Plants of the genus Polygala have been shown to possess protective effects against neuronal death... more Plants of the genus Polygala have been shown to possess protective effects against neuronal death and cognitive impairments in neurodegenerative disorders related to excitotoxicity. Moreover, previous reports from our group have shown the neuroprotective effects of the plant Polygala paniculata against methylmercury (MeHg)-induced neurotoxicity. In this work, we have examined the potential protective effects of three compounds (7-prenyloxy-6-methoxycoumarin, quercetin, and 1,5-dihidroxi-2,3-dimethoxy xanthone) from Polygala species against MeHg-and mercuric chloride (HgCl 2 )-induced disruption of mitochondrial function under in vitro conditions using mitochondrial-enriched fractions from mouse brain. MeHg and HgCl 2 (10-100 µM) significantly decreased mitochondrial viability; this phenomenon was positively correlated to mercurial-induced glutathione oxidation. Among the isolated compounds, only quercetin (100-300 µM) prevented mercurial-induced disruption of mitochondrial viability. Moreover, quercetin, which did not display any chelating effect on MeHg or HgCl 2 , prevented mercurial-induced glutathione oxidation. The present results suggest that the protective effects of quercetin against mercurialinduced mitochondrial dysfunction is related to the removal of oxidant species generated in the presence of either MeHg or HgCl 2 . Reinforcing this hypothesis, MeHg and HgCl 2 increased the production of hydrogen peroxide in the brain mitochondria, as well as the levels of malondialdehyde. These oxidative phenomena were prevented by co-incubation with quercetin or catalase. These results are the first to show the involvement of hydrogen peroxide as a crucial molecule related to the toxic effects of both organic and inorganic mercurials in brain mitochondria. In addition, the study is the first to show the protective effect of quercetin against mercurial-induced toxicity, pointing to its capability to counteract mercurial-dependent hydrogen peroxide generation as a potential molecular mechanism of protection. Taken together, these data render quercetin a promising molecule for pharmacological studies with respects to mercurials' poisoning.
S100B belongs to a family of calcium-binding proteins involved in cell cycle and cytoskeleton reg... more S100B belongs to a family of calcium-binding proteins involved in cell cycle and cytoskeleton regulation. We observed an inhibitory effect of S100B on glial fibrillary acidic protein (GFAP) phosphorylation, when stimulated by cAMP or Ca2+/calmodulin, in a cytoskeletal fraction from primary astrocyte cultures. We found that S100B has no direct effect on CaM KII activity, the major kinase in this cytoskeletal fraction able to phosphorylate GFAP. The inhibition of GFAP phosphorylation is most likely due to the binding of S100B to the phosphorylation sites on this protein and blocking the access of these sites to the protein kinases. This inhibition was dependent on Ca2+. However, Zn2+ could substitute for Ca2+. The inhibitory effect of S100B was prevented by TRTK-12, a peptide that blocks S100B interaction with several target proteins including glial fibrillary acidic protein. These data suggest a role for S100B in the assembly of intermediate filaments in astrocytes.
Physical exercise is a widely accepted behavioral strategy to enhance overall health, including m... more Physical exercise is a widely accepted behavioral strategy to enhance overall health, including mental function. However, there is controversial evidence showing brain mitochondrial dysfunction, oxidative damage and decreased neurotrophin levels after high-intensity exercise, which presumably worsens cognitive performance. Here we investigated learning and memory performance dependent on different brain regions, glutathione antioxidant system, and extracellular signal-regulated protein kinase 1/2 (ERK1/2), serine/threonine protein kinase (AKT), cAMP response element binding (CREB) and dopamine- and cyclic AMP-regulated phosphoprotein (DARPP)-32 signaling in adult Swiss mice submitted to 9 weeks of high-intensity exercise. The exercise did not alter the animals’ performance in the reference and working memory versions of the water maze task. On the other hand, we observed a significant impairment in the procedural memory (an implicit memory that depends on basal ganglia) accompanied by a reduced antioxidant capacity and ERK1/2 and CREB signaling in this region. In addition, we found increased striatal DARPP-32-Thr-75 phosphorylation in trained mice. These findings indicate an increased vulnerability of the striatum to high-intensity exercise associated with the disruption of implicit memory in mice and accompanied by alteration of signaling proteins involved in the plasticity of this brain structure.
L-DOPA alleviates the motor symptoms of Parkinson’s disease, but its long-term use is associated ... more L-DOPA alleviates the motor symptoms of Parkinson’s disease, but its long-term use is associated with undesirable dyskinesia. We now tested whether exercise can attenuate this L-DOPA-induced dyskinesia (LID). We tested the effects of exercise on LID in 6-hydroxydopamine hydrochloride-hemiparkinsonian mice. Animals were treated with L-DOPA/benserazide (25/12.5 mg/kg, i.p.) without and with possibility to exercise (running wheel) during 2 weeks. Exercise drastically prevented the development of LID, and its associated aberrant striatal signaling, namely the hyperphosphorylation of dopamine and cAMP-regulated phosphoprotein 32 kDa protein and c-Fos expression. Our results indicate that exercise can partially prevent the development of LID through the normalization of striatopallidal dopaminergic signaling.
pyrene (BaP) is an environmental contaminant produced during incomplete combustion of organic mat... more pyrene (BaP) is an environmental contaminant produced during incomplete combustion of organic material that is well known as a mutagenic and carcinogenic toxin. There are few studies addressing the molecular and cellular basis of behavioural alterations related to BaP exposure. The aim of this study was to evaluate the effect of subchronic oral administration of BaP on behavioral and neurochemical parameters. Wistar male rats received BaP (2 mg/kg) or corn oil (control), once a day for 28 days (n = 12/group). Spontaneous locomotor activity and shortand long-term memories were evaluated. Glial fibrillary acid protein and S100B content in the hippocampus, serum and CSF were measured using ELISA and total and phosphorylated forms of mitogen activated protein kinases (MAPKs) named extracellular signal-regulated kinases 1 and 2, p38 MAPK and c-Jun amino-terminal kinases 1 and 2, in the hippocampus, were evaluated by western blotting. BaP induced a significant increase on locomotor activity and a decrease in short-term memory. S100B content was increased significantly in cerebrospinal fluid. BaP induced a decrease on ERK2 phosphorylation in the hippocampus. Thus, BaP subchronic treatment induces an astroglial response and impairs both motor and cognitive behavior, with parallel inhibition of ERK2, a signaling enzyme involved in the hippocampal neuroplasticity. All these effects suggest that BaP neurotoxicity is a concern for environmental pollution.
Preclinical and clinical studies indicate that deficiency in folic acid plays a role in the patho... more Preclinical and clinical studies indicate that deficiency in folic acid plays a role in the pathophysiology of depression. Considering that alterations in the signaling pathways that regulate neuroplasticity and cellular survival are implicated in depressive disorders, the present study investigated the involvement of the phosphoinositide 3-kinase (PI3K), glycogen synthase kinase-3 (GSK-3β), and peroxisome proliferator-activated receptor-γ (PPARγ) in the antidepressant-like effect
Lectins recognize and reversibly bind to carbohydrates attached to proteins and lipids modulating... more Lectins recognize and reversibly bind to carbohydrates attached to proteins and lipids modulating a variety of signaling pathways. We previously showed that ConBr, a lectin from Canavalia brasiliensis seeds, produced an antidepressant-like effect in mice by modulating the monoaminergic neurotransmitter systems. Moreover, ConBr blocked hippocampal neurotoxicity induced by quinolinic acid in vivo and by glutamate in vitro, suggesting a neuroprotective activity of ConBr via glutamatergic system modulation. Therefore, the present study was undertaken to investigate the involvement of the N-methyl-D-aspartate (NMDA) receptor and the L-arginine-nitric oxide (NO) pathway in the antidepressant-like action displayed by ConBr in the forced swimming test (FST). With the aim of verifying the involvement of NMDA receptors in the antidepressant-like effect of ConBr (10 μg/site, i.c.v.), an intracerebroventricular (i.c.v.) pretreatment with either NMDA (0.1 pmol/site) or D-serine (30 μg/site) was carried out. The results show that both treatments blocked the effect of ConBr. Furthermore, the coadministration of subeffective doses of the NMDA receptor antagonist MK-801 (0.001 mg/kg, i.p.) or ketamine (0.1 mg/kg, i.p.; NMDA receptor antagonist) and ConBr (0.1 μg/site, i.c.v.) caused a synergistic reduction in immobility time. In order to verify the dependence of the L-arginine-NO-cGMP pathway, on the effect of ConBr in the FST, a pretreatment with the NO precursor, L-arginine (750 mg/kg, i.p.), or the PDE5 inhibitor, sildenafil (5 mg/kg, i.p.), was performed. Both drugs abolished the antidepressant-like action of ConBr. Finally, the administration of subeffective doses of the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 30 pmol/site, i.c.v.) and ConBr (0.1 μg/site, i.c.v.) produced a synergistic antidepressant-like effect in the FST. Taken together, the results suggest that the antidepressant-like effect of ConBr in the FST involves NMDA receptor inhibition and reduction in NO and cGMP synthesis.
the hippocampus (Hip) and cerebral cortex (Ctx) at different time points after pilocarpine-induce... more the hippocampus (Hip) and cerebral cortex (Ctx) at different time points after pilocarpine-induced status epilepticus (Pilo-SE) in male adult Wistar rats. Biochemical analysis was performed in the Hip and Ctx at 1, 3, 12 h (acute period), 5 days (latent period), and 50 days (chronic period) after Pilo-SE. Key findings include an increase in the phosphorylation of GluR1-Ser 845 in the Ctx and GluR1-Ser 831 in the Hip at different times during the acute period, and a decrease in the total content of the GluR1 subunit in the Ctx in the latent period. There was a down-regulation of the mRNA expression and protein levels of EAAT1 and EAAT2, and a decrease of the NR1 mRNA expression, in the Ctx during the latent period. Notably, during the chronic period, the EAAT2 mRNA expression and protein levels decreased while the NR1 mRNA levels increased in the Hip. Taken together, our findings suggest a time-and structuredependent imbalance of glutamatergic transmission in response to Pilo-SE, which might be associated with either epileptogenesis or the seizure threshold in MTLE-HS.
Resveratrol can also inhibit the activation of proinflammatory mediators and cytokines at the ear... more Resveratrol can also inhibit the activation of proinflammatory mediators and cytokines at the early gene expression stage. It is well known that lectins are sugar-binding proteins that act as both pro-and antiinflammatory molecules. Thus, the objective of this work was to verify the binding of a polyphenol compound with a lectin of Canavalia maritima (ConM) based on their ability to inhibit pro-inflammatory processes. To accomplish this, ConM was purified and crystallized, and resveratrol was soaked at 5 mM for 2 h of incubation. The crystal belongs to the monoclinic space group C2, the final refinement resulted in an R factor of 16.0% and an R free of 25.5%. Resveratrol binds in the rigid -sheet through H-bonds and hydrophobic interaction with amino acids that compose the fifth and sixth -strands of the rigid -sheet of ConM. The ConM and resveratrol inhibited DPPH oxidation, showing synergic activity with the most effective ratio of 2:3 and carbohydrate binding site is not directly related to antioxidant activity. It is the interaction between ConM and resveratrol that indicates the synergism of these two molecules in acting as free radicals scavengers and in reducing the inflammatory process through the inhibition of many pro-inflammatory events.
Lectins are proteins capable of reversible binding to carbohydrates or glycoconjugates. In the ce... more 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 lg/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.
OB causes behavioral and neurochemical changes that mimic depressive symptoms. OB induced a signi... more OB causes behavioral and neurochemical changes that mimic depressive symptoms. OB induced a significant increase in ERK1/CREB/BDNF pathway in the hippocampus. Fluoxetine effects were associated with a reduced ERK1/2 and CREB phosphorylation and BDNF levels.
The S100B protein belongs to a family of small Ca 2+ -binding proteins involved in several functi... more The S100B protein belongs to a family of small Ca 2+ -binding proteins involved in several functions including cytoskeletal reorganization. The effect of S100B on protein phosphorylation was investigated in a cytoskeletal fraction prepared from immature rat hippocampus. An inhibitory effect of 5 uM S100B on total protein phosphorylation, ranging from 25% to 40%, was observed in the presence of Ca 2+ alone, Ca 2+ plus calmodulin or Ca 2+ plus cAMP. Analysis by two dimensional electrophoresis revealed a Ca 2+ /calmodulin-dependent and a Ca 2+ /cAMP-dependent inhibitory effect of S100B, ranging from 62% to 67% of control, on the phosphorylation of the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin. The fact that S100B binds to the Nterminal domain of GFAP and that the two proteins are co-localized in astrocytes suggests a potential in vivo role for S100B in modulating the phosphorylation of intermediate filament proteins in glia.
Regional variations in protein phosphorylating activity in the rat brain were studied. Micro-slic... more Regional variations in protein phosphorylating activity in the rat brain were studied. Micro-slices (1 mm diameter) were prepared from 19 brain areas, phosphoproteins labeled by incubation with [32P]phosphate, and the tissue analyzed by nonequilibrium two-dimensional electrophoresis and autoradiography. Attention was focused on three phosphorylating systems that showed consistent variation in activity. (1) A system that phosphorylates a substrate of 47 kDa (ppH-47) whose activity was highest in the hippocampus. The next highest activity of this system was observed in the globus pallidus, followed by the periventricular gray matter of the aqueduct, lateral septum, cerebellar cortex, entorhinal cortex, hypothalamus, mammillary nuclei, amygdala, and substantia nigra. Activity was low or undetectable in the cerebral cortex, neostriatum, and the colliculi. (2) A system that phosphorylates a substrate of 50 kDa (ppC-50) whose activity was highest in the caudate nucleus. The activity of this system was roughly inversely correlated with that of the ppH-47 system. (3) The protein kinase C system that phosphorylates an 82- to 87-kDa substrate known as MARCKS. The highest activity of this system was observed in the cerebellar cortex, followed by the hypothalamus, mammillary nuclei, periventricular gray matter of the aqueduct, and the superior colliculus. Activity of this system was relatively low in several regions of the cerebral cortex, the neostriatum, and the inferior colliculus.
. In previous work we showed that phosphorylation of the astrocyte marker glial fibrillary acidic... more . In previous work we showed that phosphorylation of the astrocyte marker glial fibrillary acidic protein GFAP in hippocampal slices from adult rats is dependent on external Ca 2q , whereas in slices from immature rats aged 12-16 days postnatal 32 P incorporation into GFAP is inhibited by external Ca 2q . The nature of this late developmental change in Ca 2q sensitivity for GFAP phosphorylation was investigated in the present work by comparing in immature and adult animals phosphorylation of GFAP by endogenous protein kinase w 32 x activity in cytoskeletal fractions and tryptic phosphopeptide maps prepared from cytoskeletal fractions labelled with g-P ATP and from w 32 x slices labelled with P phosphate. Cytoskeletal fractions prepared from immature and adult hippocampus both contained endogenous protein kinase activity towards GFAP and other proteins stimulated by Ca 2q rcalmodulin and by cyclic AMP. The maps of GFAP isolated from the cytoskeletal fractions labelled in the presence of Ca 2q rcalmodulin were very similar and exhibited two major and several minor phosphopeptides. Comparison with maps derived from these fractions labelled in the presence of cyclic AMP showed that one of the major phosphopeptides was either directly or indirectly phosphorylated by Ca 2q rcalmodulin-stimulated kinase activity. Maps derived from GFAP isolated from adult slices labelled in the presence of Ca 2q and immature slices labelled in the absence of Ca 2q were qualitatively identical, with minor differences from the cytoskeletal maps. At both ages the slice maps displayed the phosphopeptide phosphorylated through the activity of a Ca 2q rcalmodulin kinase in the cytoskeletal fractions. By its migration properties this peptide appears to correspond to a sequence containing a site shown by other workers to be phosphorylated in vitro by CaM kinase II, suggesting that even in the absence of external Ca 2q , kinase activity directly or indirectly dependent on Ca 2q was occurring in the immature slices. The near identity of the phosphorylation sites at the two ages suggest that the change in Ca 2q sensitivity of GFAP phosphorylation during Ž . development is not due to a change in the balance of kinase and phosphatase activities, but rather to a change in the mechanism s whereby Ca 2q controls the relative activity of these enzymes. q 1997 Elsevier Science B.V.
The S100B protein belongs to a family of small Ca 2+ -binding proteins involved in several functi... more The S100B protein belongs to a family of small Ca 2+ -binding proteins involved in several functions including cytoskeletal reorganization. The effect of S100B on protein phosphorylation was investigated in a cytoskeletal fraction prepared from immature rat hippocampus. An inhibitory effect of 5 uM S100B on total protein phosphorylation, ranging from 25% to 40%, was observed in the presence of Ca 2+ alone, Ca 2+ plus calmodulin or Ca 2+ plus cAMP. Analysis by two dimensional electrophoresis revealed a Ca 2+ /calmodulin-dependent and a Ca 2+ /cAMP-dependent inhibitory effect of S100B, ranging from 62% to 67% of control, on the phosphorylation of the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin. The fact that S100B binds to the Nterminal domain of GFAP and that the two proteins are co-localized in astrocytes suggests a potential in vivo role for S100B in modulating the phosphorylation of intermediate filament proteins in glia.
Many health claims have been made about the medicinal benefits of drinking green tea, including n... more Many health claims have been made about the medicinal benefits of drinking green tea, including neuroprotection. This study mainly focuses on Epigallocatechin 3-gallate (EGCG), a potent antioxidant, which is abundantly found in green tea. Cadmium [Cd 2+ ] is a toxic pollutant that leads to neurotoxicity in both animals and humans. Although the entrance of Cd 2+ in the adult central nervous system is limited, developmental neurotoxicity has been evidenced as result of the blood-brain barrier (BBB) immaturity. Moreover, high Cd 2+ levels are known to impair BBB function. Furthermore, the molecular mechanisms related to its neurotoxic properties remain unknown. This study evaluates the potential protective effect of the major green tea polyphenol, EGCG, against Cd 2+ -induced mitotoxicity under in vitro conditions, using mitochondrial-enriched fractions from rat brain. Co-incubation of EGCG with Cd 2+ prevented the Cd 2+induced mitochondrial dysfunction (capacity to reduce MTT to formazan). In addition, EGCG completely prevented mitochondrial lipid peroxidation induced by Cd 2+ but did not affect non protein thiols levels. Spectroscopic studies have shown EGCG able to form a chemical complex with Cd 2+ , in an equimolar ratio. In this study we demonstrate EGCG effectiveness in protecting against Cd 2+ -induced mitochondrial dysfunction and lipid peroxidation probably due to its antioxidant and chelating effects.
Here we show that a systemic injection of the histone deacetylase inhibitor (HDACi) sodium butyra... more Here we show that a systemic injection of the histone deacetylase inhibitor (HDACi) sodium butyrate (NaB) immediately after training in a step-down inhibitory avoidance task produced an enhancement of memory consolidation that persisted across consecutive retention tests during 14 days in aged rats, while it did not significantly affect memory in young adults. Control aged and young adult rats showed comparable basal levels of memory retention. Our results suggest that HDACis can display memory-enhancing effects specific for aged animals, even in the absence of age-related memory impairment.
The molecular mechanisms mediating manganese (Mn)-induced neurotoxicity, particularly in the imma... more The molecular mechanisms mediating manganese (Mn)-induced neurotoxicity, particularly in the immature central nervous system, have yet to be completely understood. In this study, we investigated whether mitogen-activated protein kinases (MAPKs) and tyrosine hydroxylase (TH) could represent potential targets of Mn in striatal and hippocampal slices obtained from immature rats (14 days old). The aim of this study was to evaluate if the MAPK pathways are modulated after subtoxic Mn exposure, which do not significantly affect cell viability. The concentrations of manganese chloride (MnCl₂; 10-1,000 μM) caused no change in cell viability in slices exposed for 3 or 6 hours. However, Mn exposure significantly increased extracellular signal-regulated kinase (ERK) 1/2, as well as c-Jun N-terminal kinase (JNK) 1/2/3 phosphorylation at both 3 and 6 hours incubations, in both brain structures. Furthermore, Mn exposure did not change the total content or phosphorylation of TH at the serine 40 s...
Plants of the genus Polygala have been shown to possess protective effects against neuronal death... more Plants of the genus Polygala have been shown to possess protective effects against neuronal death and cognitive impairments in neurodegenerative disorders related to excitotoxicity. Moreover, previous reports from our group have shown the neuroprotective effects of the plant Polygala paniculata against methylmercury (MeHg)-induced neurotoxicity. In this work, we have examined the potential protective effects of three compounds (7-prenyloxy-6-methoxycoumarin, quercetin, and 1,5-dihidroxi-2,3-dimethoxy xanthone) from Polygala species against MeHg-and mercuric chloride (HgCl 2 )-induced disruption of mitochondrial function under in vitro conditions using mitochondrial-enriched fractions from mouse brain. MeHg and HgCl 2 (10-100 µM) significantly decreased mitochondrial viability; this phenomenon was positively correlated to mercurial-induced glutathione oxidation. Among the isolated compounds, only quercetin (100-300 µM) prevented mercurial-induced disruption of mitochondrial viability. Moreover, quercetin, which did not display any chelating effect on MeHg or HgCl 2 , prevented mercurial-induced glutathione oxidation. The present results suggest that the protective effects of quercetin against mercurialinduced mitochondrial dysfunction is related to the removal of oxidant species generated in the presence of either MeHg or HgCl 2 . Reinforcing this hypothesis, MeHg and HgCl 2 increased the production of hydrogen peroxide in the brain mitochondria, as well as the levels of malondialdehyde. These oxidative phenomena were prevented by co-incubation with quercetin or catalase. These results are the first to show the involvement of hydrogen peroxide as a crucial molecule related to the toxic effects of both organic and inorganic mercurials in brain mitochondria. In addition, the study is the first to show the protective effect of quercetin against mercurial-induced toxicity, pointing to its capability to counteract mercurial-dependent hydrogen peroxide generation as a potential molecular mechanism of protection. Taken together, these data render quercetin a promising molecule for pharmacological studies with respects to mercurials' poisoning.
S100B belongs to a family of calcium-binding proteins involved in cell cycle and cytoskeleton reg... more S100B belongs to a family of calcium-binding proteins involved in cell cycle and cytoskeleton regulation. We observed an inhibitory effect of S100B on glial fibrillary acidic protein (GFAP) phosphorylation, when stimulated by cAMP or Ca2+/calmodulin, in a cytoskeletal fraction from primary astrocyte cultures. We found that S100B has no direct effect on CaM KII activity, the major kinase in this cytoskeletal fraction able to phosphorylate GFAP. The inhibition of GFAP phosphorylation is most likely due to the binding of S100B to the phosphorylation sites on this protein and blocking the access of these sites to the protein kinases. This inhibition was dependent on Ca2+. However, Zn2+ could substitute for Ca2+. The inhibitory effect of S100B was prevented by TRTK-12, a peptide that blocks S100B interaction with several target proteins including glial fibrillary acidic protein. These data suggest a role for S100B in the assembly of intermediate filaments in astrocytes.
Physical exercise is a widely accepted behavioral strategy to enhance overall health, including m... more Physical exercise is a widely accepted behavioral strategy to enhance overall health, including mental function. However, there is controversial evidence showing brain mitochondrial dysfunction, oxidative damage and decreased neurotrophin levels after high-intensity exercise, which presumably worsens cognitive performance. Here we investigated learning and memory performance dependent on different brain regions, glutathione antioxidant system, and extracellular signal-regulated protein kinase 1/2 (ERK1/2), serine/threonine protein kinase (AKT), cAMP response element binding (CREB) and dopamine- and cyclic AMP-regulated phosphoprotein (DARPP)-32 signaling in adult Swiss mice submitted to 9 weeks of high-intensity exercise. The exercise did not alter the animals’ performance in the reference and working memory versions of the water maze task. On the other hand, we observed a significant impairment in the procedural memory (an implicit memory that depends on basal ganglia) accompanied by a reduced antioxidant capacity and ERK1/2 and CREB signaling in this region. In addition, we found increased striatal DARPP-32-Thr-75 phosphorylation in trained mice. These findings indicate an increased vulnerability of the striatum to high-intensity exercise associated with the disruption of implicit memory in mice and accompanied by alteration of signaling proteins involved in the plasticity of this brain structure.
L-DOPA alleviates the motor symptoms of Parkinson’s disease, but its long-term use is associated ... more L-DOPA alleviates the motor symptoms of Parkinson’s disease, but its long-term use is associated with undesirable dyskinesia. We now tested whether exercise can attenuate this L-DOPA-induced dyskinesia (LID). We tested the effects of exercise on LID in 6-hydroxydopamine hydrochloride-hemiparkinsonian mice. Animals were treated with L-DOPA/benserazide (25/12.5 mg/kg, i.p.) without and with possibility to exercise (running wheel) during 2 weeks. Exercise drastically prevented the development of LID, and its associated aberrant striatal signaling, namely the hyperphosphorylation of dopamine and cAMP-regulated phosphoprotein 32 kDa protein and c-Fos expression. Our results indicate that exercise can partially prevent the development of LID through the normalization of striatopallidal dopaminergic signaling.
pyrene (BaP) is an environmental contaminant produced during incomplete combustion of organic mat... more pyrene (BaP) is an environmental contaminant produced during incomplete combustion of organic material that is well known as a mutagenic and carcinogenic toxin. There are few studies addressing the molecular and cellular basis of behavioural alterations related to BaP exposure. The aim of this study was to evaluate the effect of subchronic oral administration of BaP on behavioral and neurochemical parameters. Wistar male rats received BaP (2 mg/kg) or corn oil (control), once a day for 28 days (n = 12/group). Spontaneous locomotor activity and shortand long-term memories were evaluated. Glial fibrillary acid protein and S100B content in the hippocampus, serum and CSF were measured using ELISA and total and phosphorylated forms of mitogen activated protein kinases (MAPKs) named extracellular signal-regulated kinases 1 and 2, p38 MAPK and c-Jun amino-terminal kinases 1 and 2, in the hippocampus, were evaluated by western blotting. BaP induced a significant increase on locomotor activity and a decrease in short-term memory. S100B content was increased significantly in cerebrospinal fluid. BaP induced a decrease on ERK2 phosphorylation in the hippocampus. Thus, BaP subchronic treatment induces an astroglial response and impairs both motor and cognitive behavior, with parallel inhibition of ERK2, a signaling enzyme involved in the hippocampal neuroplasticity. All these effects suggest that BaP neurotoxicity is a concern for environmental pollution.
Preclinical and clinical studies indicate that deficiency in folic acid plays a role in the patho... more Preclinical and clinical studies indicate that deficiency in folic acid plays a role in the pathophysiology of depression. Considering that alterations in the signaling pathways that regulate neuroplasticity and cellular survival are implicated in depressive disorders, the present study investigated the involvement of the phosphoinositide 3-kinase (PI3K), glycogen synthase kinase-3 (GSK-3β), and peroxisome proliferator-activated receptor-γ (PPARγ) in the antidepressant-like effect
Lectins recognize and reversibly bind to carbohydrates attached to proteins and lipids modulating... more Lectins recognize and reversibly bind to carbohydrates attached to proteins and lipids modulating a variety of signaling pathways. We previously showed that ConBr, a lectin from Canavalia brasiliensis seeds, produced an antidepressant-like effect in mice by modulating the monoaminergic neurotransmitter systems. Moreover, ConBr blocked hippocampal neurotoxicity induced by quinolinic acid in vivo and by glutamate in vitro, suggesting a neuroprotective activity of ConBr via glutamatergic system modulation. Therefore, the present study was undertaken to investigate the involvement of the N-methyl-D-aspartate (NMDA) receptor and the L-arginine-nitric oxide (NO) pathway in the antidepressant-like action displayed by ConBr in the forced swimming test (FST). With the aim of verifying the involvement of NMDA receptors in the antidepressant-like effect of ConBr (10 μg/site, i.c.v.), an intracerebroventricular (i.c.v.) pretreatment with either NMDA (0.1 pmol/site) or D-serine (30 μg/site) was carried out. The results show that both treatments blocked the effect of ConBr. Furthermore, the coadministration of subeffective doses of the NMDA receptor antagonist MK-801 (0.001 mg/kg, i.p.) or ketamine (0.1 mg/kg, i.p.; NMDA receptor antagonist) and ConBr (0.1 μg/site, i.c.v.) caused a synergistic reduction in immobility time. In order to verify the dependence of the L-arginine-NO-cGMP pathway, on the effect of ConBr in the FST, a pretreatment with the NO precursor, L-arginine (750 mg/kg, i.p.), or the PDE5 inhibitor, sildenafil (5 mg/kg, i.p.), was performed. Both drugs abolished the antidepressant-like action of ConBr. Finally, the administration of subeffective doses of the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 30 pmol/site, i.c.v.) and ConBr (0.1 μg/site, i.c.v.) produced a synergistic antidepressant-like effect in the FST. Taken together, the results suggest that the antidepressant-like effect of ConBr in the FST involves NMDA receptor inhibition and reduction in NO and cGMP synthesis.
the hippocampus (Hip) and cerebral cortex (Ctx) at different time points after pilocarpine-induce... more the hippocampus (Hip) and cerebral cortex (Ctx) at different time points after pilocarpine-induced status epilepticus (Pilo-SE) in male adult Wistar rats. Biochemical analysis was performed in the Hip and Ctx at 1, 3, 12 h (acute period), 5 days (latent period), and 50 days (chronic period) after Pilo-SE. Key findings include an increase in the phosphorylation of GluR1-Ser 845 in the Ctx and GluR1-Ser 831 in the Hip at different times during the acute period, and a decrease in the total content of the GluR1 subunit in the Ctx in the latent period. There was a down-regulation of the mRNA expression and protein levels of EAAT1 and EAAT2, and a decrease of the NR1 mRNA expression, in the Ctx during the latent period. Notably, during the chronic period, the EAAT2 mRNA expression and protein levels decreased while the NR1 mRNA levels increased in the Hip. Taken together, our findings suggest a time-and structuredependent imbalance of glutamatergic transmission in response to Pilo-SE, which might be associated with either epileptogenesis or the seizure threshold in MTLE-HS.
Resveratrol can also inhibit the activation of proinflammatory mediators and cytokines at the ear... more Resveratrol can also inhibit the activation of proinflammatory mediators and cytokines at the early gene expression stage. It is well known that lectins are sugar-binding proteins that act as both pro-and antiinflammatory molecules. Thus, the objective of this work was to verify the binding of a polyphenol compound with a lectin of Canavalia maritima (ConM) based on their ability to inhibit pro-inflammatory processes. To accomplish this, ConM was purified and crystallized, and resveratrol was soaked at 5 mM for 2 h of incubation. The crystal belongs to the monoclinic space group C2, the final refinement resulted in an R factor of 16.0% and an R free of 25.5%. Resveratrol binds in the rigid -sheet through H-bonds and hydrophobic interaction with amino acids that compose the fifth and sixth -strands of the rigid -sheet of ConM. The ConM and resveratrol inhibited DPPH oxidation, showing synergic activity with the most effective ratio of 2:3 and carbohydrate binding site is not directly related to antioxidant activity. It is the interaction between ConM and resveratrol that indicates the synergism of these two molecules in acting as free radicals scavengers and in reducing the inflammatory process through the inhibition of many pro-inflammatory events.
Lectins are proteins capable of reversible binding to carbohydrates or glycoconjugates. In the ce... more 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 lg/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.
OB causes behavioral and neurochemical changes that mimic depressive symptoms. OB induced a signi... more OB causes behavioral and neurochemical changes that mimic depressive symptoms. OB induced a significant increase in ERK1/CREB/BDNF pathway in the hippocampus. Fluoxetine effects were associated with a reduced ERK1/2 and CREB phosphorylation and BDNF levels.
The S100B protein belongs to a family of small Ca 2+ -binding proteins involved in several functi... more The S100B protein belongs to a family of small Ca 2+ -binding proteins involved in several functions including cytoskeletal reorganization. The effect of S100B on protein phosphorylation was investigated in a cytoskeletal fraction prepared from immature rat hippocampus. An inhibitory effect of 5 uM S100B on total protein phosphorylation, ranging from 25% to 40%, was observed in the presence of Ca 2+ alone, Ca 2+ plus calmodulin or Ca 2+ plus cAMP. Analysis by two dimensional electrophoresis revealed a Ca 2+ /calmodulin-dependent and a Ca 2+ /cAMP-dependent inhibitory effect of S100B, ranging from 62% to 67% of control, on the phosphorylation of the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin. The fact that S100B binds to the Nterminal domain of GFAP and that the two proteins are co-localized in astrocytes suggests a potential in vivo role for S100B in modulating the phosphorylation of intermediate filament proteins in glia.
Regional variations in protein phosphorylating activity in the rat brain were studied. Micro-slic... more Regional variations in protein phosphorylating activity in the rat brain were studied. Micro-slices (1 mm diameter) were prepared from 19 brain areas, phosphoproteins labeled by incubation with [32P]phosphate, and the tissue analyzed by nonequilibrium two-dimensional electrophoresis and autoradiography. Attention was focused on three phosphorylating systems that showed consistent variation in activity. (1) A system that phosphorylates a substrate of 47 kDa (ppH-47) whose activity was highest in the hippocampus. The next highest activity of this system was observed in the globus pallidus, followed by the periventricular gray matter of the aqueduct, lateral septum, cerebellar cortex, entorhinal cortex, hypothalamus, mammillary nuclei, amygdala, and substantia nigra. Activity was low or undetectable in the cerebral cortex, neostriatum, and the colliculi. (2) A system that phosphorylates a substrate of 50 kDa (ppC-50) whose activity was highest in the caudate nucleus. The activity of this system was roughly inversely correlated with that of the ppH-47 system. (3) The protein kinase C system that phosphorylates an 82- to 87-kDa substrate known as MARCKS. The highest activity of this system was observed in the cerebellar cortex, followed by the hypothalamus, mammillary nuclei, periventricular gray matter of the aqueduct, and the superior colliculus. Activity of this system was relatively low in several regions of the cerebral cortex, the neostriatum, and the inferior colliculus.
. In previous work we showed that phosphorylation of the astrocyte marker glial fibrillary acidic... more . In previous work we showed that phosphorylation of the astrocyte marker glial fibrillary acidic protein GFAP in hippocampal slices from adult rats is dependent on external Ca 2q , whereas in slices from immature rats aged 12-16 days postnatal 32 P incorporation into GFAP is inhibited by external Ca 2q . The nature of this late developmental change in Ca 2q sensitivity for GFAP phosphorylation was investigated in the present work by comparing in immature and adult animals phosphorylation of GFAP by endogenous protein kinase w 32 x activity in cytoskeletal fractions and tryptic phosphopeptide maps prepared from cytoskeletal fractions labelled with g-P ATP and from w 32 x slices labelled with P phosphate. Cytoskeletal fractions prepared from immature and adult hippocampus both contained endogenous protein kinase activity towards GFAP and other proteins stimulated by Ca 2q rcalmodulin and by cyclic AMP. The maps of GFAP isolated from the cytoskeletal fractions labelled in the presence of Ca 2q rcalmodulin were very similar and exhibited two major and several minor phosphopeptides. Comparison with maps derived from these fractions labelled in the presence of cyclic AMP showed that one of the major phosphopeptides was either directly or indirectly phosphorylated by Ca 2q rcalmodulin-stimulated kinase activity. Maps derived from GFAP isolated from adult slices labelled in the presence of Ca 2q and immature slices labelled in the absence of Ca 2q were qualitatively identical, with minor differences from the cytoskeletal maps. At both ages the slice maps displayed the phosphopeptide phosphorylated through the activity of a Ca 2q rcalmodulin kinase in the cytoskeletal fractions. By its migration properties this peptide appears to correspond to a sequence containing a site shown by other workers to be phosphorylated in vitro by CaM kinase II, suggesting that even in the absence of external Ca 2q , kinase activity directly or indirectly dependent on Ca 2q was occurring in the immature slices. The near identity of the phosphorylation sites at the two ages suggest that the change in Ca 2q sensitivity of GFAP phosphorylation during Ž . development is not due to a change in the balance of kinase and phosphatase activities, but rather to a change in the mechanism s whereby Ca 2q controls the relative activity of these enzymes. q 1997 Elsevier Science B.V.
The S100B protein belongs to a family of small Ca 2+ -binding proteins involved in several functi... more The S100B protein belongs to a family of small Ca 2+ -binding proteins involved in several functions including cytoskeletal reorganization. The effect of S100B on protein phosphorylation was investigated in a cytoskeletal fraction prepared from immature rat hippocampus. An inhibitory effect of 5 uM S100B on total protein phosphorylation, ranging from 25% to 40%, was observed in the presence of Ca 2+ alone, Ca 2+ plus calmodulin or Ca 2+ plus cAMP. Analysis by two dimensional electrophoresis revealed a Ca 2+ /calmodulin-dependent and a Ca 2+ /cAMP-dependent inhibitory effect of S100B, ranging from 62% to 67% of control, on the phosphorylation of the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin. The fact that S100B binds to the Nterminal domain of GFAP and that the two proteins are co-localized in astrocytes suggests a potential in vivo role for S100B in modulating the phosphorylation of intermediate filament proteins in glia.
Many health claims have been made about the medicinal benefits of drinking green tea, including n... more Many health claims have been made about the medicinal benefits of drinking green tea, including neuroprotection. This study mainly focuses on Epigallocatechin 3-gallate (EGCG), a potent antioxidant, which is abundantly found in green tea. Cadmium [Cd 2+ ] is a toxic pollutant that leads to neurotoxicity in both animals and humans. Although the entrance of Cd 2+ in the adult central nervous system is limited, developmental neurotoxicity has been evidenced as result of the blood-brain barrier (BBB) immaturity. Moreover, high Cd 2+ levels are known to impair BBB function. Furthermore, the molecular mechanisms related to its neurotoxic properties remain unknown. This study evaluates the potential protective effect of the major green tea polyphenol, EGCG, against Cd 2+ -induced mitotoxicity under in vitro conditions, using mitochondrial-enriched fractions from rat brain. Co-incubation of EGCG with Cd 2+ prevented the Cd 2+induced mitochondrial dysfunction (capacity to reduce MTT to formazan). In addition, EGCG completely prevented mitochondrial lipid peroxidation induced by Cd 2+ but did not affect non protein thiols levels. Spectroscopic studies have shown EGCG able to form a chemical complex with Cd 2+ , in an equimolar ratio. In this study we demonstrate EGCG effectiveness in protecting against Cd 2+ -induced mitochondrial dysfunction and lipid peroxidation probably due to its antioxidant and chelating effects.
Here we show that a systemic injection of the histone deacetylase inhibitor (HDACi) sodium butyra... more Here we show that a systemic injection of the histone deacetylase inhibitor (HDACi) sodium butyrate (NaB) immediately after training in a step-down inhibitory avoidance task produced an enhancement of memory consolidation that persisted across consecutive retention tests during 14 days in aged rats, while it did not significantly affect memory in young adults. Control aged and young adult rats showed comparable basal levels of memory retention. Our results suggest that HDACis can display memory-enhancing effects specific for aged animals, even in the absence of age-related memory impairment.
The molecular mechanisms mediating manganese (Mn)-induced neurotoxicity, particularly in the imma... more The molecular mechanisms mediating manganese (Mn)-induced neurotoxicity, particularly in the immature central nervous system, have yet to be completely understood. In this study, we investigated whether mitogen-activated protein kinases (MAPKs) and tyrosine hydroxylase (TH) could represent potential targets of Mn in striatal and hippocampal slices obtained from immature rats (14 days old). The aim of this study was to evaluate if the MAPK pathways are modulated after subtoxic Mn exposure, which do not significantly affect cell viability. The concentrations of manganese chloride (MnCl₂; 10-1,000 μM) caused no change in cell viability in slices exposed for 3 or 6 hours. However, Mn exposure significantly increased extracellular signal-regulated kinase (ERK) 1/2, as well as c-Jun N-terminal kinase (JNK) 1/2/3 phosphorylation at both 3 and 6 hours incubations, in both brain structures. Furthermore, Mn exposure did not change the total content or phosphorylation of TH at the serine 40 s...
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Papers by Rodrigo B Leal