Effects of intranasal guanosine administration on brain function in a rat model of ischemic stroke.

Ischemic stroke is a major cause of morbidity and mortality worldwide and only few affected patients are able to receive treatment, especially in developing countries. Detailed pathophysiology of brain ischemia has been extensively studied in order to discover new treatments with a broad therapeutic window and that are accessible to patients worldwide. The nucleoside guanosine (Guo) has been shown to have neuroprotective effects in animal models of brain diseases, including ischemic stroke. In a rat model of focal permanent ischemia, systemic administration of Guo was effective only when administered immediately after stroke induction. In contrast, intranasal administration of Guo (In-Guo) was effective even when the first administration was 3 h after stroke induction. In order to validate the neuroprotective effect in this larger time window and to investigate In-Guo neuroprotection under global brain dysfunction induced by ischemia, we used the model of thermocoagulation of pial vessels in Wistar rats. In our study, we have found that In-Guo administered 3 h after stroke was capable of preventing ischemia-induced dysfunction, such as bilateral suppression and synchronicity of brain oscillations and ipsilateral cell death signaling, and increased permeability of the blood-brain barrier. In addition, In-Guo had a long-lasting effect on preventing ischemia-induced motor impairment. Our data reinforce In-Guo administration as a potential new treatment for brain ischemia with a more suitable therapeutic window.

Calcium signaling mechanisms disrupt the cytoskeleton of primary astrocytes and neurons exposed to diphenylditelluride.

BACKGROUND: Diphenylditelluride (PhTe)2 is a potent neurotoxin disrupting the homeostasis of the cytoskeleton. METHODS: Cultured astrocytes and neurons were incubated with (PhTe)2, receptor antagonists and enzyme inhibitors followed by measurement of the incorporation of [32P]orthophosphate into intermediate filaments (IFs). RESULTS: (PhTe)2 caused hyperphosphorylation of glial fibrillary acidic protein (GFAP), vimentin and neurofilament subunits (NFL, NFM and NFH) from primary astrocytes and neurons, respectively. These mechanisms were mediated by N-methyl-d-aspartate (NMDA) receptors, L-type voltage-dependent calcium channels (L-VDCCs) as well as metabotropic glutamate receptors upstream of phospholipase C (PLC). Upregulated Ca(2+) influx activated protein kinase A (PKA) and protein kinase C (PKC) in astrocytes causing hyperphosphorylation of GFAP and vimentin. Hyperphosphorylated (IF) together with RhoA-activated stress fiber formation, disrupted the cytoskeleton leading to altered cell morphology. In neurons, the high intracellular Ca(2+) levels activated the MAPKs, Erk and p38MAPK, beyond PKA and PKC, provoking hyperphosphorylation of NFM, NFH and NFL. CONCLUSIONS: Our findings support that intracellular Ca(2+) is one of the crucial signals that modulate the action of (PhTe)2 in isolated cortical astrocytes and neurons modulating the response of the cytoskeleton against the insult. GENERAL SIGNIFICANCE: Cytoskeletal misregulation is associated with neurodegeneration. This compound could be a valuable tool to induce molecular changes similar to those found in different pathologies of the brain.

Impairment of GABAergic system contributes to epileptogenesis in glutaric acidemia type I.

OBJECTIVES: Glutaric acidemia type I (GA-I) is an inherited neurometabolic disorder caused by deficiency of glutaryl-CoA dehydrogenase (GCDH) and characterized by increased levels of glutaric, 3-OH-glutaric, and glutaconic acids in the brain parenchyma. The increment of these organic acids inhibits glutamate decarboxylase (GAD) and consequently lowers the γ-aminobutyric acid (GABA) synthesis. Untreated patients exhibit severe neurologic deficits during development, including epilepsy, especially following an acute encephalopathy outbreak. In this work, we evaluated the role of the GABAergic system on epileptogenesis in GA-I using the Gcdh-/- mice exposed to a high lysine diet (Gcdh-/- -Lys). METHODS: Spontaneous recurrent seizures (SRS), seizure susceptibility, and changes in brain oscillations were evaluated by video-electroencephalography (EEG). Cortical GABAergic synaptic transmission was evaluated using electrophysiologic and neurochemical approaches. RESULTS: SRS were observed in 72% of Gcdh-/- -Lys mice, whereas no seizures were detected in age-matched controls (Gcdh+/+ or Gcdh-/- receiving normal diet). The severity and number of PTZ-induced seizures were higher in Gcdh-/- -Lys mice. EEG spectral analysis showed a significant decrease in theta and gamma oscillations and predominant delta waves in Gcdh-/- -Lys mice, associated with increased EEG left index. Analysis of cortical synaptosomes revealed a significantly increased percentage of glutamate release and decreased GABA release in Gcdh-/- -Lys mice that were associated with a decrease in cortical GAD immunocontent and activity and confirmed by reduced frequency of inhibitory events in cortical pyramidal cells. SIGNIFICANCE: Using an experimental model with a phenotype similar to that of GA-I in humans-the Gcdh-/- mice under high lysine diet (Gcdh-/- -Lys)-we provide evidence that a reduction in cortical inhibition of Gcdh-/- -Lys mice, probably induced by GAD dysfunction, leads to hyperexcitability and increased slow oscillations associated with neurologic abnormalities in GA-I. Our findings offer a new perspective on the pathophysiology of brain damage in GA-I.

Intranasal guanosine administration presents a wide therapeutic time window to reduce brain damage induced by permanent ischemia in rats.

In addition to its intracellular roles, the nucleoside guanosine (GUO) also has extracellular effects that identify it as a putative neuromodulator signaling molecule in the central nervous system. Indeed, GUO can modulate glutamatergic neurotransmission, and it can promote neuroprotective effects in animal models involving glutamate neurotoxicity, which is the case in brain ischemia. In the present study, we aimed to investigate a new in vivo GUO administration route (intranasal, IN) to determine putative improvement of GUO neuroprotective effects against an experimental model of permanent focal cerebral ischemia. Initially, we demonstrated that IN [(3)H] GUO administration reached the brain in a dose-dependent and saturable pattern in as few as 5 min, presenting a higher cerebrospinal GUO level compared with systemic administration. IN GUO treatment started immediately or even 3 h after ischemia onset prevented behavior impairment. The behavior recovery was not correlated to decreased brain infarct volume, but it was correlated to reduced mitochondrial dysfunction in the penumbra area. Therefore, we showed that the IN route is an efficient way to promptly deliver GUO to the CNS and that IN GUO treatment prevented behavioral and brain impairment caused by ischemia in a therapeutically wide time window.

Experimental lung injury promotes alterations in energy metabolism and respiratory mechanics in the lungs of rats: prevention by exercise.

In the present study we investigated the effects of lung injury on energy metabolism (succinate dehydrogenase, complex II, cytochrome c oxidase, and ATP levels), respiratory mechanics (dynamic and static compliance, elastance and respiratory system resistance) in the lungs of rats, as well as on phospholipids in bronchoalveolar lavage fluid. The protective effect of physical exercise on the alterations caused by lung injury, including lung edema was also evaluated. Wistar rats were submitted to 2 months of physical exercise. After this period the lung injury was induced by intratracheal instillation of lipopolysaccharide. Adult Wistar rats were submitted to 2 months of physical exercise and after this period the lung injury was induced by intratracheal instillation of lipopolysaccharide in dose 100 µg/100 g body weight. The sham group received isotonic saline instillation. Twelve hours after the injury was performed the respiratory mechanical and after the rats were decapitated and samples were collected. The rats subjected to lung injury presented a decrease in activities of the enzymes of the electron transport chain and ATP levels in lung, as well as the formation of pulmonary edema. A decreased lung dynamic and static compliance, as well as an increase in respiratory system resistance, and a decrease in phospholipids content were observed. Physical exercise was able to totally prevent the decrease in succinate dehydrogenase and complex II activities and the formation of pulmonary edema. It also partially prevented the increase in respiratory system resistance, but did not prevent the decrease in dynamic and static compliance, as well as in phospholipids content. These findings suggest that the mitochondrial dysfunction may be one of the important contributors to lung damage and that physical exercise may be beneficial in this pathology, although it did not prevent all changes present in lung injury.

Cytoskeleton of cortical astrocytes as a target to proline through oxidative stress mechanisms.

Hyperprolinemia is an inherited disorder of proline (Pro) metabolism and patients affected by this disease may present neurological manifestations. However, the mechanisms of neural excitotoxicity elicited by hyperprolinemia are far from being understood. Considering the pivotal role of cytoskeletal remodeling in several neurodegenerative pathologies and the potential links between cytoskeleton, reactive oxygen species production and cell death, the aim of the present work was to study the effects of Pro on astrocyte and neuron cytoskeletal remodeling and the possible oxidative stress involvement. Pro induced a shift of actin cytoskeleton in stress fibers together with increased RhoA immunocontent and ERK1/2 phosphorylation/activation in cortical astrocytes. Unlike astrocytes, results evidenced little susceptibility of neuron cytoskeleton remodeling, since Pro-treated neurons presented unaltered neuritogenesis. We observed increased hydrogen peroxide production characterizing oxidative stress together with decreased superoxide dismutase (SOD) and catalase (CAT) activities in cortical astrocytes after Pro treatment, while glutathione peroxidase (GSHPx) activity remained unaltered. However, coincubation with Pro and Trolox/melatonin prevented decreased SOD and CAT activities in Pro-treated astrocytes. Accordingly, these antioxidants were able to prevent the remodeling of the actin cytoskeleton, RhoA increased levels and ERK1/2 phosphorylation in response to high Pro exposure. Taken together, these findings indicated that the cytoskeleton of cortical astrocytes, but not of neurons in culture, is a target to Pro and such effects could be mediated, at least in part, by redox imbalance, RhoA and ERK1/2 signaling pathways. The vulnerability of astrocyte cytoskeleton may have important implications for understanding the effects of Pro in the neurotoxicity linked to inborn errors of Pro metabolism.

Mild hyperhomocysteinemia reduces the activity and immunocontent, but does not alter the gene expression, of catalytic α subunits of cerebral Na+,K+-ATPase.

Na(+),K(+)-ATPase is a membrane protein which plays a key role in the maintenance of ion homeostasis that is necessary to neuronal excitability, secondary transport and neurotransmitter uptake. Mild hyperhomocysteinemia leads to several clinical manifestations and particularly cerebral diseases; however, little is known about the mechanisms of homocysteine on cerebral Na(+),K(+)-ATPase. In the present study, we investigated the effect of mild hyperhomocysteinemia on the activity, the immunocontent of catalytic subunits (α1, α2, and α3) and the gene expression of this enzyme. We used the experimental model of mild hyperhomocysteinemia that was induced by homocysteine administration (0.03 µmol/g of body weight) twice a day, from the 30th to the 60th postpartum day. Controls received saline in the same volumes. Results showed that mild hyperhomocysteinemia significantly decreased the activity and the immunocontent of the α 1 and α 2 subunits of the Na(+),K(+)-ATPase in cerebral cortex and hippocampus of adult rats. On the other hand, we did not observe any change in levels of Na(+),K(+)-ATPase mRNA transcripts in such cerebral structures of rats after chronic exposure to homocysteine. The present findings support that the homocysteine modulates the Na(+),K(+)-ATPase and this could be associated, at least in part, with the risk to the development of cerebral diseases in individuals with mild hyperhomocysteinemia.

Methylglyoxal-induced cytotoxicity in neonatal rat brain: a role for oxidative stress and MAP kinases.

Carbonyl compounds such as methylglyoxal (MGO) seem to play an important role in complications resulting from diabetes mellitus, in aging and neurodegenerative disorders. In this study, we are showing, that MGO is able to suppress cell viability and induce apoptosis in the cerebral cortex and hippocampus of neonatal rats ex-vivo. These effects are partially related with ROS production, evaluated by DCFH-DA assay. Coincubation of MGO and reduced glutathione (GSH) or Trolox (vitamin E) totally prevented ROS production but only partially prevented the MGO-induced decreased cell viability in the two brain structures, as evaluated by the MTT assay. Otherwise, L-NAME, a nitric oxide (NO) inhibitor, partially prevented ROS production in the two structures but partially prevented cytotoxicity in the hippocampus. Pharmacological inhibition of Erk, has totally attenuated MGO-induced ROS production and cytotoxicity, suggesting that MEK/Erk pathway could be upstream of ROS generation and cell survival. Otherwise, p38MAPK and JNK failed to prevent ROS generation but induced decreased cell survival consistent with ROS-independent mechanisms. We can propose that Erk, p38MAPK and JNK are involved in the cytotoxicity induced by MGO through different signaling pathways. While Erk could be an upstream effector of ROS generation, p38MAPK and JNK seem to be associated with ROS-independent cytotoxicity in neonatal rat brain. The cytotoxic damage progressed to apoptotic cell death at MGO concentration higher than those described for adult brain, suggesting that the neonatal brain is resistant to MGO-induced cell death. The consequences of MGO-induced brain damage early in life, remains to be clarified. However, it is feasible that high MGO levels during cortical and hippocampal development could be, at least in part, responsible for the impairment of cognitive functions in adulthood.

In vivo treatment with diphenyl ditelluride induces neurodegeneration in striatum of young rats: implications of MAPK and Akt pathways.

In the present report 15day-old Wistar rats were injected with 0.3µmol of diphenyl ditelluride (PhTe)(2)/kg body weight and parameters of neurodegeneration were analyzed in slices from striatum 6days afterwards. We found hyperphosphorylation of intermediate filament (IF) proteins from astrocyte (glial fibrillary acidic protein-GFAP and vimentin) and from neuron (low-, medium- and high molecular weight neurofilament subunits: NF-L, NF-M and NF-H, respectively) and increased MAPK (Erk, JNK and p38MAPK) as well as PKA activities. The treatment induced reactive astrogliosis in the striatum, evidenced by increased GFAP and vimentin immunocontent as well as their mRNA overexpression. Also, (PhTe)(2) significantly increased the propidium iodide (PI) positive cells in NeuN positive population without altering PI incorporation into GFAP positive cells, indicating that in vivo exposure to (PhTe)(2) provoked neuronal damage. Immunohistochemistry showed a dramatic increase of GFAP staining characteristic of reactive astrogliosis. Moreover, increased caspase 3 in (PhTe)(2) treated striatal slices suggested apoptotic cell death. (PhTe)(2) exposure decreased Akt immunoreactivity, however phospho-GSK-3-ß (Ser9) was unaltered, suggesting that this kinase is not directly implicated in the neurotoxicity of this compound. Therefore, the present results shed light into the mechanisms of (PhTe)(2)-induced neurodegeneration in rat striatum, evidencing a critical role for the MAPK and Akt signaling pathways and disruption of cytoskeletal homeostasis, which could be related with apoptotic neuronal death and astrogliosis.

Chronic hyperhomocysteinemia increases inflammatory markers in hippocampus and serum of rats.

This study investigated the effects of chronic homocysteine administration on some parameters of inflammation, such as cytokines (TNF-α, IL-1ß and IL-6), chemokine CCL(2) (MCP-1), nitrite and prostaglandin E(2) levels, as well as on immunocontent of NF-κB/p65 subunit in hippocampus and/or serum of rats. Since acetylcholinesterase has been associated with inflammation, we also evaluated the effect of homocysteine on this enzyme activity in hippocampus of rats. Wistar rats received daily subcutaneous injections of homocysteine (0.3-0.6 µmol/g body weight) or saline (control) from the 6th to the 28th days-of-age. One or 12 h after the last injection, rats were euthanized and hippocampus and serum were used. Results showed that chronic hyperhomocysteinemia significantly increased pro-inflammatory cytokines (TNF-α, IL-1ß and IL-6), chemokine CCL(2) (MCP-1) and prostaglandin E(2) in hippocampus and serum of rats at 1 and 12 h after the last injection of homocysteine. Nitrite levels increased in hippocampus, but decreased in serum at 1 h after chronic hyperhomocysteinemia. Acetylcholinesterase activity and immunocontent of citoplasmic and nuclear NF-κB/p65 subunit were increased in hippocampus of rats subjected to hyperhomocysteinemia at 1 h, but did not alter at 12 h after the last injection of homocysteine. According to our results, chronic hyperhomocysteinemia increases inflammatory parameters, suggesting that this process might be associated, at least in part, with the cerebrovascular and vascular dysfunctions characteristic of some homocystinuric patients.

Alterations of PI3K and Akt signaling pathways in the hippocampus and hypothalamus of Wistar rats treated with highly palatable food.

BACKGROUND/OBJECTIVES: Highly palatable food (HPF), which is enriched in simple sugars and saturated fat, contributes to obesity and insulin resistance in humans. These metabolic changes are associated with serious complications of the central nervous system, including an elevated risk of cognitive dysfunction. We, herein, treated rats with HPF and then examined the insulin-signaling pathway, in particular, the levels of phosphatidylinositol-3 kinase (PI3K), Akt, and insulin receptor substrate-1 (IRS-1) in the hippocampus and hypothalamus. METHODS: Adult Wistar rats fed with HPF (heated or not during preparation) for 4 months and then measured the levels of PI3K, Akt, and IRS-1 in the hippocampus and hypothalamus, by western blotting and quantitative real-time polymerase chain reaction. RESULTS: We observed changes in body weight, glucose intolerance, and lipidemia, confirming that peripheral metabolic alterations were induced using this model. Hippocampal PI3K and hypothalamic Akt were affected in rats that are submitted to chronic exposure to an HPF diet. Moreover, heated HPF caused differentiated alterations in the regulatory subunit of PI3K in the hippocampus. DISCUSSION: Our data suggest that this diet alters insulin signaling differentially in each brain region, and that hippocampal changes induced by this diet could contribute to the understanding of cognitive impairments that are dependent on the hippocampus.

Diphenyl ditelluride induces hypophosphorylation of intermediate filaments through modulation of DARPP-32-dependent pathways in cerebral cortex of young rats.

We studied the effect of different concentrations of diphenyl ditelluride (PhTe)(2) on the in vitro phosphorylation of glial fibrillary acidic protein (GFAP) and neurofilament (NF) subunits from cerebral cortex and hippocampus of rats during development. (PhTe)(2)-induced hypophosphorylation of GFAP and NF subunits only in cerebral cortex of 9- and 15-day-old animals but not in hippocampus. Hypophosphorylation was dependent on ionotropic glutamate receptors, as demonstrated by the specific inhibitors 10 µM DL-AP5 and 50 µM MK801, 100 µM CNQX and 100 µM DNQX. Also, 10 µM verapamil and 10 µM nifedipine, two L-voltage-dependent Ca(2+) channels (L-VDCC) blockers; 50 µM dantrolene, a ryanodine channel blocker, and the intracellular Ca(2+) chelator Bapta-AM (50 µM) totally prevented this effect. Results obtained with 0.2 µM calyculin A (PP1 and PP2A inhibitor), 1 µM Fostriecin a potent protein phosphatase 2A (PP2A) inhibitor, 100 µM FK-506 or 100 µM cyclosporine A, specific protein phosphatase 2B inhibitors, pointed to PP1 as the protein phosphatase directly involved in the hypophosphorylating effect of (PhTe)(2). Finally, we examined the activity of DARPP-32, an important endogenous Ca(2+)-mediated inhibitor of PP1 activity. Western blot assay using anti-DARPP-32, anti-pThr34DARPP-32, and anti-pThr75DARPP-32 antibodies showed a decreased phosphorylation level of the inhibitor at Thr34, compatible with inactivation of protein kinase A (PKA) by pThr75 DARPP-32. Decreased cAMP and catalytic subunit of PKA support that (PhTe)(2) acted on neuron and astrocyte cytoskeletal proteins through PKA-mediated inactivation of DARPP-32, promoting PP1 release and hypophosphorylation of IF proteins of those neural cells. Moreover, in the presence of Bapta, the level of the PKA catalytic subunit was not decreased by (PhTe)(2), suggesting that intracellular Ca(2+) levels could be upstream the signaling pathway elicited by this neurotoxicant and targeting the cytoskeleton.

Cross-talk among intracellular signaling pathways mediates the diphenyl ditelluride actions on the hippocampal cytoskeleton of young rats.

In the present report, we showed that diphenyl ditelluride (PhTe)(2) induced in vitro hyperphosphorylation of glial fibrillary acidic protein (GFAP), vimentin and neurofilament (NF) subunits in hippocampus of 21 day-old rats. Hyperphosphorylation was dependent on L-voltage dependent Ca(2+) channels (L-VDCC), N-methyl-d-aspartate (NMDA) and metabotropic glutamate receptors, as demonstrated by the specific inhibitors verapamil, DL-AP5 and MCPG, respectively. Also, dantrolene, a ryanodine channel blocker, EGTA and Bapta-AM, extra and intracellular Ca(2+) chelators respectively, totally prevented this effect. Activation of metabotropic glutamate receptors by (PhTe)(2) upregulates phospholipase C (PLC), producing inositol 1, 4, 5-trisphosphate (IP(3)) and diacylglycerol (DAG). Therefore, high Ca(2+) levels and DAG directly activate Ca(2+)/calmodulin-dependent protein kinase (PKCaMII) and protein kinase C (PCK), resulting in the hyperphosphorylation of Ser-57 in the carboxyl-terminal tail domain of the low molecular weight NF subunit (NF-L). Also, the activation of Erk1/2, and p38MAPK resulted in hyperphosphorylation of KSP repeats of the medium molecular weight NF subunit (NF-M). It is noteworthy that PKCaMII and PKC inhibitors prevented (PhTe)(2)-induced Erk1/2MAPK and p38MAPK activation as well as hyperphosphorylation of KSP repeats on NF-M, suggesting that PKCaMII and PKC could be upstream of this activation. Taken together, our results highlight the role of Ca(2+) as a mediator of the (PhTe)(2)-elicited signaling targeting specific phosphorylation sites on IF proteins of neural cells of rat hippocampus. Interestingly, this action shows a significant cross-talk among signaling pathways elicited by (PhTe)(2), connecting glutamate metabotropic cascade with activation of Ca(2+) channels. The extensively phosphorylated amino- and carboxyl- terminal sites could explain, at least in part, the neural dysfunction associated with (PhTe)(2) exposure.

Effect of Proline on Cell Death, Cell Cycle, and Oxidative Stress in C6 Glioma Cell Line.

Since proline metabolism has been implicated to play an underlying role in apoptotic signaling and cancer, and hyperprolinemic patients present susceptibility to tumors development, this study investigated the effect of proline on cell death, cell cycle, antioxidant enzymes activities, and immunocontent/activity of proteins involved in cell death/survival signaling pathways in C6 glioma cells. C6 cells were incubated with proline (0-5 mM) for 1 h, 24 h, 48 h, 72 h, or 7 days. Proline in high concentrations slightly decreased LDH release, and no cytotoxic effect was seen by Annexin-PI staining. Superoxide dismutase and catalase activities were increased by proline (1 mM) after 72 h, suggesting an increase in reactive species levels. Acetylcholinesterase activity was inhibited by proline at 1, 3, and 5 mM. The cell cycle progression was not altered. Results from Western blot analyses showed that proline at 1 mM after 72 h increased p-NF-ĸB and decreased acetylcholinesterase immunocontent but did not altered AKT, p-AKT, GSK3ß, and p-GSK3ß. Taken together, the data suggest that high proline levels seems to favor the signaling pathways towards cell proliferation, since acetylcholinesterase, which may act as tumor suppressor, is inhibited by proline. Also, p-NF-κB is increased by proline treatment and its activation is related to tumor cell proliferation and cellular response to oxidants. Proline also induced oxidative stress, but it appears to be insufficient to induce a significant change in cell cycle progression. These data may be related, at least in part, to the increased susceptibility to tumor development in hyperprolinemic individuals.

Antidepressant-Like Effects of Chronic Guanosine in the Olfactory Bulbectomy Mouse Model.

Major depressive disorder (MDD) leads to pervasive changes in the health of afflicted patients. Despite advances in the understanding of MDD and its treatment, profound innovation is needed to develop fast-onset antidepressants with higher effectiveness. When acutely administered, the endogenous nucleoside guanosine (GUO) shows fast-onset antidepressant-like effects in several mouse models, including the olfactory bulbectomy (OBX) rodent model. OBX is advocated to possess translational value and be suitable to assess the time course of depressive-like behavior in rodents. This study aimed at investigating the long-term behavioral and neurochemical effects of GUO in a mouse model of depression induced by bilateral bulbectomy (OBX). Mice were submitted to OBX and, after 14 days of recovery, received daily (ip) administration of 7.5 mg/kg GUO or 40 mg/kg imipramine (IMI) for 45 days. GUO and IMI reversed the OBX-induced hyperlocomotion and recognition memory impairment, hippocampal BDNF increase, and redox imbalance (ROS, NO, and GSH levels). GUO also mitigated the OBX-induced hippocampal neuroinflammation (IL-1, IL-6, TNF-α, INF-γ, and IL-10). Brain microPET imaging ([18F]FDG) shows that GUO also prevented the OBX-induced increase in hippocampal FDG metabolism. These results provide additional evidence for GUO antidepressant-like effects, associated with beneficial neurochemical outcomes relevant to counteract depression.

Homocysteine induces hypophosphorylation of intermediate filaments and reorganization of actin cytoskeleton in C6 glioma cells.

In this study, we investigated the actions of high homocysteine (Hcy) levels (100 and 500 microM) on the cytoskeleton of C6 glioma cells. Results showed that the predominant cytoskeletal response was massive formation of actin-containing filopodia at the cell surface that could be related with Cdc42 activation and increased vinculin immunocontent. In cells treated with 100 microM Hcy, folic acid, trolox, and ascorbic acid, totally prevented filopodia formation, while filopodia induced by 500 microM Hcy were prevented by ascorbic acid and attenuated by folic acid and trolox. Moreover, competitive NMDA ionotropic antagonist DL-AP5 totally prevented the formation of filopodia in both 100 and 500 microM Hcy treated cells, while the metabotropic non-selective group I/II antagonist MCPG prevented the effect of 100 microM Hcy but only slightly attenuated the effect induced by of 500 microM Hcy on actin cytoskeleton. The competitive non-NMDA ionotropic antagonist CNQX was not able to prevent the effects of Hcy on the reorganization of actin cytoskeleton in the two concentrations used. Also, Hcy-induced hypophosphorylation of vimentin and glial fibrillary acidic protein (GFAP) and this effect was prevented by DL-AP5, MCPG, and CNQX. In conclusion, our results show that Hcy target the cytoskeleton of C6 cells probably by excitoxicity and/or oxidative stress mechanisms. Therefore, we could propose that the dynamic restructuring of the actin cytoskeleton of glial cells might contribute to the response to the injury provoked by elevated Hcy levels in brain.

Homocysteine activates calcium-mediated cell signaling mechanisms targeting the cytoskeleton in rat hippocampus.

Homocysteine is considered to be neurotoxic and a risk factor for neurodegenerative diseases. Despite the increasing evidences of excitotoxic mechanisms of homocysteine (Hcy), little is known about the action of Hcy on the cytoskeleton. In this context, the aim of the present work was to investigate the signaling pathways involved in the mechanism of action of Hcy on cytoskeletal phosphorylation in cerebral cortex and hippocampus of rats during development. Results showed that 100 microM Hcy increased the intermediate filament (IF) phosphorylation only in 17-day-old rat hippocampal slices without affecting the cerebral cortex from 9- to 29-day-old animals. Stimulation of (45)Ca(2+) uptake supported the involvement of NMDA receptors and voltage-dependent channels in extracellular Ca(2+) flux, as well as Ca(2+) release from intracellular stores through inositol-3-phosphate and ryanodine receptors. Moreover, the mechanisms underlying the Hcy effect on hippocampus cytoskeleton involved the participation of phospholipase C, protein kinase C, mitogen-activated protein kinase, phosphoinositol-3 kinase and calcium/calmodulin-dependent protein kinase II. The Hcy-induced IF hyperphosphorylation was also related to G(i) protein and inhibition of cAMP levels. These findings demonstrate that Hcy at a concentration described to induce neurotoxicity activates the IF-associated phosphorylating system during development in hippocampal slices of rats through different cell signaling mechanisms. These results probably suggest that hippocampal rather than cortical cytoskeleton is susceptible to neurotoxical concentrations of Hcy during development and this could be involved in the neural damage characteristic of mild homocystinuric patients.

Effects of in vivo treatment with diphenyl ditelluride on the phosphorylation of cytoskeletal proteins in cerebral cortex and hippocampus of rats.

In this work, we investigated the effect of a single subcutaneous injection of diphenyl ditelluride (PheTe)(2) in 15-day-old Wistar rats (0.3 micromol/kg body weight) on the phosphorylation of intermediate filament (IF) proteins in cerebral cortex and hippocampus, 1, 3 or 6 days after injection. Results showed that 3 and 6 days after injection with (PheTe)(2), animals presented loss of body weight and cortical hyperphosphorylation of neurofilament subunits, glial fibrillary acidic protein (GFAP) and vimentin (Vim), the neuronal and glial intermediate filaments, respectively. Otherwise, in hippocampus, only GFAP and Vim were hyperphosphorylated and this effect was evidenced 6 days after injection. In cerebral cortex, hyperphosphorylation was accompanied by increased immunocontent of these proteins both in tissue homogenate and in cytoskeletal fraction, while in hippocampus only the immunocontent of cytoskletal-associated GFAP was increased. Moreover, hyperphosphorylation of cortical IF proteins, induced by (PheTe)(2), was totally reversed by a single subcutaneous injection of diphenyl diselenide (PheSe)(2) (5mumol/kg body weight) 24h after (PheTe)(2) administration. Taken together, our results suggest that cortical cytoskeleton is more susceptible to (PheTe)(2) than hippocampal cytoskeleton. Moreover, cytoskeletal dysfunction in cortical and hippocampal cells could be involved in the neurotoxicity induced by acute treatment with (PheTe)(2).

Branched-chain amino acids accumulating in maple syrup urine disease induce morphological alterations in C6 glioma cells probably through reactive species.

In the present study, we investigated the effects of the branched-chain amino acids (BCAA) leucine (Leu), isoleucine (Ile) and valine (Val), which accumulate in maple syrup urine disease (MSUD), on C6 glioma cell morphology and cytoskeletal reorganization by exposing the cultured cells to 1 and 5 mM BCAA. We observed that cells showed a fusiform shape with processes after 3 h treatment. Cell death was also observed when cells were incubated in the presence of the BCAA for 3 and 24 h. Val-treated cells presented the most dramatic morphological alterations. Immunocytochemistry with anti-actin and anti-GFAP antibodies revealed that all BCAA induced reorganization of actin and GFAP cytoskeleton. Although phosphorylation regulates intermediate filament (IF) assembly/disassembly, we verified that the BCAA did not change the in vitro phosphorylation of IF proteins either in C6 cells or in slices of cerebral cortex of rats during development (9-, 12-, 17- and 21-day-old). Furthermore, we observed that 3 h cell exposure to 5 mM of each BCAA resulted in a marked reduction of reduced glutathione (GSH) levels and significantly increased nitric oxide production. Finally, we observed that the morphological features caused by the BCAA on C6 cells were prevented by the use of the antioxidants GSH (1 mM) and N(omega)-nitro-L-arginine methyl ester (L-NAME, 0.5 mM). On the basis of the present results, we conclude that free radical attack might be involved in the cell morphological alterations, as well as, in the cytoskeletal reorganization elicited by the BCAA. It is therefore presumed that these findings could be involved in the neuropathological features observed in patients affected by MSUD.

Guanosine Anxiolytic-Like Effect Involves Adenosinergic and Glutamatergic Neurotransmitter Systems.

Accumulating evidences indicate that endogenous modulators of excitatory synapses in the mammalian brain are potential targets for treating neuropsychiatric disorders. Indeed, glutamatergic and adenosinergic neurotransmissions were recently highlighted as potential targets for developing innovative anxiolytic drugs. Accordingly, it has been shown that guanine-based purines are able to modulate both adenosinergic and glutamatergic systems in mammalian central nervous system. Here, we aimed to investigate the potential anxiolytic-like effects of guanosine and its effects on the adenosinergic and glutamatergic systems. Acute/systemic guanosine administration (7.5 mg/kg) induced robust anxiolytic-like effects in three classical anxiety-related paradigms (elevated plus maze, light/dark box, and round open field tasks). These guanosine effects were correlated with an enhancement of adenosine and a decrement of glutamate levels in the cerebrospinal fluid. Additionally, pre-administration of caffeine (10 mg/kg), an unspecific adenosine receptors' antagonist, completely abolished the behavioral and partially prevented the neuromodulatory effects exerted by guanosine. Although the hippocampal glutamate uptake was not modulated by guanosine (both ex vivo and in vitro protocols), the synaptosomal K+-stimulated glutamate release in vitro was decreased by guanosine (100 µM) and by the specific adenosine A1 receptor agonist, 2-chloro-N 6-cyclopentyladenosine (CCPA, 100 nM). Moreover, the specific adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 100 nM) fully reversed the inhibitory guanosine effect in the glutamate release. The pharmacological modulation of A2a receptors has shown no effect in any of the evaluated parameters. In summary, the guanosine anxiolytic-like effects seem closely related to the modulation of adenosinergic (A1 receptors) and glutamatergic systems.