Carvalho Rônan Vivian, da Silva Ferreira Fernanda, Heimfarth Luana, Pierozan Paula, Fernandes Carolina, Pessoa-Pureur Regina
Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Rua Ramiro Barcelos 2600 anexo, Porto Alegre, RS, CEP 90035-003, Brazil.
Neurotox Res. 2016 Aug;30(2):138-49. doi: 10.1007/s12640-016-9607-7. Epub 2016 Mar 2.
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.
在本研究中，我们研究了有毒氨水平对神经细胞细胞骨架的影响，重点关注与中间丝（IFs）相关的磷酸化系统的稳态。我们使用了10日龄和21日龄大鼠急性高氨血症的体内和体外模型。在体内模型中，给动物腹腔注射醋酸铵（7 mmol/Kg），并在注射后30分钟和60分钟分析大脑皮层和海马中细胞骨架蛋白的磷酸化水平。注射的氨改变了21日龄大鼠大脑皮层中星形胶质细胞（胶质纤维酸性蛋白和波形蛋白）和神经元（分别为低、中、高分子量的神经丝亚基：神经丝轻链、神经丝中链和神经丝重链）的IF磷酸化。这是注射后30分钟观察到的一种短暂效应，之后30分钟恢复。10日龄幼崽的大脑皮层中磷酸化未改变。在所研究的年龄和时间，海马IFs的稳态得以维持。在体外模型中，将10日龄和21日龄大鼠的皮层切片与0.5、1或5 mM氯化铵孵育，并在30分钟后分析IF蛋白的磷酸化水平。10日龄大鼠的皮层切片中IF磷酸化未改变；然而，在21日龄幼崽的皮层切片中，5 mM氯化铵诱导胶质纤维酸性蛋白和波形蛋白的低磷酸化，同时维持神经丝磷酸化水平。低磷酸化由蛋白磷酸酶1（PP1）和2B（PP2B）介导，并且该事件与通过N-甲基-D-天冬氨酸（NMDA）谷氨酸受体的Ca(2+)内流有关。本研究的目的是表明急性氨毒性以发育调节的方式靶向大鼠大脑皮层中IFs的磷酸化系统，并且NMDA介导的Ca(2+)信号在该机制中起核心作用。我们提出细胞骨架稳态的破坏可能是发育中大脑急性高氨血症的一个终点。我们相信这些结果有助于更好地理解大脑中氨毒性的分子基础。
