Regina Pessoa Pureur

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.

Reduction of glutamate uptake into cerebral cortex of developing rats by the branched-chain alpha-keto acids accumulating in maple syrup urine disease

In the current study we investigated the effect of the branched-chain alpha-keto acids (BCKA) α-ketoisocaproic (KIC), α-keto-β-methylvaleric (KMV), and α-ketoisovaleric (KIV) acids, which accumulate in maple syrup urine disease (MSUD), on the in vitro uptake of [3H]glutamate by cere-bral cortical slices from rats aged 9, 21, and 60 days of life. We initially observed that glutamate uptake into cerebral cortex of 9- and 21-day-old rats was significantly higher, as compared to that of 60-day-old rats. Furthermore, KIC inhibited this uptake by tissue slices at all ages studied, whereas KMV and KIV produced the same effect only in cortical slices of 21- and 60-day-old rats. Kinetic assays showed that KIC significantly inhibited glutamate uptake in the presence of high glutamate concentrations (50 μM and greater). We also verified that the reduction of glutamate uptake was not due to cellular death, as evidenced by tetrazolium salt and lactate dehydrogenase viability tests of cortical slices in the presence of the BCKA. It is therefore presumed that the reduced glutamate uptake caused by the BCKA accumulating in MSUD may lead to higher extracellular glutamate levels and potentially to excitotoxicity, which may contribute to the neurological dysfunction of the affected individuals.

Diminished concentration of the NF-H subunit of neurofilaments in cerebral cortex of rats chronically treated with proline, methylmalonate and phenylalanine plus α-methylphenylalanine

SummaryWistar rats from the same litter were randomly divided into four groups and received subcutaneously from the 6th to 28th daypost partum one of the following drugs:l-proline, methylmalonate,l-phenylalanine plus α-methylphenylalanine, or equivalent volumes of 0.9% (w/v) saline (controls). On day 30, the animals were killed, the brain was removed and the cerebral cortex and cerebellum was immediately dissected.Total intermediate filament fraction (IF) was obtained from cerebral cortex and cerebellum by using a high-salt phosphate-buffered solution supplemented by 1% Triton X-100. The pellet contained the bulk of the IF proteins. Following SDS-polyacrylamide gel electrophoresis, these proteins were identified as the 200, 150 and 68 kD subunits of neurofilaments (NF-H, NF-M and NF-L, respectively), the 66 kDa associated protein, the 57 kDa intermediate filament-like protein and the 52 kDa glial fibrillary acidic protein (GFAP). They were further scanned through densitometry from enriched fractions of controls and of animals treated with the various drugs in order to determine the effects of the treatments on their concentration. Our results showed that the concentration of IF protein in cerebellum was not affected by the treatments, whereas chronic administration of all drugs significantly decreased NF-H subunit concentration in rat cerebral cortex.

Delayed neurochemical effects of prenatal exposure to MeHg in the cerebellum of developing rats

Human fetuses and neonates are particularly vulnerable to methylmercury (MeHg)-induced brain damage and are sensitive even to low exposure levels. Previous work of our group evidence that prenatal exposure to MeHg causes cognitive and behavioral alterations and disrupt hippocampus signaling. The current study aimed to investigate the effect of gestational exposure of rats to MeHg at low doses (1 or 2 mg/kg) on parameters of redox imbalance and key signaling pathways in the cerebellum of their offspring. Pregnant females received MeHg (treated group) or 0.9% saline water (control group) by gavage in alternated days from gestational day 5 (GD5) until parturition and analyzes were proceed in the cerebellum of 30-day-old pups. We found increased lipid peroxidation and protein carbonylation levels as well as decreased SH content in pups prenatally exposed to 2 mg/kg MeHg. In addition, misregulated SOD/catalase activities supported imbalanced redox equilibrium. We found decreased GSK3β(Ser9) phosphorylation, suggesting activation of this enzyme and dephosphorylation/inhibition of ERK1/2 and JNK pathways. Increased PKAα catalytic subunit could be upstream of hyperphosphorylated c-Raf(Ser259) and downregulated MAPK pathway. In addition, we found raised levels of the Ca2+-dependent protein phosphatase 2 B (PP2B). We also found preserved immunohistochemical staining for both glial fibrillary acidic protein (GFAP) and NeuN in MeHg-exposed pups. Western blot analysis showed unaltered levels of BAX/BCL-XL, BAD/BCL-2 and active caspase 3. Together, these findings support absence of reactive astrocytes, neuronal damage and apoptotic cell death in the cerebellum of MeHg treated pups. The present study provides evidence that prenatal exposure to MeHg leads to later redox imbalance and disrupted signaling mechanisms in the cerebellum of 30-day-old pups potentially predisposing them to long-lasting neurological impairments in CNS.