Glutamatergic system and mTOR-signaling pathway participate in the antidepressant-like effect of inosine in the tail suspension test.

Glutamatergic system and mTOR signaling pathway have been proposed to be important targets for pharmacological treatment of major depressive disorder. Previous studies have shown that inosine, an endogenous purine, is able to exert a remarkable antidepressant-like effect in mice. Nevertheless, the role of glutamatergic system and mTOR in this effect was not previously determined. This study was designed to investigate the possible modulation of NMDA receptors (NMDAR), AMPA receptors (AMPAR) and mTOR complex 1 (mTORC1) signaling pathway in the inosine anti-immobility effect in the tail suspension test (TST) in mice. Pre-treatment of mice with NMDA (0.1 pmol/mouse, NMDAR agonist, i.c.v.) and D-serine (30 µg/mouse, NMDAR co-agonist, i.c.v.) prevented inosine (10 mg/kg, i.p.) anti-immobility effect in the TST. In addition, a synergistic antidepressant-like effect was observed when a sub-effective dose of inosine (0.1 mg/kg, i.p.) was combined with sub-effective doses of NMDAR antagonists MK-801 (0.001 mg/kg, p.o.) or ketamine (0.1 mg/kg, i.p.). Conversely, the antidepressant-like effect elicited by inosine was not altered by pre-treatment with AMPAR antagonist, DNQX (2.5 µg/mouse, i.c.v.). The mTORC1 inhibitor rapamycin (0.2 nmol/mouse, i.c.v.) prevented the inosine anti-immobility effect in the TST. Noteworthy, inosine treatment did not change the immunocontent of the synaptic proteins PSD95, GluA1 and synapsin I. Mice locomotor activity assessed by open-field test, was not altered by treatments. Taken together, this study shows a pivotal role of NMDAR inhibition and mTORC1 activation for inosine antidepressant-like effect and extends the knowledge concerning the molecular mechanism and potential of inosine for antidepressant strategies.

Signaling pathways underlying the antidepressant-like effect of inosine in mice.

Inosine is a purine nucleoside formed by the breakdown of adenosine that elicits an antidepressant-like effect in mice through activation of adenosine A1 and A2A receptors. However, the signaling pathways underlying this effect are largely unknown. To address this issue, the present study investigated the influence of extracellular-regulated protein kinase (ERK)1/2, Ca2+/calmoduline-dependent protein kinase (CaMKII), protein kinase A (PKA), phosphoinositide 3-kinase (PI3K)/Akt, and glycogen synthase kinase 3beta (GSK-3ß) modulation in the antiimmobility effect of inosine in the tail suspension test (TST) in mice. In addition, we attempted to verify if inosine treatment was capable of altering the immunocontent and phosphorylation of the transcription factor cyclic adenosine monophosphatate (cAMP) response-binding element protein (CREB) in mouse prefrontal cortex and hippocampus. Intracerebroventricular administration of U0126 (5 µg/mouse, MEK1/2 inhibitor), KN-62 (1 µg/mouse, CaMKII inhibitor), H-89 (1 µg/mouse, PKA inhibitor), and wortmannin (0.1 µg/mouse, PI3K inhibitor) prevented the antiimmobility effect of inosine (10 mg/kg, intraperitoneal (i.p.)) in the TST. Also, administration of a sub-effective dose of inosine (0.1 mg/kg, i.p.) in combination with a sub-effective dose of AR-A014418 (0.001 µg/mouse, GSK-3ß inhibitor) induced a synergic antidepressant-like effect. None of the treatments altered locomotor activity of mice. Moreover, 24 h after a single administration of inosine (10 mg/kg, i.p.), CREB phosphorylation was increased in the hippocampus. Our findings provided new evidence that the antidepressant-like effect of inosine in the TST involves the activation of PKA, PI3K/Akt, ERK1/2, and CaMKII and the inhibition of GSK-3ß. These results contribute to the comprehension of the mechanisms underlying the purinergic system modulation and indicate the intracellular signaling pathways involved in the antidepressant-like effect of inosine in a preclinical test of depression.

Differential Activation of Mitogen-Activated Protein Kinases, ERK 1/2, p38(MAPK) and JNK p54/p46 During Postnatal Development of Rat Hippocampus.

Mitogen-activated protein kinases (MAPKs) are a group of serine-threonine kinases, including p38(MAPK), ERK 1/2 and JNK p54/p46, activated by phosphorylation in response to extracellular stimuli. The early postnatal period is characterized by significant changes in brain structure as well as intracellular signaling. In the hippocampus MAPKs have been involved in the modulation of development and neural plasticity. However, the temporal profile of MAPK activation throughout the early postnatal development is incomplete. An understanding of this profile is important since slight changes in the activity of these enzymes, in response to environmental stress in specific developmental windows, might alter the course of development. The present study was undertaken to investigate the hippocampal differential activation of MAPK during postnatal period. MAPK activation and total content were evaluated by Western blotting of hippocampal tissue obtained from male Wistar rats at postnatal days (P) 1, 4, 7, 10, 14, 21, 30 and 60. The total content and phosphorylation of each MAPK was expressed as mean ± SEM and then calculates as a percentile compared to P1 (set at 100 %). The results showed: (1) phosphorylation peaks of p38(MAPK) at PN4 (p = 0.036) and PN10 to PN60; (2) phosphorylation of ERK1 and ERK2 were increased with age (ERK1 p = 0.0000005 and ERK2 p = 0.003); (3) phosphorylation profile of JNK p54/p46 was not changed during the period analyzed (JNKp56 p = 0.716 and JNKp46 p = 0.192). Therefore, the activity profile of ERK 1/2 and p38(MAPK) during postnatal development of rat hippocampus are differentially regulated. Our results demonstrate that ERK 1/2 and p38(MAPK) are dynamically regulated during postnatal neurodevelopment, suggesting temporal correlation of MAPK activity with critical periods when programmed cell death and synaptogenesis are occurring. This suggests an important role for these MAPKs in postnatal development of rat hippocampus.

Time course evaluation of behavioral impairments in the pilocarpine model of epilepsy.

Epilepsy is a brain function disorder characterized by unpredictable and recurrent seizures. The majority of patients with temporal lobe epilepsy (TLE), which is the most common type of epilepsy, have to live not only with seizures but also with behavioral alterations, including anxiety, psychosis, depression, and impaired cognitive functioning. The pilocarpine model has been recognized as an animal model of TLE. However, there are few studies addressing behavioral alterations in the maturation phase when evaluating the time course of the epileptogenic process after pilocarpine administration. Therefore, the present work was designed to analyze the neurobehavioral impairments of male adult Wistar rats during maturation and chronic phases in the pilocarpine model of epilepsy. Behavioral tests included: open-field tasks, olfactory discrimination, social recognition, elevated plus maze, and the forced swimming test. The main behavioral alterations observed in both maturation and chronic phases of the pilocarpine model were olfactory and short-term social memory deficits and decrease in the immobility time in the forced swimming test. Moreover, increased anxiety-like responses were only observed in the maturation phase. These findings indicate that early behavioral impairments can be observed in the pilocarpine model during the maturation phase, and these behavioral deficits also occur during the acquired epilepsy (chronic phase). Several of the neurobehavioral impairments that are associated with epilepsy in humans were observed in the pilocarpine-treated rats, thus, rendering this animal model a useful tool to study neuroprotective strategies as well as neurobiological and psychopathological mechanisms associated with epileptogenesis.

Effects of pentylenetetrazole kindling on mitogen-activated protein kinases levels in neocortex and hippocampus of mice.

The epileptogenesis process involves cell signaling events associated with neuroplasticity. The mitogen-activated protein kinases (MAPKs) integrate signals originating from a variety of extracellular stimuli and may regulate cell differentiation, survival, cell death and synaptic plasticity. Here we compared the total and phosphorylated MAPKs (ERK1/2, JNK1/2 and p38(MAPK)) levels in the neocortex and hippocampus of adult Swiss male mice quantified by western blotting analysis 48 h after the last injection of pentylenetetrazole (PTZ), according to the kindling protocol (35 mg/kg, i.p., on alternated days, with a total of eight injections). The total levels of the investigated MAPKs and the phospho-p38(MAPK) in the neocortex and hippocampus were not affected by the PTZ injections. The MAPKs phosphorylation levels remain unaltered in PTZ-treated animals without convulsive seizures. The phospho-JNK2 phosphorylation, but not the phospho-JNK1, was increased in the hippocampus of PTZ-treated animals showing 1-3 days with convulsive seizures, whereas no significant changes were observed in those animals with more than 3 days with convulsive seizures. The phospho-ERK1/2 phosphorylation decreased in the neocortex and increased in the hippocampus of animals with 1-4 days with convulsive seizures and became unaltered in mice that showed convulsive seizures for more than 4 days. These findings indicate that resistance to PTZ kindling is associated with unaltered ERK1/2, JNK1/2 and p38(MAPK) phosphorylation levels in the neocortex and hippocampus. Moreover, when the PTZ kindling-induced epileptogenesis manifests behaviorally, the activation of the different MAPKs sub-families shows a variable and non-linear pattern in the neocortex and hippocampus.

Vatairea macrocarpa lectin (VML) induces depressive-like behavior and expression of neuroinflammatory markers in mice.

Lectins are proteins capable of reversible binding to the carbohydrates in glycoconjugates that can regulate many physiological and pathological events. Galectin-1, a ß-galactoside-binding lectin, is expressed in the central nervous system (CNS) and exhibits neuroprotective functions. Additionally, lectins isolated from plants have demonstrated beneficial action in the CNS. One example is a lectin with mannose-glucose affinity purified from Canavalia brasiliensis seeds, ConBr, which displays neuroprotective and antidepressant activity. On the other hand, the effects of the galactose-binding lectin isolated from Vatairea macrocarpa seeds (VML) on the CNS are largely unknown. The aim of this study was to verify if VML is able to alter neural function by evaluating signaling enzymes, glial and inflammatory proteins in adult mice hippocampus, as well as behavioral parameters. VML administered by intracerebroventricular (i.c.v) route increased the immobility time in the forced swimming test (FST) 60 min after its injection through a carbohydrate recognition domain-dependent mechanism. Furthermore, under the same conditions, VML caused an enhancement of COX-2, GFAP and S100B levels in mouse hippocampus. However, phosphorylation of Akt, GSK-3ß and mitogen-activated protein kinases named ERK1/2, JNK1/2/3 and p38(MAPK), was not changed by VML. The results reported here suggest that VML may trigger neuroinflammatory response in mouse hippocampus and exhibit a depressive-like activity. Taken together, our findings indicate a dual role for galactose binding lectins in the modulation of CNS function.

Redox toxicology of environmental chemicals causing oxidative stress.

Living organisms are surrounded with heavy metals such as methylmercury, manganese, cobalt, cadmium, arsenic, as well as pesticides such as deltamethrin and paraquat, or atmospheric pollutants such as quinone. Extensive studies have demonstrated a strong link between environmental pollutants and human health. Redox toxicity is proposed as one of the main mechanisms of chemical-induced pathology in humans. Acting as both a sensor of oxidative stress and a positive regulator of antioxidants, the nuclear factor erythroid 2-related factor 2 (NRF2) has attracted recent attention. However, the role NRF2 plays in environmental pollutant-induced toxicity has not been systematically addressed. Here, we characterize NRF2 function in response to various pollutants, such as metals, pesticides and atmospheric quinones. NRF2 related signaling pathways and epigenetic regulations are also reviewed.

Neurotoxicity of e-cigarettes.

It appears that electronic cigarettes (EC) are a less harmful alternative to conventional cigarette (CC) smoking, as they generate substantially lower levels of harmful carcinogens and other toxic compounds. Thus, switching from CC to EC may be beneficial for smokers. However, recent accounts of EC- or vaping-associated lung injury (EVALI) has raised concerns regarding their adverse health effects. Additionally, the increasing popularity of EC among vulnerable populations, such as adolescents and pregnant women, calls for further EC safety evaluation. In this state-of-the-art review, we provide an update on recent findings regarding the neurological effects induced by EC exposure. Moreover, we discuss possible neurotoxic effects of nicotine and numerous other chemicals which are inherent both to e-liquids and EC aerosols. We conclude that in recognizing pertinent issues associated with EC usage, both government and scientific researchers must address this public health issue with utmost urgency.

Post-translational modifications in MeHg-induced neurotoxicity.

Mercury (Hg) exposure remains a major public health concern due to its widespread distribution in the environment. Organic mercurials, such as MeHg, have been extensively investigated especially because of their congenital effects. In this context, studies on the molecular mechanism of MeHg-induced neurotoxicity are pivotal to the understanding of its toxic effects and the development of preventive measures. Post-translational modifications (PTMs) of proteins, such as phosphorylation, ubiquitination, and acetylation are essential for the proper function of proteins and play important roles in the regulation of cellular homeostasis. The rapid and transient nature of many PTMs allows efficient signal transduction in response to stress. This review summarizes the current knowledge of PTMs in MeHg-induced neurotoxicity, including the most commonly PTMs, as well as PTMs induced by oxidative stress and PTMs of antioxidant proteins. Though PTMs represent an important molecular mechanism for maintaining cellular homeostasis and are involved in the neurotoxic effects of MeHg, we are far from understanding the complete picture on their role, and further research is warranted to increase our knowledge of PTMs in MeHg-induced neurotoxicity.

Molecular Pathways Associated With Methylmercury-Induced Nrf2 Modulation.

Methylmercury (MeHg) is a potent neurotoxin that affects particularly the developing brain. Since MeHg is a potent electrophilic agent, a wide range of intracellular effects occur in response to its exposure. Yet, the molecular mechanisms associated with MeHg-induced cell toxicity have yet to be fully understood. Activation of cell defense mechanisms in response to metal exposure, including the up-regulation of Nrf2- (nuclear factor erythroid 2-related factor 2)-related genes has been previously shown. Nrf2 is a key regulator of cellular defenses against oxidative, electrophilic and environmental stress, regulating the expression of antioxidant proteins, phase-II xenobiotic detoxifying enzymes as well phase-III xenobiotic transporters. Analogous to other electrophiles, MeHg activates Nrf2 through modification of its repressor Keap1 (Kelch-like ECH-associated protein 1). However, recent findings have also revealed that Keap1-independent signal pathways might contribute to MeHg-induced Nrf2 activation and cytoprotective responses against MeHg exposure. These include, Akt phosphorylation (Akt/GSK-3ß/Fyn-mediated Nrf2 activation pathway), activation of the PTEN/Akt/CREB pathway and MAPK-induced autophagy and p62 expression. In this review, we summarize the state-of-the-art knowledge regarding Nrf2 up-regulation in response to MeHg exposure, highlighting the modulation of signaling pathways related to Nrf2 activation. The study of these mechanisms is important in evaluating MeHg toxicity in humans, and can contribute to the identification of the molecular mechanisms associated with MeHg exposure.

Agmatine produces antidepressant-like effects by activating AMPA receptors and mTOR signaling.

The activation of AMPA receptors and mTOR signaling has been reported as mechanisms underlying the antidepressant effects of fast-acting agents, specially the NMDA receptor antagonist ketamine. In the present study, oral administration of agmatine (0.1mg/kg), a neuromodulator that has been reported to modulate NMDA receptors, caused a significant reduction in the immobility time of mice submitted to the tail suspension test (TST), an effect prevented by the administration of DNQX (AMPA receptor antagonist, 2.5µg/site, i.c.v.), BDNF antibody (1µg/site, i.c.v.), K-252a (TrkB receptor antagonist, 1µg/site, i.c.v.), LY294002 (PI3K inhibitor, 10nmol/site, i.c.v.) or rapamycin (selective mTOR inhibitor, 0.2nmol/site, i.c.v.). Moreover, the administration of lithium chloride (non-selective GSK-3ß inhibitor, 10mg/kg, p.o.) or AR-A014418 (selective GSK-3ß inhibitor, 0.01µg/site, i.c.v.) in combination with a sub-effective dose of agmatine (0.0001mg/kg, p.o.) reduced the immobility time in the TST when compared with either drug alone. Furthermore, increased immunocontents of BDNF, PSD-95 and GluA1 were found in the prefrontal cortex of mice just 1h after agmatine administration. These results indicate that the antidepressant-like effect of agmatine in the TST may be dependent on the activation of AMPA and TrkB receptors, PI3K and mTOR signaling as well as inhibition of GSK-3ß, and increase in synaptic proteins. The results contribute to elucidate the complex signaling pathways involved in the antidepressant effect of agmatine and reinforce the pivotal role of these molecular targets for antidepressant responses.

Region-specific alterations of AMPA receptor phosphorylation and signaling pathways in the pilocarpine model of epilepsy.

Disturbances in glutamatergic transmission and signaling pathways have been associated with temporal lobe epilepsy (TLE) in humans. However, the profile of these alterations within specific regions of the hippocampus and cerebral cortex has not yet been examined. The pilocarpine model in rodents reproduces the main features of TLE in humans. The present study aims to characterize specific alterations of the glutamatergic transmission and signaling pathways in the dorsal (DH) and ventral hippocampus (VH) and temporal cortex (Ctx) of male adult Wistar rats 60 days after pilocarpine treatment (chronic period). The western blotting analyzes show a decrease of AMPA glutamate receptor subunit (GluA1)-Ser(845) phosphorylation; reduction of ERK1 and PKA activity; up-regulation of GFAP and down-regulation of the glutamate transporter EAAT2 expression in the DH. In contrast, in the VH it was observed a decrease of GluA1-Ser(831) phosphorylation and JNKp54 and PKC activity. In the Ctx, only ERK1 phosphorylation/activity decreased. The level of GluA1-Ser(845) phosphorylation and PKA activity (DH) and the level of GluA1-Ser(831) phosphorylation and PKC activity (VH) appear to be correlated, respectively. These findings suggest a differential imbalance of the signaling pathways involved in the site-specific phosphorylation of AMPA receptor in the hippocampus. Furthermore, we suggest that dorsal hippocampus is probably more susceptible to the impairment of glutamate uptake and gliose, since only this area displayed a significant decrease of EAAT2 and increment of GFAP. Taken together, our study suggests that specific neurochemical alterations take place in hippocampal sub regions. This approach may be valuable for understanding the onset of seizures and the alterations of neuronal excitability in specific regions and may help to establish therapeutic targets for treatment of this neuropathology.

In vitro manganese exposure disrupts MAPK signaling pathways in striatal and hippocampal slices from immature rats.

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 site in striatal slices. Thus, Mn at concentrations that do not disrupt cell viability causes activation of MAPKs (ERK1/2 and JNK1/2/3) in immature hippocampal and striatal slices. These findings suggest that altered intracellular MAPKs signaling pathways may represent an early event concerning the effects of Mn in the immature brain.