Samanta Oliveira Loureiro

In vitro phosphorylation of cytoskeletal proteins from cerebral cortex of rats

Procedures for the preparation of high- and low-salt Triton insoluble cytoskeletal fractions from rat brain suitable for studying in vitro phosphorylation by endogenous kinases and phosphatases are described. The high-salt Triton insoluble cytoskeletal fraction is enriched in neurofilament subunits (NF-H, NF-M and NF-L), vimentin and glial fibrillary acidic protein (GFAP), while the low-salt Triton insoluble cytoskeletal fraction contains detergent insoluble cytoskeletal elements such as intermediate filament subunits and tubulins. One of our approaches is to incubate cerebral cortex slices with [32P]orthophosphate before the cytoskeletal fraction extraction, which allows the in vitro phosphorylation of cytoskeletal constituents in an intact intracellular environment. On the other hand, we also incubate low- or high-salt cytoskeletal fractions previously prepared with [gamma(32)P]ATP. By doing so, we are able to study the direct effects of substances on the kinase and phosphatase activities associated with the cytoskeletal fraction. Moreover by using specific activators or inhibitors of protein kinases and phosphatases we can obtain more detailed information on the alterations provoked by these substances. These approaches are useful for the investigation of the neurotoxic effects of various drugs and metabolites affecting the cytoskeletal-associated phosphorylation system in the brain.

Acute intrastriatal administration of quinolinic acid provokes hyperphosphorylation of cytoskeletal intermediate filament proteins in astrocytes and neurons of rats.

In the present study we investigated the effect of in vivo intrastriatal injection of quinolinic acid (QA) on cytoskeletal proteins in astrocytes and neurons of young rats at early stage (30 min) after infusion. QA (150 nmoles/0.5 microL) significantly increased the in vitro phosphorylation of the low molecular weight neurofilament subunit (NFL) and the glial fibrillary acidic protein (GFAP) of neurons and astrocytes, respectively. This effect was mediated by cAMP-dependent protein kinase A (PKA), protein kinase C (PKC) and Ca(2+)/calmodulin-dependent protein kinase II (PKCaMII). In contrast, mitogen activated protein kinases were not activated by QA infusion. Furthermore, the specific N-methyl-D-aspartate (NMDA) antagonist MK-801 (0.25 mg/kg i.p), the antioxidant L-NAME (60 mg\kg\day), and diphenyldisselenide (PheSe)(2) (0.625 mg\kg\day) injected prior to QA infusion totally prevented QA-induced cytoskeletal hyperphosphorylation. We also observed that QA-induced hyperphosphorylation was targeted at the Ser55 phosphorylating site on NFL head domain, described as a regulatory site for NF assembly in vivo. This effect was fully prevented by MK801, by the PKA inhibitor H89 and by (PheSe)(2), whereas staurosporine (PKC inhibitor) only partially prevented Ser55 phosphorylation. The PKCaMII inhibitor (KN93) and the antioxidant L-NAME failed to prevent the hyperphosphorylation of Ser55 by QA infusion. Therefore, we presume that QA-elicited hyperphosphorylation of the neural cytoskeleton, and specially of NFLSer55, achieved by intrastriatal QA injection could represent an early step in the pathophysiological cascade of deleterious events exerted by QA in rat striatum. Our observations also indicate that NMDA-mediated Ca(2+) events and oxidative stress may be related to the altered protein cytoskeleton hyperphosphorylation observed with important implications for brain function.

Homocysteine induces cytoskeletal remodeling and production of reactive oxygen species in cultured cortical astrocytes

Homocysteine (Hcy) is an excitatory amino acid which markedly enhances the vulnerability of neuronal cells to excitotoxicity and oxidative injury. Patients with severe hyperhomocysteinemia exhibit a wide range of clinical manifestations including neurological abnormalities such as mental retardation, cerebral atrophy, and seizures. In this study we treated cortical astrocytes and neurons in culture with 10 and 100 μM Hcy and after 24h exposure cytoskeletal remodeling was analyzed by immunocytochemistry. We observed dramatically altered actin cytoskeleton in astrocytes exposed to 100 μM Hcy, with concomitant change of morphology to fusiform and/or flattened cells with retracted cytoplasm. Moreover, we observed disruption of the glial fibrillary acidic protein (GFAP) meshwork, supporting misregulation of actin cytoskeleton. Induction of reactive oxygen species (ROS) in astrocytes showed fluctuating levels along 24h exposure to both Hcy concentrations. Actin remodeling induced by 100 μM Hcy was prevented by the antioxidants folate (5 μM) or trolox (80 μM). Unlike astrocyte cytoskeleton, results evidence little susceptibility of neuron cytoskeleton until 24h of treatment, since immunocytochemical analysis showed that 10 and 100 μM Hcy-treated neurons presented unaltered neurite arborization. Moreover, alterations in astrocyte and neuron viability were not observed along the 24h of exposure to Hcy. Neuron/astrocyte co-cultures evidence an anchorage dependence for neuronal survival over long exposure to Hcy. Taken together, these findings indicate, that the cytoskeleton of cortical astrocytes, but not of neurons in culture, is a target to Hcy and such effects are mediated by redox signaling. Astrocytes were able to respond to Hcy (100 μM) reorganizing their cytoskeleton, surviving, and protecting neurons from Hcy damage. Moreover our results suggest a protective role for astrocytes remodeling the cytoskelon, and probably generating signals that would assure neuronal survival in response to the damage induced by Hcy.

α-Ketoisocaproic acid regulates phosphorylation of intermediate filaments in postnatal rat cortical slices through ionotropic glutamatergic receptors

In this study we investigated the effects of alpha-ketoisocaproic acid (KIC), the main keto acid accumulating in the inherited neurometabolic disorder maple syrup urine disease (MSUD), on the in vitro incorporation of 32P into intermediate filament (IF) proteins from cerebral cortex of rats during development. KIC decreased the in vitro incorporation of 32P into the IF proteins studied up to day 12, had no effect on day 15, and increased this phosphorylation in cortical slices of 17- and 21-day-old rats. A similar effect on IF phosphorylation was achieved along development by incubating cortical slices with glutamate. Furthermore, the altered phosphorylation caused by the presence of KIC in the incubation medium was mediated by the ionotropic receptors NMDA, AMPA and kainate up to day 12 and by NMDA and AMPA in tissue slices from 17- and 21-day-old rats. The results suggest that alterations of IF phosphorylation may be associated with the neuropathology of MSUD.

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 μM 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 45Ca2+ uptake supported the involvement of NMDA receptors and voltage‐dependent channels in extracellular Ca2+ flux, as well as Ca2+ 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 Gi protein and inhibition of cAMP levels. These findings demonstrate that Hcy at a concentration described to induce neurototoxicity 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 succeptible to neurotoxical concentrations of Hcy during development and this could be involved in the neural damage characteristic of mild homocystinuric patients.

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.

Disrupted cytoskeletal homeostasis, astrogliosis and apoptotic cell death in the cerebellum of preweaning rats injected with diphenyl ditelluride.

In the present report 15 day-old rats were injected with 0.3μmol of diphenyl ditelluride (PhTe)(2)/kg body weight and parameters of neurodegeneration were analyzed in slices from cerebellum 3 and 6 days afterwards. The earlier responses, at day 3 after injection, included hyperphosphorylation of intermediate filament (IF) proteins from astrocyte (glial fibrillary acidic protein - GFAP - and vimentin) and neuron (low-, medium- and high molecular weight neurofilament subunits: NF-L, NF-M and NF-H); increased mitogen-activated protein kinase (MAPK) (Erk and p38MAPK) and cAMP-dependent protein kinase (PKA) activities. Also, reactive astrogliosis takes part of the early responses to the insult with (PhTe)(2), evidenced by upregulated GFAP in Western blot, PCR and immunofluorescence analysis. Six days after (PhTe)(2) injection we found persistent astrogliosis, increased propidium iodide (PI) positive cells in NeuN positive population evidenced by flow cytometry and reduced immunofluorescence for NeuN, suggesting that the in vivo exposure to (PhTe)(2) progressed to neuronal death. Moreover, activated caspase 3 suggested apoptotic neuronal death. Neurodegeneration was related with decreased [(3)H]glutamate uptake and decreased Akt immunoreactivity, however phospho-GSK-3-β (Ser9) was not altered in (PhTe)(2) injected rat. Therefore, the present results show that the earlier cerebellar responses to (PhTe)(2) include disruption of cytoskeletal homeostasis that could be related with MAPK and PKA activation and reactive astrogliosis. Akt inhibition observed at this time could also play a role in the neuronal death evidenced afterwards. The later events of the neurodegenerative process are characterized by persistent astrogliosis and activation of apoptotic neuronal death through caspase 3 mediated mechanisms, which could be related with glutamate excitotoxicity. The progression of these responses are therefore likely to be critical for the outcome of the neurodegeneration provoked by (PhTe)(2) in rat cerebellum.

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-β (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.

Acute ethanol exposure disrupts actin cytoskeleton and generates reactive oxygen species in c6 cells

Central nervous system dysfunctions are among the most significant effects of exposure to ethanol and the glial cells that play an important role in maintaining neuronal function, are extremely involved with these effects. The actin cytoskeleton plays a crucial role in a wide variety of cellular functions, especially when there is some injury. Therefore the aim of the present study was to analyze the short-term effects of ethanol (50, 100 and 200 mM) on the cytoskeleton of C6 glioma cells. Here we report that acute ethanol exposure profoundly disrupts the actin cytoskeleton in C6 cells decreasing stress fiber formation and downregulating RhoA and vinculin immunocontent. In contrast, microtubule and GFAP networks were not altered. We further demonstrate that anti-oxidants prevent ethanol-induced actin alterations, suggesting that the actions of ethanol on the actin cytoskeleton are related with generation of reactive oxygen species (ROS) in these cells. Our results show that ethanol at concentrations described to be toxic to the central nervous system was able to target the cytoskeleton of C6 cells and this effect could be related with increased ROS generation. Therefore, we propose that the dynamic restructuring of the cytoskeleton of glial cells might contribute to the response to the injury provoked by binge-like ethanol exposure in brain.

Effect of propionic and methylmalonic acids on the in vitro phosphorylation of intermediate filaments from cerebral cortex of rats during development

In this study we investigated the in vivo and in vitro effects of methylmalonic (MMA) and propionic acids (PA), at concentrations usually found in methylmalonic acidemia and propionic acidemia respectively, on the phosphorylation of intermediate filament proteins in cerebral cortex of rats during development. Rats of 9, 12, and 17 days were acutely injected with the acids and sacrificed 90 min after injection. The cerebral cortex was dissected, and slices were incubated with 32P-orthophosphate. The cytoskeletal fraction was extracted and the radioactivity incorporated into intermediate filament subunits was measured. In addition, cortical slices from nontreated rats of 9, 12, 15, 17, 21, and 60 days of life were incubated with the acids in the presence of 32P-orthophosphate, the cytoskeletal fraction was extracted and the radioactivity was measured. Results demonstrated that MMA and PA significantly decreased the radioactivity incorporated into intermediate filament proteins at day 12, both in vivo and in tissue slices. In contrast, PA increased the in vitro phosphorylation of the cytoskeletal proteins in slices of 21-day-old animals. It acts through PP2A and PP2B in 12-day-old rats and through PKA and PKCaMII in 21-day-old animals. We propose that alteration of cytoskeletal protein phosphorylation caused by methylmalonic and propionic acids may be related to the neurological dysfunction characteristic of propionic and methylmalonic acidemia.