Nongenomic actions of thyroxine modulate intermediate filament phosphorylation in cerebral cortex of rats.

The developmental effects of thyroid hormones (TH) in mammalian brain are mainly mediated by nuclear receptors regulating gene expression. However, there are increasing evidences of nongenomic mechanisms of these hormones associated with kinase- and calcium-activated signaling pathways. In this context, the aim of the present work was to investigate the signaling pathways involved in the mechanism of action of TH on cytoskeletal phosphorylation in cerebral cortex of 15-day-old male rats. Results showed that L-thyroxine (L-T4) increased the intermediate filament (IF) phosphorylation independently of protein synthesis, without altering the total immunocontent of these proteins. Otherwise, neither 3,5,3'-triiodo-L-thyronine (L-T3) nor neurotransmitters (GABA, ATP, L-glutamate or epinephrine) acted on the IF-associated phosphorylation level. We also demonstrated that the mechanisms underlying the L-T4 effect on the cytoskeleton involve membrane initiated actions through Gi protein-coupled receptor. This evidence was reinforced by the inhibition of cyclic adenosine 5'-monophosphate (cAMP) levels. Moreover, we showed the participation of phospholipase C, protein kinase C, mitogen-activated protein kinase, calcium/calmodulin-dependent protein kinase II, intra- and extracellular Ca2+ mediating the effects of L-T4 on the cytoskeleton. Stimulation of 45Ca2+ uptake by L-T4 was also demonstrated. These findings demonstrate that L-T4 has important physiological roles modulating the cytoskeleton of neural cells during development.

Cytoskeleton as a potential target in the neuropathology of maple syrup urine disease: insight from animal studies.

In this short review we provide evidence that the branched-chain keto acids accumulating in the neurometabolic disorder maple syrup urine disease disturb rat cerebral cytoskeleton in a developmentally regulated manner. Alterations of protein phosphorylation leading to brain cytoskeletal misregulation and neural cell death caused by these metabolites are associated with energy deprivation, oxidative stress and excitotoxicity that may ultimately disrupt normal cell function and viability.

Diphenyl ditelluride- and methylmercury-induced hyperphosphorilation of the high molecular weight neurofilament subunit is prevented by organoselenium compounds in cerebral cortex of young rats.

Organotellurides are important intermediates in organic synthesis and, consequently, the occupational exposure to them is a constant risk for laboratory workers. These compounds can elicit many neurotoxic events in the central nervous system (CNS) that are associated with several neurological symptoms. In contrast, organoselenium compounds are considered to exert neuroprotective actions on such effects. Neurofilaments (NF) are important cytoskeletal proteins and phosphorylation/dephosphorylation of NF is important to stabilize the cytoskeleton. In this work we investigated the potential protective ability of the selenium compounds ebselen and diphenyl diselenide (PhSe)(2) against the effect of diphenyl ditelluride (PhTe)(2) and methylmercury (MeHg) on the total (phosphorylated plus nonphosphorylated) and phosphorylated immunocontent of the high molecular weight neurofilament subunit (NF-H) from slices of cerebral cortex of 17-day-old rats. We observed that 1muM MeHg induced hyperphosphorylation, increasing the total immunocontent of this subunit of the high-salt Triton insoluble NF-H. Otherwise, 15muM (PhTe)(2) induced hyperphosphorylation of the high-salt Triton insoluble NF-H without altering the total immunocontent of this protein into the cytoskeletal fraction. Concerning the selenium compounds, 15muM (PhSe)(2) and 5muM ebselen did not induce alteration per se on the in vitro phosphorylation of NF-H. In addition, (PhSe)(2) and ebselen at these concentrations, presented a protective effect against the action of (PhTe)(2) and MeHg, on the immunoreactivity of NF-H. Considering that hyperphosphorylation of NF-H is associated with neuronal dysfunction it is probable that the effects of (PhTe)(2) and MeHg could be related to the remarkable neurotoxicity of these organocalcogenides. Furthermore the neuroprotective action of selenium compounds against (PhTe)(2) and MeHg effects could be a promising route to be exploited for a possible treatment of calcogenides poisoning.

Selenium compounds prevent the effects of methylmercury on the in vitro phosphorylation of cytoskeletal proteins in cerebral cortex of young rats.

In this study we investigated the protective ability of the selenium compounds ebselen and diphenyldiselenide against the effect of methylmercury on the in vitro incorporation of 32P into intermediate filament (IF) proteins from the cerebral cortex of 17-day-old rats. We observed that methylmercury in the concentrations of 1 and 5 microM was able to inhibit the phosphorylating system associated with IF proteins without altering the immunocontent of these proteins. Concerning the selenium compounds, diselenide (1, 15, and 50 microM) did not induce alteration of the in vitro phosphorylation of IF proteins. Conversely, 15 microM diselenide was effective in preventing the toxic effects induced by methylmercury. Otherwise, ebselen induced an altered in vitro phosphorylation of the cytoskeletal proteins in a dose-dependent manner. Ebselen at intermediate concentrations (15 and 30 microM) increased the in vitro phosphorylation. However, at low (5 microM) or high (50 and 100 microM) concentrations it was ineffective in altering the cytoskeletal-associated phosphorylating system. Furthermore, 5 microM ebselen presented a protective effect against the action of methylmercury on the phosphorylating system. In conclusion, our results indicate that the selenium compounds ebselen and diselenide present protective actions toward the alterations of the phosphorylating system associated with the IF proteins induced by methylmercury in slices of the cerebral cortex of rats.

Organoselenium compounds prevent hyperphosphorylation of cytoskeletal proteins induced by the neurotoxic agent diphenyl ditelluride in cerebral cortex of young rats.

In this work we investigated the protective ability of the selenium compounds ebselen and diphenyl diselenide against the effect of diphenyl ditelluride on the in vitro incorporation of 32P into intermediate filament (IF) proteins from slices of cerebral cortex of 17-day-old rats. We observed that ditelluride in the concentrations of 1, 15 and 50 microM induced hyperphosphorylation of the high-salt Triton insoluble neurofilament subunits (NF-M and NF-L), glial fibrillary acidic protein (GFAP) and vimentin, without altering the immunocontent of these proteins. Concerning the selenium compounds, diselenide (1,15 and 50 microM) did not induce alteration of the in vitro phosphorylation of the IF proteins. Otherwise, ebselen induced an altered in vitro phosphorylation of the cytoskeletal proteins in a dose-dependent manner. At intermediate concentrations (15 and 30 microM) it increased the in vitro phosphorylation even though, at low (5 microM) or high (50 and 100 microM) concentrations this compound was ineffective in altering the activity of the cytoskeletal-associated phosphorylating system. In addition, 15 microM diselenide and 5 microM ebselen, presented a protective effect against the action of ditelluride, on the phosphorylation of the proteins studied. Considering that hyperphosphorylation of cytoskeletal proteins is associated with neuronal dysfunction and neurodegeneration, it is probable that the effects of ditelluride could be related to the remarkable neurotoxicity of this organic form of tellurium. Furthermore the neuroprotective action of selenium compounds against tellurium effects could be a promising route to be exploited for a possible treatment of organic tellurium poisoning.

Propionic and methylmalonic acids inhibit the in vitro phosphorylation of a 85 kDa cytoskeletal protein from cerebral cortex of rats.

In this study we examine the action of methylmalonic (MMA) and propionic (PA) acids, metabolites which accumulate in methylmalonic and propionic acidemias respectively, on the endogenous phosphorylating system associated with the cytoskeletal fraction of cerebral cortex of young rats. Chronic treatment with PA and treatment of tissue slices with MMA or PA are effective in decreasing the in vitro phosphorylation into a 85 kDa cytoskeletal associated protein. We tested the effect of the acids on the endogenous kinase activities by using specific kinase activators and inhibitors. Results demonstrated that the acids interfere with the endogenous cAMP-dependent and Ca2+/calmodulin-dependent kinase activities. Furthermore, in vitro dephosphorylation of the 85 kDa protein was totally inhibited in brain slices treated with the acids. Considering the importance of protein phosphorylation to cellular function, we speculate that alteration in the phosphorylating level of cytoskeletal associated phosphoproteins induced by MMA and PA treatments may somehow be involved in steps leading to brain damage.

Effects of acute and chronic administration of methylmalonic and propionic acids on the in vitro incorporation of 32P into cytoskeletal proteins from cerebral cortex of young rats.

We studied the effects of acute and chronic administration of methylmalonic (MMA) and propionic (PA) acids on the in vitro incorporation of 32P into neurofilament subunits (NF-M and NF-L), alpha and beta tubulins, from cerebral cortex of rats. In the chronic treatment, drugs were administered subcutaneously from day 6-17 post-partum (MMA 0.76-0.89 micromol/g body weight and PA 0.93 micromol/g body weight). In the acute treatment MMA and PA were injected (MMA 3.78 micromol/g body weight and PA 3.90 micromol/g body weight). Control animals received saline in the same volumes. The Triton-insoluble cytoskeletal fraction of control in treated animals was isolated and incubated with 32P-ATP. Our results demonstrate that both drugs were able to inhibit 32P in vitro incorporation into neurofilaments and tubulins. The acute administration of MMA decreased the in vitro 32P incorporation into NF-L and alpha-tubulin subunit, whereas PA administration decreased the 32P in vitro incorporation into NF-M, NF-L, and tubulins. On the other hand, chronic MMA administration induced a decreased 32P in vitro incorporation into NF-M, while chronic treatment with propionate decreased the in vitro phosphorylation of NF-M and alpha-tubulin. This study provides consistent evidence that a decreased phosphorylation of cytoskeletal proteins is induced by MMA and PA metabolites which accumulate in methylmalonic and propionic acidemias respectively. Therefore, it is possible that an altered brain cytoskeletal metabolism could be related with the structural alterations of CNS observed in these disorders.

Malnutrition increases insoluble-to-soluble tubulin ratio and in vitro incorporation of 32ATP in rat cerebral cortex.

Wistar rats were fed a normal protein (25% casein) or an isoenergetic low protein (8% casein) diet from the day of birth to weaning on day 21. Litters were killed at weaning and cerebral cortex was removed. Tubulin was prepared by centrifugation at 100,000 g, 4 degrees C, as described by Shelansky et al. [Proc. Natn. Acad. Sci. U.S.A. 70, 765-768 (1973)]. Cold-insoluble tubulin was recovered in the pellet (P1) fraction and cold-soluble tubulin in the supernatant (S1) fraction. Alpha and beta tubulin were quantified by electrophoretic and immunological methods in both fractions. Our results indicated that malnutrition enhanced the ratio of cold-insoluble-tubulin-to-cold-soluble-tubulin. Furthermore malnutrition induced an increased in vitro incorporation of 32P into both soluble and insoluble tubulins. Although tubulin phosphorylation has been related to tubulin stability properties, we cannot unequivocally ascribe the increased insoluble/soluble tubulin ratio with malnutrition to increased in vitro incorporation of 32P.

Effect of phenylalanine and alpha-methylphenylalanine on in vitro incorporation of 32P into cytoskeletal cerebral proteins.

We studied the effects of L-phenylalanine and alpha-methylphenylalanine on 32P in vitro incorporation into cytoskeletal proteins from cerebral cortex of 17-day-old rats. Slices of cerebral cortex were incubated in the absence or presence of increasing concentrations of L-phenylalanine, alpha-methylphenylalanine or L-phenylalanine plus alpha-methylphenylalanine for 1 h. The cytoskeletal fraction obtained from slices was incubated in the presence of the same drugs and the 32P in vitro incorporation into cytoskeletal proteins was measured. Addition of alpha-methylphenylalanine did not change 32P in vitro incorporation into the cytoskeletal proteins, but phenylalanine decreased the in vitro phosphorylation of beta tubulin. Furthermore, addition of L-phenylalanine plus alpha-methylphenylalanine decreased the in vitro phosphorylation of both 160 kDa neurofilaments and alpha-tubulin.

Alpha-ketoisocaproate increases the in vitro 32P incorporation into intermediate filaments in cerebral cortex of rats.

In this study we investigated the effects of alpha-ketoisocaproic (KIC), alpha-ketoisovaleric (KIV) and alpha-keto-beta-methylvaleric (KMV) acids on the phosphorylation of intermediate filament (IF) proteins of cerebral cortex of rats. Tissue slices were incubated with [32P] orthophosphate in the presence or absence of the acids. The intermediate filament enriched cytoskeletal fraction was isolated and the radioactivity incorporated into neurofilament subunits, vimentin and glial fibrillary acidic protein was measured. Results demonstrated that KIC significantly increased phosphorylation of these proteins whereas the other acids had no effect. Experiments using protein kinase inhibitors indicated that the effect of KIC was mediated by Ca2+/calmodulin- and cAMP-dependent protein kinases. This study provides evidence that KIC, a key metabolite accumulating in maple syrup urine disease, increases phosphorylation of IF proteins.

Methylmalonic and propionic acids increase the in vitro incorporation of 32P into cytoskeletal proteins from cerebral cortex of young rats through NMDA glutamate receptors.

In this study we investigated the effects of methylmalonic acid (MMA) and propionic acid (PA) on the phosphorylation of cytoskeletal proteins of cerebral cortex of rats. Slices of tissue were incubated with 32P-orthophosphate in the presence or absence of glutamate, MMA, PA and ionotropic or metabotropic glutamate receptor agonists. The cytoskeletal fraction was isolated and the radioactivity incorporated into the cytoskeletal proteins was measured. Results demonstrated that the acids, glutamate and NMDA increased the phosphorylation of the proteins studied. However, this effect was not observed for non-NMDA ionotropic agonists or metabotropic agonists. Experiments using glutamate receptor antagonists confirmed that MMA and PA at the same concentrations as found in tissues from propionic or methylmalonic acidemic children increase the phosphorylation of cytoskeletal proteins, possibly via NMDA glutamate receptors. Therefore, it is feasible that these findings may be related to the neurological dysfunction characteristic of these disorders.

Methylmalonic acid reduces the in vitro phosphorylation of cytoskeletal proteins in the cerebral cortex of rats.

The present work was undertaken to determine the action of methylmalonic acid (MMA), a metabolite, which accumulates in high amounts in methylmalonic acidemia, on the endogenous phosphorylating system associated with the cytoskeletal fraction proteins of cerebral cortex of young rats. We demonstrated that pre-treatment of cerebral cortex slices of young rats with 2.5 mM buffered methylmalonic acid (MMA) is effective in decreasing in vitro incorporation of [32P]ATP into neurofilament subunits (NF-M and NF-L) and alpha- and beta-tubulins. Based on the fact that this system contains cAMP-dependent protein kinase (PKA), Ca2+/calmodulin-dependent protein kinase II (CaMKII) and protein phosphatase 1 (PP1), we first tested the effect of MMA on the kinase activities by using the specific activators cAMP and Ca2+/calmodulin or the inhibitors PKAI or KN-93 for PKA and CaMKII, respectively. We observed that MMA totally inhibited the stimulatory effect of cAMP and interfered with the inhibitory effect of PKAI. In addition, the metabolite partially prevented the stimulatory effect of Ca2+/calmodulin and interfered with the effect of KN-93. Furthermore, in vitro dephosphorylation of neurofilament subunits and tubulins was totally inhibited in brain slices pre-treated with MMA. Taken together, these results suggest that MMA, at the same concentrations found in tissues of methylmalonic acidemic children, inhibits the in vitro activities of PKA, CaMKII and PP1 associated with the cytoskeletal fraction of the cerebral cortex of rats, a fact that may be involved with the pathogenesis of the neurological dysfunction characteristic of methylmalonic acidemia.

The effects of behavioral tasks on the in vitro phosphorylation of intermediate filament subunits of rat hippocampus are mediated by CaMKII and PKA.

Neurofilaments (NF) are the most abundant constituents of the neuronal cytoskeleton, while glial fibrillary acidic protein (GFAP) is a major component of the glial astrocyte cytoskeleton. These proteins can be phosphorylated by different protein kinases and they are regulated in a complex way by phosphorylation. Using a hippocampal cytoskeletal fraction we demonstrated that the behavioral tasks of inhibitory avoidance and habituation can differently alter the in vitro phosphorylation of the 150 kDa (NF-M) and the 68 kDa (NF-L) neurofilament subunits and of the GFAP. In order to verify the effect of habituation and inhibitory avoidance training on the phosphatase activity, we performed the time course-dephosphorylation assay (5-30 min of incubation of the cytoskeletal fraction with 32P-ATP). Subsequently we investigated the effect of these behavioral tasks on the protein kinase activities associated with the cytoskeletal fraction, carring out the 32P incorporation assays in the presence of specific kinase inhibitors. Results suggest that phosphatase activity is not altered in the cytoskeletal fraction by the behavioral tasks and that the increased in vitro phosphorylation of NF-M and NF-L caused by habituation is probably mediated by the Ca2+/calmodulin dependent protein kinase (CaMKII). However, the inhibition of GFAP in vitro phosphorylation caused by inhibitory avoidance training is probably related to the cAMP dependent protein kinase (PKA).

Developmentally regulated in vitro phosphorylation of a 85 kDa triton-insoluble protein of the cerebral cortex of rats.

We studied the ontogeny of concentration and in vitro phosphorylation of an 85 kDa Triton-insoluble protein from cerebral cortex of 7, 15, 21 and 90 day old rats. The Triton-insoluble cytoskeletal fraction contains an 85 kDa basic phosphoprotein different from synapsin 1, as determined by nonequilibrium pH gradient electrophoresis and phosphopeptide mapping with V8 protease. The concentration of the 85 kDa cytoskeletal associated phosphoprotein was analyzed during development. Results indicated that the concentration of this protein oscillated during suckling, presenting a maximal value at day 15 and decreasing again to stabilize at values near those of 7 day old rats, remaining constant in 21 and 90 day old animals. However, in vitro 32P incorporation, expressed as cpm/microgram, presented a developmentally regulated pattern, with maximal values in young rats, declining with age to negligible values in 90 day old animals. The endogenous phosphorylating system responsible for in vitro 32P incorporation into the 85 kDa protein was determined by the addition of specific activators of second-messenger protein kinases (cAMP, Ca2+/ calmodulin and Ca2+/phosphatidylserine/phorbol ester) and a protein phosphatase inhibitor (okadaic acid) to the incubation system. Results suggested that the in vitro phosphorylation system is composed of protein kinase A, Ca2+/calmodulin dependent protein kinase and protein phosphatase 1.

Ca(2+)-mediated phosphorylation and proteolysis activity associated with the cytoskeletal fraction from cerebral cortex of rats.

We describe a Triton-insoluble cytoskeletal fraction extracted from cerebral cortex of young rats retaining an endogenous Ca(2+)-mediated mechanism acting in vitro on Ca2+/calmodulin-dependent protein kinase II (CaM-KII) activity and on phosphorylation and proteolysis of the 150 kDa neurofilament subunit (NF-M), alpha and beta tubulin. Exogenous Ca2+ induced a 70% decrease in the in vitro phosphorylation of the NF-M and tubulins and a 30-50% decrease in the total amount of these proteins. However, when calpastatin was added basal phosphorylation and NF-M and tubulin content were recovered. Furthermore, exogenous Ca2+/calmodulin induced increased in vitro phosphorylation of the cytoskeletal proteins and CaM-KII activity only in the presence of calpastatin, suggesting the presence of Ca(2+)-induced calpain-mediated proteolysis. This fraction could be an interesting model to further studies concerning the in vitro effects of Ca(2+)-mediated protein kinases and proteases associated with the cytoskeletal fraction.

Developmentally regulated in vitro phosphorylation of cytoskeletal proteins of the cerebral cortex of normal and malnourished rats.

In this investigation we studied developmentally regulated endogenous protein kinase activity in cytoskeletal proteins in the cerebral cortex of rats and the effect of early malnutrition imposed on dams on the pattern of 32P incorporation into the cytoskeleton of pups. Our results indicated that in vitro incorporation was maximum in 7-day-old pups for both normal and malnourished groups, decreasing with development, and reaching minimum values in adult animals. However, 32P incorporation into NF-M and tubulin was significantly lower in 7-day-old malnourished pups than in normal pups.

Effect of hyperphenylalaninemia chemically induced on in vitro incorporation of 32P into cytoskeletal proteins from cerebral cortex of developing rats.

We studied the effect of hyperphenylalaninemia on in vitro incorporation of 32P into cytoskeletal proteins from cerebral cortex of rats by injecting l-phenylalanine plus alpha-methylphenylalanine subcutaneously from the 6th to the 14th day postpartum. Chronic hyperphenylalaninemia induced an increased in vitro phosphorylation of the 150-kDa neurofilament subunit and tubulins present in the cytoskeletal fraction at the end of the treatment and 3 days after treatment discontinuation. In addition, when in vitro phosphorylation of the cytoskeletal proteins from treated animals was performed in the presence of the drugs we observed a decreased in vitro incorporation of 32P into these proteins. Thus, the effect of l-phenylalanine plus alpha-methylphenylalanine on the endogenous protein kinase and phosphatase activities was examined and the results demonstrated that these drugs have an inhibitory effect on calcium/calmodulin-dependent protein kinase II and protein phosphatase type 1.

Cytoskeletal-associated protein kinase and phosphatase activities from cerebral cortex of young rats.

We describe the phosphorylation system associated with the Triton-insoluble cytoskeletal fraction that phosphorylates in vitro the 150 kDa neurofilament subunit (NF-M) and alpha and beta tubulin from cerebral cortex of rats. The protein kinase activities were determined in the presence of 20 microM cyclic AMP (cAMP), 1 mM calcium and 1 microM calmodulin (Ca2+/calmodulin) or 1 mM calcium, 0.2 mM phosphatidylserine and 0.5 microM phorbol 12,13-dibutyrate (Ca2+/PS/PDBu). Phosphorylation of these cytoskeletal proteins increased approximately 35% and 65% in the presence of cAMP and Ca2+/calmodulin, respectively, but was unaffected in the presence of Ca2+/PS/PDBu. Basal phosphorylation of these proteins studied increased approximately 35% and 72% in the presence of 0.5 microM okadaic acid and 0.01 microM microcystin-LR, respectively, suggesting the presence of phosphatase type 1. Results suggest that at least two protein kinases and one protein phosphatase are associated with the Triton-insoluble cytoskeletal fraction from cerebral cortex of rats.

Effect of the branched-chain alpha-ketoacids accumulating in maple syrup urine disease on the high molecular weight neurofilament subunit (NF-H) in rat cerebral cortex.

In this study we investigated the effects of the branched chain alpha-ketoacids accumulating in maple syrup urine disease (MSUD) on the concentrations of the high molecular weight neurofilament subunit (NF-H) associated with the cytoskeletal fraction of the cerebral cortex of 12-day-old rats. Cortical slices were incubated with alpha-ketoisocaproic acid (KIC), alpha-keto beta-methylvaleric acid (KMV) and alpha-ketoisovaleric acid (KIV) at concentrations ranging from 0.5 to 1.0 mM. The cytoskeletal fraction was extracted and the immunoreactivity for phosphorylated and total NF-H was analyzed by immunoblotting. The in vitro 32P incorporation into NF-H was also determined. Results showed that treatment of tissue slices induced with KMV increased Triton-insoluble phosphorylated NF-H immunoreactivity, with no alteration in total NF-H immunoreactivity. Furthermore, KIC treatment drastically increased the total amount of NF-H, whereas KIV did not change either phosphorylated or total NF-H immunoreactivity. KMV also increased the in vitro 32P incorporation into NF-H, confirming the highly phosphorylated NF-H levels detected in the immunoblot. These findings demonstrate that KIC and KMV alter the dynamic regulation of NF-H assembly in the cytoskeletal fraction. Therefore we may suggest that cytoskeletal disorganization may be one of the factors associated with the neurodegeneration characteristic of MSUD disease.

Phenylalanine inhibition of the phosphorylation of cytoskeletal proteins from cerebral cortex of young rats is prevented by alanine.

BACKGROUND: Phenylalanine has been considered the main responsible agent for the brain damage that occurs in phenylketonuria. METHODS AND RESULTS: In this work we studied the effect of this amino acid on the in vitro phosphorylation of cytoskeletal proteins of the cerebral cortex of rats. We observed that 2 mM phenylalanine, a concentration usually found in the plasma of phenylketonuric patients, decreased the in vitro 32P incorporation into these proteins. In addition, we investigated the effect of alanine on the inhibition of 32P incorporation into cytoskeletal proteins caused by phenylalanine. We observed that 0.5 mM alanine did not alter 32P incorporation but prevented the inhibition provoked by phenylalanine. CONCLUSION: In case the inhibition of cytoskeletal protein phosphorylation by phenylalanine also occurs in human phenylketonuria, it is possible that alanine supplementation to the phenylalanine-restricted diet may be beneficial to these patients.