Priscila de Lima Pelaez

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.

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.

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.

α-Keto-β-methylvaleric acid increases the in vitro phosphorylation of intermediate filaments in cerebral cortex of young rats through the gabaergic system

Abstract In this study we investigated the effects of α-ketoisovaleric (KIV) and α-keto-β-methylvaleric acids (KMV), metabolites 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 young rats during development (9–21 days of age) We observed that KMV significantly increased the in vitro incorporation of 32P into the IF proteins studied in cortical slices of 12-day-old rats through the PKA and PKCaMII, with no alteration at the other ages. In contrast, KIV was ineffective in altering the phosphorylating system associated with IF proteins at all ages examined. A similar effect on IF phosphorylation was achieved by incubating cortical slices with γ-aminobutiric acid (GABA). Furthermore, by using specific GABA antagonists, we verified that KMV induced a stimulatory effect on IF phosphorylation of tissue slices from 12-day-old rats mediated by GABAA and GABAB receptors. In conclusion, our results indicate the involvement of the GABAergic system in the alterations of IF phosphorylation caused by KMV, one of the branched-chain keto acids accumulating in MSUD.

Hyperhomocysteinemia selectively alters expression and stoichiometry of intermediate filament and induces glutamate- and calcium-mediated mechanisms in rat brain during development

The aim of the present work was to investigate the actions of a chemically induced chronic hyperhomocysteinemia model on intermediate filaments (IFs) of cortical and hippocampal neural cells and explore signaling mechanisms underlying such effects. Results showed that in hyperhomocysteinemic rats the expression of neural IF subunits was affected. In cerebral cortex, glial fibrillary acidic protein (GFAP) expression was donwregulated while in hippocampus high and middle molecular weight neurofilament subunits (NF‐H and NF‐M, respectively) were up‐regulated. Otherwise, the immunocontent of IF proteins was unaltered in cerebral cortex while in hippocampus the immunocontent of cytoskeletal‐associated low molecular weight neurofilament (NF‐L) and NF‐H subunits suggested a stoichiometric ratio consistent with a decreased amount of core filaments enriched in lateral projections. These effects were not accompanied by an alteration in IF phosphorylation. In vitro results showed that 500 μM Hcy‐induced protein phosphatases 1‐, 2A‐ and 2B‐mediated hypophosphorylation of NF subunits and GFAP in hippocampal slices of 17‐day‐old rats without affecting the cerebral cortex, showing a window of vulnerability of cytoskeleton in developing hippocampus. Ionotropic and metabotropic glutamate receptors were involved in this action, as well as Ca2+ release from intracellular stores through ryanodine receptors. We propose that the mechanisms observed in the hippocampus of 17‐day‐old rats could support the neural damage observed in these animals.

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 Nω‐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.

Cytoskeleton of Human Mononuclear Cells as a Possible Peripheral Marker for Phenylalanine Neurotoxicity in PKU

In this work we tested human mononuclear cells as a peripheral marker to study neurotoxicity of phenylalanine (Phe). Slices of cerebral cortex of rats or human mononuclear cells were incubated with different concentrations of Phe and/or Ala in the presence of 32P-orthophosphate, the cytoskeletal fraction was extracted, and the radioactivity incorporated into intermediate filament proteins was measured. Our results show that 2 mM Phe as well as 1 mM Ala are effective in increasing the 32P in vitro incorporation into IFs in both tissues. When cerebral cortex slices or mononuclear cells were incubated with different concentrations of Phe and/or Ala, the effects on the 32P in vitro incorporation into IF proteins was compatible with an antagonistic mechanism of action of the two amino acids on the enzymes of the phosphorylating system. In addition, these blood cells may be a possible peripheral marker to study neurotoxicity of Phe in patients with PKU.