Acute Hyperammonemia Induces NMDA-Mediated Hypophosphorylation of Intermediate Filaments Through PP1 and PP2B in Cerebral Cortex of Young Rats.

In the present work, we studied the effects of toxic ammonia levels on the cytoskeleton of neural cells, with emphasis in the homeostasis of the phosphorylating system associated with the intermediate filaments (IFs). We used in vivo and in vitro models of acute hyperammonemia in 10- and 21-day-old rats. In the in vivo model, animals were intraperitoneally injected with ammonium acetate (7 mmol/Kg), and the phosphorylation level of the cytoskeletal proteins was analyzed in the cerebral cortex and hippocampus 30 and 60 min after injection. The injected ammonia altered the IF phosphorylation of astrocytes (GFAP and vimentin) and neurons (neurofilament subunits of low, middle, and high molecular weight, respectively: NFL, NFM, and NFH) from cerebral cortex of 21-day-old rats. This was a transitory effect observed 30 min after injection, recovering 30 min afterward. Phosphorylation was not altered in the cerebral cortex of 10-day-old pups. The homeostasis of hippocampal IFs was preserved at the studied ages and times. In the in vitro model, cortical slices of 10- and 21-day-old rats were incubated with 0.5, 1, or 5 mM NH4Cl, and the phosphorylation level of the IF proteins was analyzed after 30 min. The IF phosphorylation was not altered in cortical slices of 10-day-old rats; however, in cortical slices of 21-day-old pups, 5 mM NH4Cl induced hypophosphorylation of GFAP and vimentin, preserving neurofilament phosphorylation levels. Hypophosphorylation was mediated by the protein phosphatases 1 (PP1) and 2B (PP2B), and this event was associated with Ca(2+) influx via N-methyl-D-aspartate (NMDA) glutamate receptors. The aim of this study is to show that acute ammonia toxicity targets the phosphorylating system of IFs in the cerebral cortex of rats in a developmentally regulated manner, and NMDA-mediated Ca(2+) signaling plays a central role in this mechanism. We propose that the disruption of cytoskeletal homeostasis could be an endpoint of the acute hyperammonemia in the developing brain. We believe that these results contribute for better understanding the molecular basis of the ammonia toxicity in brain.

High postnatal susceptibility of hippocampal cytoskeleton in response to ethanol exposure during pregnancy and lactation.

Ethanol exposure to offspring during pregnancy and lactation leads to developmental disorders, including central nervous system dysfunction. In the present work, we have studied the effect of chronic ethanol exposure during pregnancy and lactation on the phosphorylating system associated with the astrocytic and neuronal intermediate filament (IF) proteins: glial fibrillary acidic protein (GFAP), and neurofilament (NF) subunits of low, medium, and high molecular weight (NFL, NFM, and NFH, respectively) in 9- and 21-day-old pups. Female rats were fed with 20% ethanol in their drinking water during pregnancy and lactation. The homeostasis of the IF phosphorylation was not altered in the cerebral cortex, cerebellum, or hippocampus of 9-day-old pups. However, GFAP, NFL, and NFM were hyperphosphorylated in the hippocampus of 21-day-old pups. PKA had been activated in the hippocampus, and Ser55 in the N-terminal region of NFL was hyperphosphorylated. In addition, JNK/MAPK was activated and KSP repeats in the C-terminal region of NFM were hyperphosphorylated in the hippocampus of 21-day-old pups. Decreased NFH immunocontent but an unaltered total NFH/phosphoNFH ratio suggested altered stoichiometry of NFs in the hippocampus of ethanol-exposed 21-day-old pups. In contrast to the high susceptibility of hippocampal cytoskeleton in developing rats, the homeostasis of the cytoskeleton of ethanol-fed adult females was not altered. Disruption of the cytoskeletal homeostasis in neural cells supports the view that regions of the brain are differentially vulnerable to alcohol insult during pregnancy and lactation, suggesting that modulation of JNK/MAPK and PKA signaling cascades target the hippocampal cytoskeleton in a window of vulnerability in 21-day-old pups. Our findings are relevant, since disruption of the cytoskeleton in immature hippocampus could contribute to later hippocampal damage associated with ethanol toxicity.