]CGS21680. These results demonstrate changes in A2AR functionality in mice following NMDA preconditioning.

Guanosine modulates SUMO2/3-ylation in neurons and astrocytes via adenosine receptors.

SUMOylation is a post-translational modification (PTM) whereby members of the Small Ubiquitin-like MOdifier (SUMO) family of proteins are conjugated to lysine residues in target proteins. SUMOylation has been implicated in a wide range of physiological and pathological processes, and much attention has been given to its role in neurodegenerative conditions. Due to its reported role in neuroprotection, pharmacological modulation of SUMOylation represents an attractive potential therapeutic strategy in a number of different brain disorders. However, very few compounds that target the SUMOylation pathway have been identified. Guanosine is an endogenous nucleoside with important neuromodulatory and neuroprotective effects. Experimental evidence has shown that guanosine can modulate different intracellular pathways, including PTMs. In the present study we examined whether guanosine alters global protein SUMOylation. Primary cortical neurons and astrocytes were treated with guanosine at 1, 10, 100, 300, or 500 µM at four time points, 1, 6, 24, or 48 h. We show that guanosine increases global SUMO2/3-ylation in neurons and astrocytes at 1 h at concentrations above 10 µM. The molecular mechanisms involved in this effect were evaluated in neurons. The guanosine-induced increase in global SUMO2/3-ylation was still observed in the presence of dipyridamole, which prevents guanosine internalization, demonstrating an extracellular guanosine-induced effect. Furthermore, the A1 adenosine receptor antagonist DPCPX abolished the guanosine-induced increase in SUMO2/3-ylation. The A2A adenosine receptor antagonist ZM241385 increased SUMOylation per se, but did not alter guanosine-induced SUMOylation, suggesting that guanosine may modulate SUMO2/3-ylation through an A1-A2A receptor interaction. Taken together, this is the first report to show guanosine as a SUMO2/3-ylation enhancer in astrocytes and neurons.

Guanosine prevents oxidative damage and glutamate uptake impairment induced by oxygen/glucose deprivation in cortical astrocyte cultures: involvement of A1 and A2A adenosine receptors and PI3K, MEK, and PKC pathways.

Glial cells are involved in multiple cerebral functions that profoundly influence brain tissue viability during ischemia, and astrocytes are the main source of extracellular purines as adenosine and guanosine. The endogenous guanine-based nucleoside guanosine is a neuromodulator implicated in important processes in the brain, such as modulation of glutamatergic transmission and protection against oxidative and inflammatory damage. We evaluated if the neuroprotective effect of guanosine is also observed in cultured cortical astrocytes subjected to oxygen/glucose deprivation (OGD) and reoxygenation. We also assessed the involvement of A1 and A2A adenosine receptors and phosphatidylinositol-3 kinase (PI3K), MAPK, and protein kinase C (PKC) signaling pathways on the guanosine effects. OGD/reoxygenation decreased cell viability and glutamate uptake and increased reactive oxygen species (ROS) production in cultured astrocytes. Guanosine treatment prevented these OGD-induced damaging effects. Dipropyl-cyclopentyl-xanthine (an adenosine A1 receptor antagonist) and 4-[2-[[6-amino-9-(N-ethyl-ß-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl] benzenepropanoic acid hydrochloride (an adenosine A2A receptor agonist) abolished guanosine-induced protective effects on ROS production, glutamate uptake, and cell viability. The PI3K pathway inhibitor 2-morpholin-4-yl-8-phenylchromen-4-one, the extracellular-signal regulated kinase kinase (MEK) inhibitor 2'-amino-3'-methoxyflavone, or the PKC inhibitor chelerythrine abolished the guanosine effect of preventing OGD-induced cells viability reduction. PI3K inhibition partially prevented the guanosine effect of reducing ROS production, whereas MEK and PKC inhibitions prevented the guanosine effect of restoring glutamate uptake. The total immunocontent of the main astrocytic glutamate transporter glutamate transporter-1 (GLT-1) was not altered by OGD and guanosine. However, MEK and PKC inhibitions also abolished the guanosine effect of increasing cell-surface expression of GLT-1 in astrocytes subjected to OGD. Then, guanosine prevents oxidative damage and stimulates astrocytic glutamate uptake during ischemic events via adenosine A1 and A2A receptors and modulation of survival signaling pathways, contributing to microenvironment homeostasis that culminates in neuroprotection.

Guanosine and GMP increase the number of granular cerebellar neurons in culture: dependence on adenosine A2A and ionotropic glutamate receptors.

The guanine-based purines (GBPs) have essential extracellular functions such as modulation of glutamatergic transmission and trophic effects on neurons and astrocytes. We previously showed that GBPs, such as guanosine-5'-monophosphate (GMP) or guanosine (GUO), promote the reorganization of extracellular matrix proteins in astrocytes, and increase the number of neurons in a neuron-astrocyte co-culture protocol. To delineate the molecular basis underlying these effects, we isolated cerebellar neurons in culture and treated them with a conditioned medium derived from astrocytes previously exposed to GUO or GMP (GBPs-ACM) or, directly, with GUO or GMP. Agreeing with the previous studies, there was an increase in the number of ß-tubulin III-positive neurons in both conditions, compared with controls. Interestingly, the increase in the number of neurons in the neuronal cultures treated directly with GUO or GMP was more prominent, suggesting a direct interaction of GBPs on cerebellar neurons. To investigate this issue, we assessed the role of adenosine and glutamate receptors and related intracellular signaling pathways after GUO or GMP treatment. We found an involvement of A2A adenosine receptors, ionotropic glutamate N-methyl-D-aspartate (NMDA), and non-NMDA receptors in the increased number of cerebellar neurons. The signaling pathways extracellular-regulated kinase (ERK), calcium-calmodulin-dependent kinase-II (CaMKII), protein kinase C (PKC), phosphatidilinositol-3'-kinase (PI3-K), and protein kinase A (PKA) are also potentially involved with GMP and GUO effect. Such results suggest that GMP and GUO, and molecules released in GBPs-ACM promote the survival or maturation of primary cerebellar neurons or both via interaction with adenosine and glutamate receptors.

Involvement of glutamatergic neurotransmission in the antidepressant-like effect of zinc in the chronic unpredictable stress model of depression.

Stress and excessive glutamatergic neurotransmission have been implicated in the pathophysiology of depression. Therefore, this study was aimed at investigating the influence of zinc on depressive-like behavior induced by chronic unpredictable stress (CUS), on alterations in glutamate-induced toxicity and immunocontent of proteins involved in the control of glutamatergic neurotransmission in the hippocampus of mice. Mice were subjected to CUS procedure for 14 days. From the 8th to the 14th day, mice received zinc chloride (ZnCl2) (10 mg/kg) or fluoxetine (10 mg/kg, positive control) once a day by oral route. CUS caused a depressive-like behavior evidenced by the increased immobility time in the tail suspension test (TST), which was prevented by treatment with ZnCl2 or fluoxetine. Ex vivo exposure of hippocampal slices to glutamate (10 mM) resulted in a significant decrease on cell viability; however, neither CUS procedure nor drug treatments altered this reduction. No alterations in the immunocontents of GLT-1 and GFAP or p-Akt were observed in any experimental group. The ratio of p-Akt/AKT was also not altered in any group. However, Akt immunocontent was increased in stressed mice and in animals treated with ZnCl2 (stressed or non-stressed mice) and EAAC1 immunocontent was increased in stressed mice treated with ZnCl2, fluoxetine or vehicle and in non-stressed mice treated with ZnCl2 and fluoxetine. These findings indicate a robust effect of zinc in reversing behavioral alteration induced by CUS in mice, through a possible modulation of the glutamatergic neurotransmission, extending literature data regarding the mechanisms underlying its antidepressant-like action.

Guanosine prevents nitroxidative stress and recovers mitochondrial membrane potential disruption in hippocampal slices subjected to oxygen/glucose deprivation.

Guanosine, the endogenous guanine nucleoside, prevents cellular death induced by ischemic events and is a promising neuroprotective agent. During an ischemic event, nitric oxide has been reported to either cause or prevent cell death. Our aim was to evaluate the neuroprotective effects of guanosine against oxidative damage in hippocampal slices subjected to an in vitro ischemia model, the oxygen/glucose deprivation (OGD) protocol. We also assessed the participation of nitric oxide synthase (NOS) enzymes activity on the neuroprotection promoted by guanosine. Here, we showed that guanosine prevented the increase in ROS, nitric oxide, and peroxynitrite production induced by OGD. Moreover, guanosine prevented the loss of mitochondrial membrane potential in hippocampal slices subjected to OGD. Guanosine did not present an antioxidant effect per se. The protective effects of guanosine were mimicked by inhibition of neuronal NOS, but not of inducible NOS. The neuroprotective effect of guanosine may involve activation of cellular mechanisms that prevent the increase in nitric oxide production, possibly via neuronal NOS.

The modulation of NMDA receptors and L-arginine/nitric oxide pathway is implicated in the anti-immobility effect of creatine in the tail suspension test.

The modulation of N-methyl-D-aspartate receptor (NMDAR) and L-arginine/nitric oxide (NO) pathway is a therapeutic strategy for treating depression and neurologic disorders that involves excitotoxicity. Literature data have reported that creatine exhibits antidepressant and neuroprotective effects, but the implication of NMDAR and L-arginine/nitric oxide (NO) pathway in these effects is not established. This study evaluated the influence of pharmacological agents that modulate NMDAR/L-arginine-NO pathway in the anti-immobility effect of creatine in the tail suspension test (TST) in mice. The NOx levels and cellular viability in hippocampal and cerebrocortical slices of creatine-treated mice were also evaluated. The anti-immobility effect of creatine (10 mg/kg, po) in the TST was abolished by NMDA (0.1 pmol/mouse, icv), D-serine (30 µg/mouse, icv, glycine-site NMDAR agonist), arcaine (1 mg/kg, ip, polyamine site NMDAR antagonist), L-arginine (750 mg/kg, ip, NO precursor), SNAP (25 µg/mouse, icv, NO donor), L-NAME (175 mg/kg, ip, non-selective NOS inhibitor) or 7-nitroindazole (50 mg/kg, ip, neuronal NOS inhibitor), but not by DNQX (2.5 µg/mouse, icv, AMPA receptor antagonist). The combined administration of sub-effective doses of creatine (0.01 mg/kg, po) and NMDAR antagonists MK-801 (0.001 mg/kg, po) or ketamine (0.1 mg/kg, ip) reduced immobility time in the TST. Creatine (10 mg/kg, po) increased cellular viability in hippocampal and cerebrocortical slices and enhanced hippocampal and cerebrocortical NO x levels, an effect potentiated by L-arginine or SNAP and abolished by 7-nitroindazole or L-NAME. In conclusion, the anti-immobility effect of creatine in the TST involves NMDAR inhibition and enhancement of NO levels accompanied by an increase in neural viability.

Purine receptors are required for DHA-mediated neuroprotection against oxygen and glucose deprivation in hippocampal slices.

Docosahexaenoic acid (DHA) is important for central nervous system function during pathological states such as ischemia. DHA reduces neuronal injury in experimental brain ischemia; however, the underlying mechanisms are not well understood. In the present study, we investigated the effects of DHA on acute hippocampal slices subjected to experimental ischemia by transient oxygen and glucose deprivation (OGD) and re-oxygenation and the possible involvement of purinergic receptors as the mechanism underlying DHA-mediated neuroprotection. We observed that cellular viability reduction induced by experimental ischemia as well as cell damage and thiobarbituric acid reactive substances (TBARS) production induced by glutamate (10 mM) were prevented by hippocampal slices pretreated with DHA (5 µM). However, glutamate uptake reduction induced by OGD and re-oxygenation was not prevented by DHA. The beneficial effect of DHA against cellular viability reduction induced by OGD and re-oxygenation was blocked with PPADS (3 µM), a nonselective P2X1-5 receptor antagonist as well as with a combination of TNP-APT (100 nM) plus brilliant blue (100 nM), which blocked P2X1, P2X3, P2X2/3, and P2X7 receptors, respectively. Moreover, adenosine receptors blockade with A1 receptor antagonist DPCPX (100 nM) or with A2B receptor antagonist alloxazine (100 nM) inhibited DHA-mediated neuroprotection. The addition of an A2A receptor antagonist ZM241385 (50 nM), or A3 receptor antagonist VUF5574 (1 µM) was ineffective. Taken together, our results indicated that neuroprotective actions of DHA may depend on P2X, A1, and A2B purinergic receptors activation. Our results reinforce the notion that dietary DHA may act as a local purinergic modulator in order to prevent neurodegenerative diseases.

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.

Fluorescence resonance energy transfer-based technologies in the study of protein-protein interactions at the cell surface.

Understanding of the molecular mechanisms of protein-protein interactions (PPIs) at the cell surface of living cells is fundamental to comprehend the functional meaning of a large number of cellular processes. Here we discuss how new methodological strategies derived from non-invasive fluorescence-based approaches (i.e. fluorescence resonance energy transfer, FRET) have been successfully developed to characterize plasma membrane PPIs. Importantly, these technologies alone - or in concert with complementary methods (i.e. SNAP-tag/TR-FRET, TIRF/FRET) - can become extremely powerful approaches for visualizing cell surface PPIs, even between more than two proteins and also in native tissues. Interestingly, these methods would also be relevant in drug discovery in order to develop new high-throughput screening approaches or to identify new therapeutic targets. Accordingly, herein we provide a thorough assessment on all biotechnological aspects, including strengths and weaknesses, of these fluorescence-based methodologies when applied in the study of PPIs occurring at the cell surface of living cells.

NMDA receptor-mediated modulation on glutamine synthetase and glial glutamate transporter GLT-1 is involved in the antidepressant-like and neuroprotective effects of guanosine.

Guanosine has been reported to elicit antidepressant-like responses in rodents, but if these actions are associated with its ability to afford neuroprotection against glutamate-induced toxicity still needs to be fully understood. Therefore, this study investigated the antidepressant-like and neuroprotective effects elicited by guanosine in mice and evaluated the possible involvement of NMDA receptors, glutamine synthetase, and GLT-1 in these responses. We found that guanosine (0.05 mg/kg, but not 0.01 mg/kg, p. o.) was effective in producing an antidepressant-like effect and protecting hippocampal and prefrontocortical slices against glutamate-induced damage. Our results also unveiled that ketamine (1 mg/kg, but not 0.1 mg/kg, i. p, an NMDA receptor antagonist) effectively elicited antidepressant-like actions and protected hippocampal and prefrontocortical slices against glutamatergic toxicity. Furthermore, the combined administration of sub-effective doses of guanosine (0.01 mg/kg, p. o.) with ketamine (0.1 mg/kg, i. p.) promoted an antidepressant-like effect and augmented glutamine synthetase activity and GLT-1 immunocontent in the hippocampus, but not in the prefrontal cortex. Our results also showed that the combination of sub-effective doses of ketamine and guanosine, at the same protocol schedule that exhibited an antidepressant-like effect, effectively abolished glutamate-induced damage in hippocampal and prefrontocortical slices. Our in vitro results reinforce that guanosine, ketamine, or sub-effective concentrations of guanosine plus ketamine protect against glutamate exposure by modulating glutamine synthetase activity and GLT-1 levels. Finally, molecular docking analysis suggests that guanosine might interact with NMDA receptors at the ketamine or glycine/d-serine co-agonist binding sites. These findings provide support for the premise that guanosine has antidepressant-like effects and should be further investigated for depression management.

ConBr, a lectin from Canavalia brasiliensis seeds, protects against quinolinic acid-induced seizures in mice.

Lectins are proteins capable of reversible binding to carbohydrates or glycoconjugates. In the central nervous system of mammals, lectins with affinity for mannose/glucose or galactose can modulate cellular communication. ConBr, a lectin isolated from the seeds of Canavalia brasiliensis, previously showed antidepressant effect in the forced swimming test in mice, with involvement of the monoaminergic system. In this study, we investigated the neuroprotective effects of ConBr against quinolinic acid (QA), a well-known NMDA agonist that produces severe neurotoxicity when administered in vivo. ConBr (10 µg/site) administered via intracerebroventricular (i.c.v.) showed a neuroprotective activity against seizures induced by QA (36.8 nmol/site; i.c.v.) when administered 15 min prior to QA, with a percentage of protection around 50%. ConBr was also able to significantly decrease the severity of the seizures but without changes in the latency of the first convulsion or the duration of the seizures. This effect was dependent on the structural integrity of the ConBr protein and its binding capacity to oligosaccharides residues. ConA, a lectin with high similarity to ConBr, did not reverse the QA-induced seizures. Moreover, ConBr was able to protect against hippocampal cell death caused by QA, which was measured by propidium iodide incorporation. QA caused activation of JNK2 and improved the phosphorylation of Ser831 and 845 on the AMPA receptor GluR1 subunit, and both of these effects were counteracted by ConBr. Our data suggest that the lectin ConBr may exert a modulatory action on NMDA receptors, which inhibits its activity in response to QA.

Neuroprotective effect of guanosine against glutamate-induced cell death in rat hippocampal slices is mediated by the phosphatidylinositol-3 kinase/Akt/ glycogen synthase kinase 3ß pathway activation and inducible nitric oxide synthase inhibition.

Excitotoxicity and cell death induced by glutamate are involved in many neurodegenerative disorders. We have previously demonstrated that excitotoxicity induced by millimolar concentrations of glutamate in hippocampal slices involves apoptotic features and glutamate-induced glutamate release. Guanosine, an endogenous guanine nucleoside, prevents excitotoxicity by its ability to modulate glutamate transport. In this study, we have evaluated the neuroprotective effect of guanosine against glutamate-induced toxicity in hippocampal slices and the mechanism involved in such an effect. We have found that guanosine (100 µM) was neuroprotective against 1 mM glutamate-induced cell death through the inhibition of glutamate release induced by glutamate. Guanosine also induced the phosphorylation and, thus, activation of protein kinase B (PKB/Akt), a downstream target of phosphatidylinositol-3 kinase (PI3K), as well as phosphorylation of glycogen synthase kinase 3ß, which has been reported to be inactivated by Akt after phosphorylation at Ser9. Glutamate treated hippocampal slices showed increased inducible nitric oxide synthase (iNOS) expression that was prevented by guanosine. Slices preincubated with SNAP (an NO donor), inhibited the protective effect of guanosine. LY294002 (30 µM), a PI3K inhibitor, attenuated guanosine-induced neuroprotection, guanosine prevention of glutamate release, and guanosine-induced GSK3ß(Ser9) phosphorylation but not guanosine reduction of glutamate-induced iNOS expression. Taken together, the results of this study show that guanosine protects hippocampal slices by a mechanism that involves the PI3K/Akt/GSK3ß(Ser9) pathway and prevention of glutamate-induced glutamate release. Furthermore, guanosine also reduces glutamate-induced iNOS by a PI3K/Akt-independent mechanism.

N-methyl-D-aspartate preconditioning improves short-term motor deficits outcome after mild traumatic brain injury in mice.

Traumatic brain injury (TBI) causes impairment of fine motor functions in humans and nonhuman mammals that often persists for months after the injury occurs. Neuroprotective strategies for prevention of the sequelae of TBI and understanding the molecular mechanisms and cellular pathways are related to the glutamatergic system. It has been suggested that cellular damage subsequent to TBI is mediated by the excitatory neurotransmitters, glutamate and aspartate, through the excessive activation of the N-methyl-D-aspartate (NMDA) receptors. Thus, preconditioning with a low dose of NMDA was used as a strategy for protection against locomotor deficits observed after TBI in mice. Male adult mice CF-1 were preconditioned with NMDA (75 mg/kg) 24 hr before the TBI induction. Under anesthesia with O(2)/N(2)O (33%: 66%) inhalation, the animals were subjected to the experimental model of trauma that occurs by the impact of a 25 g weight on the skull. Sensorimotor gating was evaluated at 1.5, 6, or 24 hr after TBI induction by using footprint and rotarod tests. Cellular damage also was assessed 24 hr after occurrence of cortical trauma. Mice preconditioned with NMDA were protected against all motor deficits revealed by footprint tests, but not those observed in rotarod tasks. Although mice showed motor deficits after TBI, no cellular damage was observed. These data corroborate the hypothesis that glutamatergic excitotoxicity, especially via NMDA receptors, contributes to severity of trauma. They also point to a putative neuroprotective mechanism induced by a sublethal dose of NMDA to improve motor behavioral deficits after TBI.

Atorvastatin Improves Mitochondrial Function and Prevents Oxidative Stress in Hippocampus Following Amyloid-ß1-40 Intracerebroventricular Administration in Mice.

Amyloid-ß (Aß) peptides play a significant role in the pathogenesis of Alzheimer's disease (AD). Neurotoxic effects promoted by Aß peptides involve glutamate transmission impairment, decrease of neurotrophic factors, mitochondrial dysfunction, oxidative stress, synaptotoxicity, and neuronal degeneration. Here, we assessed the early events evoked by Aß1-40 on the hippocampus. Additionally, we sought to unravel the molecular mechanisms of atorvastatin preventive effect on Aß-induced hippocampal damage. Mice were treated orally (p.o.) with atorvastatin 10 mg/kg/day during 7 consecutive days before the intracerebroventricular (i.c.v.) infusion of Aß1-40 (400 pmol/site). Twenty-four hours after Aß1-40 infusion, a reduced content of mature BDNF/proBDNF ratio was observed in Aß-treated mice. However, there is no alteration in synaptophysin, PSD-95, and doublecortin immunocontent in the hippocampus. Aß1-40 promoted an increase in reactive oxygen species (ROS) and nitric oxide (NO) generation in hippocampal slices, and atorvastatin prevented this oxidative burst. Mitochondrial OXPHOS was measured by high-resolution respirometry. At this time point, Aß1-40 did not alter the O2 consumption rates (OCR) related to phosphorylating state associated with complexes I and II, and the maximal OCR. However, atorvastatin increased OCR of phosphorylating state associated with complex I and complexes I and II, maximal OCR of complexes I and II, and OCR associated with mitochondrial spare capacity. Atorvastatin treatment improved mitochondrial function in the rodent hippocampus, even after Aß infusion, pointing to a promising effect of improving brain mitochondria bioenergetics. Therefore, atorvastatin could act as an adjuvant in battling the symptoms of AD to preventing or delaying the disease progression.

Subthreshold doses of guanosine plus ketamine elicit antidepressant-like effect in a mouse model of depression induced by corticosterone: Role of GR/NF-κB/IDO-1 signaling.

Augmentative treatment is considered the best second-option when a first-choice drug has partial limitations, particularly by allowing antidepressant dose reduction. Considering that ketamine has significant knock-on effects, this study investigated the effects of a single coadministration with subthreshold doses of ketamine plus guanosine in a corticosterone (CORT)-induced animal model of depression and the role of anti-inflammatory and antioxidant pathways. CORT administration (20 mg/kg, p.o. for 21 days) increased the immobility time in the tail suspension test (TST) and the grooming latency in the splash test (SPT), as well as reduced the total time of grooming in the SPT. These behavioral alterations were accompanied by impaired hippocampal slices viability, elevated immunocontent of nuclear factor-kappa B (NF-κB) and indoleamine-2,3-dioxygenase 1 (IDO-1), and reduced immunocontent of glucocorticoids receptor (GR), glutamate transporter (GLT-1), nuclear factor-erythroid 2-related factor 2 (Nrf2), and heme oxygenase-1 (HO-1) in the hippocampus. CORT also decreased the thioredoxin reductase activity in the hippocampus, while reduced the glutathione reductase activity and non-protein thiols levels in both hippocampus and prefrontal cortex. In addition, elevated content of malondialdehyde and protein carbonyl was also observed in the hippocampus and prefrontal cortex of CORT-treated mice. Of note, a single administration of ketamine (0.1 mg/kg, i.p.) plus guanosine (0.01 mg/kg, p.o.) attenuated the depressive-like behavior and hippocampal slices impairments induced by CORT. The behavioral response obtained by the combined administration of these drugs was paralleled by the reestablishment of the CORT-induced molecular alterations on hippocampal GR, NF-κB, IDO-1, and GLT-1 immunocontent. Moreover, CORT-induced alterations on the antioxidant enzyme activity and oxidative stress markers were partially restored by ketamine plus guanosine treatment. Taken together, these findings suggest that guanosine might potentiate the effects of ketamine on inflammatory and oxidative markers that are elevated in depression.