Renata Bezerra Meirelles

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.

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

In this study we investigated the effects of α-ketoisocaproic (KIC), α-ketoisovaleric (KIV) and α-keto-β-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.

Effects of neonatal cerebral hypoxia-ischemia on the in vitro phosphorylation of synapsin 1 in rat synaptosomes.

Synapsins are phosphoproteins related to the anchorage of synaptic vesicles to the actin skeleton. Hypoxia-ischemia causes an increased calcium influx into neurons through ionic channels gated by activation of glutamate receptors. In this work seven-day-old Wistar rats were submitted to hypoxia-ischemia and sacrificed after 21 hours, 7, 30, or 90 days. Synaptosomal fractions were obtained by Percoll gradients and incubated with 32P (10μCi/g). Proteins were analysed by SDS-PAGE and radioactivity incorporated into synapsin 1 was counted by liquid scintillation. Twenty-one hours after hypoxia-ischemia we observed a reduction on the in vitro phosphorylation of synapsin 1, mainly due to hypoxia, rather than to ischemia; this effect was reversed at day 7 after the insult. There was another decrease in phosphorylation 30 days after the event interpreted as a late effect of hypoxia-ischemia. No changes were observed at day 90. Our results suggest that decreased phosphorylation of synapsin 1 could be related to neuronal death that follows hypoxia-ischemia.