Signaling Mechanisms and Disrupted Cytoskeleton in the Diphenyl Ditelluride Neurotoxicity

Evidence from our group supports that diphenyl ditelluride (PhTe)2 neurotoxicity depends on modulation of signaling pathways initiated at the plasma membrane. The (PhTe)2-evoked signal is transduced downstream of voltage-dependent Ca2+ channels (VDCC), N-methyl-D-aspartate receptors (NMDA), or metabotropic glutamate receptors activation via different kinase pathways (protein kinase A, phospholipase C/protein kinase C, mitogen-activated protein kinases (MAPKs), and Akt signaling pathway). Among the most relevant cues of misregulated signaling mechanisms evoked by (PhTe)2 is the cytoskeleton of neural cells. The in vivo and in vitro exposure to (PhTe)2 induce hyperphosphorylation/hypophosphorylation of neuronal and glial intermediate filament (IF) proteins (neurofilaments and glial fibrillary acidic protein, resp.) in different brain structures of young rats. Phosphorylation of IFs at specific sites modulates their association/disassociation and interferes with important physiological roles, such as axonal transport. Disrupted cytoskeleton is a crucial marker of neurodegeneration and is associated with reactive astrogliosis and apoptotic cell death. This review focuses the current knowledge and important results on the mechanisms of (PhTe)2 neurotoxicity with special emphasis on the cytoskeletal proteins and their differential regulation by kinases/phosphatases and Ca2+-mediated mechanisms in developmental rat brain. We propose that the disrupted cytoskeletal homeostasis could support brain damage provoked by this neurotoxicant

Exposure of young rats to diphenyl ditelluride during lactation affects the homeostasis of the cytoskeleton in neural cells from striatum and cerebellum

AbstractIn the present report we examined the effect of maternal exposure to diphenyl ditelluride (PhTe)2 (0.01mg/kg body weight) during the first 14 days of lactational period on the activity of some protein kinases targeting the cytoskeleton of striatum and cerebellum of their offspring. We analyzed the phosphorylating system associated with glial fibrillary acidic protein (GFAP), and neurofilament of low, medium and high molecular weight (NF-L, NF-M and NF-H, respectively) of pups on PND 15, 21, 30 and 45. We found that (PhTe)2 induced hyperphosphorylation of all the proteins studied on PND 15 and 21, recovering control values on PND 30 and 45. The immunocontent of GFAP, NF-L, NF-M and NF-H in the cerebellum of 15-day-old pups was increased. Western blot assays showed activation/phosphorylation of Erk1/2 on PND 21 and activation/phosphorylation of JNK on PND 15. Otherwise, p38MAPK was not activated in the striatum of (PhTe)2 exposed pups. On the other hand, the cerebellum of pups exposed to (PhTe)2 presented activated/phosphorylated Erk1/2 on PND 15 and 21 as well as activated/phosphorylated p38MAPK on PND 21, while JNK was not activated. Western blot assays showed that both in the striatum and in the cerebellum of (PhTe)2 exposed pups, the immunocontent of the catalytic subunit of PKA (PKAcα) was increased on PND 15. Western blot showed that the phosphorylation level of NF-L Ser55 and NF-M/NF-H KSP repeats was increased in the striatum and cerebellum of both 15- and 21-day-old pups exposed to (PhTe)2. Diphenyl diselenide (PhSe)2, the selenium analog of (PhTe)2, prevented (PhTe)2-induced hyperphosphorylation of striatal intermediate filament (IF) proteins but it failed to prevent the action of (PhTe)2 in cerebellum. Western blot assay showed that the (PhSe)2 prevented activation/phosphorylation of Erk1/2, JNK and PKAcα but did not prevent the stimulatory effect of (PhTe)2 on p38MAPK in cerebellum at PND 21. In conclusion, this study demonstrated that dam exposure to low doses of (PhTe)2 can alter cellular signaling targeting the cytoskeleton of striatum and cerebellum in the offspring in a spatiotemporal manner, which can be related to the neurotoxic effects of (PhTe)2

Dual action of chronic ethanol treatment on LPS-induced response in C6 glioma cells

AbstractIn this study we investigated the anti-inflammatory effects of chronic ethanol (EtOH) treatment on lipopolysaccharide (LPS)-stimulated C6 glioma cells. The cells were chronically treated with 200mM EtOH; coincubation with LPS and EtOH was obtained upon addition of 2μg/ml LPS to the incubation medium in the last 24h of EtOH exposure. We found that EtOH prevented the LPS-induced production of tumor necrosis factor α (TNFα) without decreasing cell viability. Either LPS treated or EtOH plus LPS treated cells presented upregulated glial fibrillary acidic protein (GFAP) and downregulated vimentin levels characterizing a program of reactive astrogliosis. Also, EtOH plus LPS stimulation greatly increased the oxidative stress generation evaluated by DCF-DA measurement, while either EtOH alone or LPS alone was unable to induce oxidative stress. Western blot analysis indicated that either EtOH, LPS or EtOH plus LPS treatments are unable to affect Akt/GSK3β signaling pathway. However, LPS alone and EtOH plus LPS co-treatment inhibited Erk phosphorylation. A dramatic loss of stress fibers was found in EtOH exposed cells, evaluated by cytochemistry using phalloidin-fluorescein. However, LPS alone was not able to disrupt actin organization. Furthermore, cells co-incubated with LPS and EtOH presented reversion of the disrupted stress fibers provoked by EtOH. Supporting this action, RhoA and vinculin immunocontent were upregulated in response to EtOH plus LPS. Interestingly, EtOH suppresses the inflammatory cascade (TNFα production) in response to LPS. Concomitantly it sustains Erk inhibition, increases oxidative stress generation and induces reactive astrogliosis in the presence of LPS, conditions associated with neurotoxicity. The effects observed were not supported by actin reorganization. Altogether, these findings suggest that Erk signaling inhibition could play a role in both suppressing TNFα production and inducing oxidative stress generation and astrogliosis, therefore modulating a dual action of EtOH plus LPS in glial cells

High fat diet-induced obesity causes a reduction in brain tyrosine hydroxylase levels and non-motor features in rats through metabolic dysfunction, neuroinflammation and oxidative stress

Obesity is a health problem that has been associated with neuroinflammation, decreased cognitive functions and development of neurodegenerative diseases. Parkinson’s disease (PD) is a chronic neurodegenerative condition characterized by motor and non-motor abnormalities, increased brain inflammation, α-synuclein protein aggregation and dopaminergic neuron loss that is associated with decreased levels of tyrosine hydroxylase (TH) in the brain. Diet-induced obesity is a global epidemic and its role as a risk factor for PD is not clear. Herein, we showed that 25 weeks on a high-fat diet (HFD) promotes significant alterations in the nigrostriatal axis of Wistar rats. Obesity induced by HFD exposure caused a reduction in TH levels and increased TH phosphorylation at serine 40 in the ventral tegmental area. These effects were associated with insulin resistance, increased tumor necrosis factor-α levels, oxidative stress, astrogliosis and microglia activation. No difference was detected in the levels of α-synuclein. Obesity also induced impairment of locomotor activity, total mobility and anxiety-related behaviors that were identified in the open-field and light/dark tasks. There were no changes in motor coordination or memory. Together, these data suggest that the reduction of TH levels in the nigrostriatal axis occurs through an α-synuclein-independent pathway and can be attributed to brain inflammation, oxidative/nitrosative stress and metabolic disorders induced by obesit

Envolvimento da via PLC no mecanismo de ação do T4 sobre a fosforilação das proteínas do citoesqueleto de córtex cerebral de ratos

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Efeito da homocisteína sobre a fosforilação dos filamentos intermediários de hipocampo de ratos

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Efeito do ditelureto de difenila sobre as células neurais de ratos jovens : vias de sinalização, homeostase do citoesqueleto e neurodegeneração

O telúrio é um elemento raro usado como componente industrial de muitas ligas. Estudos in vivo e in vitro demonstraram que compostos orgânicos do telúrio são neurotóxicos, entre eles podemos destacar o ditelureto de difenila [(PhTe)2]. Os efeitos provocados por esse organotelureto nos diferentes sistemas são importantes, no entanto, os observados no SNC são particularmente marcantes. Relatos têm demonstrado que o (PhTe)2 pode causar alterações no estado de fosforilação dos filamentos intermediários (FIs) neuronais e gliais, sendo os neurônios e astrócitos importantes alvos desse neurotoxicante. Considerando que o citoesqueleto é um importante alvo de neurotoxinas, nosso trabalho estudou o efeito do (PhTe)2 sobre alguns parâmetros bioquímicos do citoesqueleto neural. Ratos submetidos a uma injeção subcutânea de (PhTe)2 apresentaram alteração na fosforilação e/ou expressão das subunidades dos neurofilamentos (NF-H, NF-M e NF-L), da GFAP e da vimentina, bem como ativação das vias das MAPK e da PKA. Essas modificações são dependentes da estrutura estudada (cerebelo ou estriado) e da idade do animal. Além disso, uma única administração de (PhTe)2 em ratos jovens provocou morte neuronal e astrogliose. Quando o (PhTe)2 é administrado nas ratas mães durante os primeiros 14 dias de lactação verificamos que ocorre uma modificação no sistema fosforilante associado aos FIs nos filhotes, novamente de uma maneira estrutura (córtex cerebral, hipocampo, estrido ou cerebelo) e idade dependente. Além disso, verificou-se que esse organocalcogênio injetado nas ratas mães também age sobre as vias das MAPK e da PKA. Estudos in vitro mostraram que esse composto orgânico do telúrio causa hipofosforilação dos FIs neuronais e gliais no córtex cerebral. Esse efeito é mediado por alteração homeostase do cálcio e do glutamato, bem como pela inibição da PKA, alteração da fosforilação da proteína DARPP-32(Thr34) e ativação da PP1. No hipocampo temos uma ativação dos receptores glutamatérgicos ionotrópicos e metabotrópicos, acarretando uma alteração na homeostase do cálcio e ativação da PKC, PKCaMII e via das MAPK, conduzindo a um aumento de fosforilação dos FIs neuronais e gliais. Nossos resultados mostraram, portanto, que o (PhTe)2 altera a homeostase do citoesqueleto cerebral e que essa modificação é dependente do desenvolvimento do animal, da estrutura cerebral estudada, bem como da maneira de contato com esse neurotoxicante. Além disso, verificou-se o envolvimento de várias vias de sinalização nas ações desencadeadas pelo composto orgânico do telúrio sobre os FIs, estando elas muitas vezes interligadas. Esses resultados podem contribuir para o melhor entendimento dos mecanismos envolvidos em uma intoxicação com compostos de telúrio, sendo que o desequilíbrio do citoesqueleto pode estar associado à neurotoxicidade desse organotelureto.Tellurium is a rare element used as a component of many industrial alloys. In vivo and in vitro studies have demonstrated that organic tellurium compounds are neurotoxic. We can highlight the organic coumpoud diphenyl ditelluride [(PhTe)2]. The effects caused by this organoteluride in different systems are important, however, the effects observed in the CNS are particularly striking. Reports have shown that (PhTe)2 can induces changes in the phosphorylation state of neuronal and glial intermediate filaments (IFs). The neurons and astrocytes are important targets of this neurotoxin. Considering that the cytoskeleton is an important target for neurotoxins, the aim of the present study was studied the effect of (PhTe)2 on some biochemical parameters of the neural cytoskeleton. Rats treated with a single subcutaneous (s.c.) injection of (PhTe)2 showed changes in the phosphorylation and / or expression of neurofilament subunit (NF-H, NF-M and NF-L), GFAP and vimentin, as well as activation of the MAPK pathway and PKA. These changes are dependent on the structure studied (cerebellum or striatum) and age of the animal. Furthermore, a single administration (PhTe)2 in young rats caused neuronal death and astrogliosis. When (PhTe)2 is administered in dams during the first 14 days of lactation we observed a change in the phosphorylating system associated with IFs in pups. This effect is dependent of the structure studied and the developmental stages of the pups. Furthermore, it was found that (PhTe)2 is administered in dams during the first 14 days of lactation also acts on the MAPK pathway and in the PKA. In vitro studies showed that the organic tellurium causes hipophosphorylation of the neuronal and glial IFs proteins in the cerebral cortex. This effect is mediated by change in calcium and glutamate homeostasis, inhibition of PKA, desphosphorylation of the protein DARPP-32 (Thr34) and activation of PP1. In the hippocampus have an activation of ionotropic and metabotropic glutamatergic receptors, causing an alteration in calcium homeostasis and activation of PKC, PKCaMII and MAPK pathway, leading to increased phosphorylation of neuronal and glial IFs. The results of this work showed that the (PhTe)2 changes cytoskeletal homeostasis in brain. This modification is dependent of the animal development, the brain structure studied, as well as the way of contact with the neurotoxin. Moreover, there is the involvement of different signaling pathways in the action of (PhTe)2 and they are often interconnected. These results may contribute to a better understanding of mechanisms involved in intoxication with tellurium compounds. The imbalance of the cytoskeleton may be associated with neurotoxicity of this organochalcogenide

Efeito do tratamento in vivo com ditelureto de difenila sobre a fosforilação das proteínas do citoesqueleto e sobre a atividade da Na+, K+-ATPase em córtex cerebral e/ou hipocampo de ratos jovens

O telúrio é um elemento raro usado na indústria eletrônica como componente industrial de muitas ligas. Tanto as formas orgânicas quando as inorgânicas do telúrio são altamente tóxicas para o SNC de roedores. Neste trabalho, nós inicialmente realizamos uma curva de dose, através de uma única administração subcutânea do composto orgânico de telúrio, ditelureto de difenila, em ratos de 15 dias de idade, estabelecendo a perda de massa corporal como critério de toxicidade do composto utilizado. A partir disso, o objetivo principal do nosso trabalho foi investigar o efeito de uma única administração subcutânea do ditelureto de difenila sobre a fosforilação dos filamentos intermediários (FI) de córtex cerebral e hipocampo de ratos Wistar jovens (15 dias de idade), 1, 3 ou 6 dias após a exposição à droga. Verificamos também a capacidade do composto orgânico de selênio, o disseleneto de difenila de reverter esse efeito. Além disso, também determinamos o efeito do tratamento in vivo com o ditelureto de difenila sobre a atividade da Na+, K+ -ATPase. Os resultados obtidos mostraram que nos dias 3 e 6 após a injeção da droga, os animais injetados com 0,3 μmol/Kg de peso corporal, ou doses maiores da neurotoxina, apresentaram uma perda significativa de massa corporal, quando comparados com os animais controles. Com base nesses dados, a concentração de 0,3 μmol/Kg de peso corporal foi escolhida para a realização dos estudos posteriores. Nesse período ocorreu também um aumento da fosforilação dos neurofilamentos (FI neuronais), da proteína glial fibrilar ácida (GFAP) e da vimentina (FI gliais), no córtex cerebral dos ratos. O efeito do ditelureto de difenila sobre a fosforilação dos FI em córtex cerebral foi acompanhado por um aumento do imunoconteúdo das proteínas NF-M, NF-L e GFAP na fração citoesquelética, sugerindo um aumento de polimerização destas proteínas. Por outro lado, o aumento do imunoconteúdo da NF-L e GFAP no homogeneizado total 3 dias após a administração do ditelureto de difenila, é compatível com um aumento de expressão dessas proteínas. Porém, 6 dias após a injeção do composto verificou-se apenas um aumento do imunoconteúdo da GFAP na fração citoesquelética, assim como da subunidade de alto peso molecular dos neurofilamentos (NFH) e da GFAP no homogeneizado total de córtex cerebral. Por outro lado, no hipocampo, ocorreu um aumento de fosforilação apenas da GFAP e da vimentina, proteínas típicas de astrócitos, sendo que esse efeito só foi observado no sexto dia após a injeção da neurotoxina. Esse efeito foi acompanhado por um aumento do imunoconteúdo da GFAP na fração citoesquelética 6 dias após a exposição ao ditelureto de difenila. O efeito sobre a fosforilação das proteínas do citoesqueleto foi completamente prevenido pelo disseleneto de difenila (5 μmol/Kg de peso corporal) injetado uma única vez, 24 h após a administração do ditelureto de difenila. Além disso, também demostramos nesse trabalho que, ele é capaz de inibir a atividade da Na+, K+ -ATPase 6 dias após a administração da droga. Nossos resultados mostraram, portanto, que o ditelureto de difenila, quando administrado de forma aguda em ratos jovens, é capaz de aumentar a expressão de proteínas de FI e de afetar o sistema fosforilante associado a essas proteínas. Os dados mostraram ainda que o aparecimento desses efeito tem uma latência de 3 a 6 dias após a injeção da toxina e indicaram que o córtex cerebral é mais sensível do que o hipocampo aos efeitos do composto orgânico do telúrio. Além disso, o ditelureto de difenila é capaz de inibir a enzima Na+, K+ - ATPase cortical, reforçando a susceptibilidade do córtex cerebral à ação desta neurotoxina. Mostramos ainda neste trabalho, que o aumento de fosforilação das proteínas de FIs pode ser prevenida pelo disseleneto de difenila Com base nos resultados obtidos, pode-se supor que as alterações no citoesqueleto e a inibição da atividade da Na+, K+ -ATPase em células corticais podem estar envolvidas com a neurotoxicidade desse composto.Tellurium is a rare element used as an industrial component of many alloys and in the electronic industry. This element also is one important intermediate and/or reagent in organic synthesis. Inorganic and organic tellurium compounds are highly toxic to the CNS of rodents. In this work we initially established the toxic dose and the time-action characteristics of diphenyl ditelluride acutely injected using body weight measurements as the end point of toxicity. Considering these findings, the aim of this work was to investigate the effect of a single subcutaneous injection of organic tellurium compounds, diphenyl ditelluride, on the intermediate filament (IF) phosphorylation in cerebral cortex and hippocampus from young Wistar rats (15 day-old), 1, 3 or 6 days after injection. We also investigated if diphenyl diselenide would be able to prevent this effect. Furthermore, we verified the effect of in vivo treatment with diphenyl ditelluride on Na+-K+-ATPase activity. Results showed that at days 3 and 6 animals injected with 0.3 μmol/Kg body weight or higher doses of diphenyl ditelluride presented loss of body mass when compared with control animals. Considering this finding, we have chosen the concentration of 0.3 μmol/Kg body weight for the next experiments. We observed an hyperphosphorylation of neurofilaments (the neuronal IF) and of glial fibrillary acidic protein (GFAP) and of vimentin (Vim) (astrocyte IFs) in cerebral cortex. The effect of diphenyl ditelluride 3 days of injection on IF phosphorylation was accompanied by an increased NF-M, NF-L and GFAP immunocontent in IF-associated cytoskeletal fraction, suggesting an increase in protein polymerization. Otherwise, the stimulated GFAP, NF-L immunoreactivity in tissue homogenate suggests an increased protein synthesis. However, 6 days after the neurotoxin administration, only GFAP immunocontent increased in IF-associated cytoskeletal fraction, while GFAP and NF-H immunoreactivity was stimulated in tissue homogenate from cerebral cortex. On the other hand, in hippocampus, we observed that astrocyte IF (GFAP and Vimentin) hyperphosphorylation was accompanied by increased immunocontent of these proteins in cytoskeletal fraction at day 6 after tellurium injection. The cortical IF hyperphosphorylation induced by diphenyl ditelluride, was totally prevented by a single subcutaneous injection of diphenyl disselenide (5μmol/kg body weigth) 24h after diphenyl ditelluride administration. We also showed that beyond the effects of the in vivo treatment with diphenyl ditelluride on the cytoskeleton of cortical cells, this neurotoxin inhibited Na+-K+-ATPase activity at day 6 after drug injection. So, our results showed that a single subcutaneous injection of (PheTe)2 in young rats is able to stimulate the phosphorylation and expression of IF proteins. Moreover, our data demonstrated that effects of diphenyl ditelluride were time- and brain structure-dependent. We also verified in this work that cerebral cortex is more susceptible than hippocampus to neurotoxin injury. In addition, the diphenyl ditelluride was also able to inhibit the Na+-K+- ATPase activity. We showed also in this work that cortical IF hyperphosphorylation induced by diphenyl ditelluride was totally prevented by diphenyl disselenide Therefore, we can suppose that the observed alterations in cytoskeleton and the inhibition of the activity Na+-K+- ATPase in cortical cells may be related with the neurotoxicity of this substance