Section of Biología y Patología Celular, Centro Investigación, Hospital La Fe, E-46009 Valencia, Spain.
Toxicol Sci. 2010 Dec;118(2):602-12. doi: 10.1093/toxsci/kfq260. Epub 2010 Sep 9.
The organization and dynamics of microtubules (MTs) and the actin cytoskeleton are critical for the correct development and functions of neurons, including intracellular traffic and signaling. In vitro ethanol exposure impairs endocytosis, exocytosis, and nucleocytoplasmic traffic in astrocytes and alters endocytosis in cultured neurons. In astrocytes, these effects relate to changes in the organization and/or function of MTs and the actin cytoskeleton. To evaluate this possibility in hippocampal cultured neurons, we analyzed if chronic ethanol exposure affects the levels, assembly, and cellular organization of both cytoskeleton elements and the possible underlying mechanisms of these effects by morphological and biochemical methods. In the experiments described below, we provide the first evidence that chronic alcohol exposure decreases the amount of both filamentous actin and polymerized tubulin in neurons and that the number of MTs in dendrites lowers in treated cells. Alcohol also diminishes the MT-associated protein-2 levels, which mainly localizes in the somatodendritic compartment in neurons. Ethanol decreases the levels of total Rac, Cdc42, and RhoA, three small guanosine triphosphatases (GTPases) involved in the organization and dynamics of the actin cytoskeleton and MTs. Yet when alcohol decreases the levels of the active forms (GTP bound) of Rac1 and Cdc42, it does not affect the active form of RhoA. We also investigated the levels of several effector and regulator molecules of these GTPases to find that alcohol induces heterogeneous results. In conclusion, our results show that MT, actin cytoskeleton organization, and Rho GTPase signaling pathways are targets for the toxic effects of ethanol in neurons.
微管 (MTs) 和肌动蛋白细胞骨架的组织和动态对于神经元的正确发育和功能至关重要，包括细胞内运输和信号转导。体外乙醇暴露会损害星形胶质细胞的内吞作用、胞吐作用和核质运输，并改变培养神经元中的内吞作用。在星形胶质细胞中，这些效应与 MTs 和肌动蛋白细胞骨架的组织和/或功能的变化有关。为了在海马培养神经元中评估这种可能性，我们通过形态学和生化方法分析了慢性乙醇暴露是否会影响细胞骨架元素的水平、组装和细胞组织，以及这些效应的潜在机制。在下面描述的实验中，我们提供了第一个证据，表明慢性酒精暴露会降低神经元中丝状肌动蛋白和聚合微管蛋白的含量，并且处理细胞中的树突中的 MT 数量降低。酒精还会降低微管相关蛋白-2 的水平，该蛋白主要定位于神经元的体树突区。乙醇降低了总 Rac、Cdc42 和 RhoA 的水平，这三种小分子鸟苷三磷酸酶 (GTPase) 参与肌动蛋白细胞骨架和 MT 的组织和动态。然而，当酒精降低 Rac1 和 Cdc42 的活性形式 (GTP 结合) 的水平时，它不会影响 RhoA 的活性形式。我们还研究了这些 GTPase 的几个效应物和调节剂分子的水平，发现酒精诱导了异质结果。总之，我们的结果表明，MT、肌动蛋白细胞骨架组织和 Rho GTPase 信号通路是神经元中乙醇毒性作用的靶点。
