Center for Neuropharmacology & Neuroscience, Albany Medical College, 47 New Scotland Ave., Albany, NY 12208, USA.
Alcohol Clin Exp Res. 2011 Jul;35(7):1321-30. doi: 10.1111/j.1530-0277.2011.01468.x.
The effects of ethanol on development of postmitotic neurons include altered neurite outgrowth and differentiation, which may contribute to neuropathology associated with fetal alcohol spectrum disorders. We previously reported that ethanol exposure alters axon growth dynamics in dissociated cultures of rat hippocampal pyramidal neurons. Given the important regulatory role of small Rho guanosine triphosphatases (GTPases) in cytoskeletal reorganization associated with axon growth, and reports that ethanol alters whole cell Rho GTPase activity in other cell types, this study explored the hypothesis that ethanol alters Rho GTPase activity specifically in axonal growth cones.
Fetal rat hippocampal pyramidal neurons were maintained in dissociated cultures for 1 day in control medium or medium containing 11 to 43 mM ethanol. Some cultures were also treated with brain-derived neurotrophic factor (BDNF), an activator of Rac1 and Cdc42 GTPases that promotes axon extension. Levels of active Rho GTPases in growth cones were measured using in situ binding assays for GTP-bound Rac1, Cdc42, and RhoA. Axon length, growth cone area, and growth cone surface expression of tyrosine kinase B (TrkB), the receptor for BDNF, were assessed by digital morphometry and immunocytochemistry.
Although ethanol increased the surface area of growth cones, the levels of active Rho GTPases in axonal growth cones were not affected in the absence of exogenous BDNF. In contrast, ethanol exposure inhibited BDNF-induced Rac1/Cdc42 activation in a dose-dependent manner and increased RhoA activation at the highest concentration tested. Similar TrkB expression was observed on the surface of axonal growth cones of control and ethanol-treated neurons.
These results reveal an inhibitory effect of ethanol on growth cone signaling via small Rho GTPases during early stages of hippocampal development in vitro, and suggest a mechanism whereby ethanol may disrupt neurotrophic factor regulation of axon growth and guidance.
乙醇对有丝分裂后神经元发育的影响包括改变突起的生长和分化，这可能导致与胎儿酒精谱系障碍相关的神经病理学。我们之前报道过，乙醇暴露会改变海马锥体神经元分离培养物中的轴突生长动力学。鉴于小 Rho 鸟苷三磷酸酶（GTPases）在与轴突生长相关的细胞骨架重排中具有重要的调节作用，并且有报道称乙醇会改变其他细胞类型中的全细胞 Rho GTPase 活性，因此本研究探讨了乙醇是否特异性改变轴突生长锥中的 Rho GTPase 活性的假说。
将胎鼠海马锥体神经元在对照培养基或含 11 至 43mM 乙醇的培养基中维持在分离培养物中 1 天。一些培养物还接受脑源性神经营养因子（BDNF）处理，BDNF 是 Rac1 和 Cdc42 GTPases 的激活剂，可促进轴突延伸。使用原位结合测定法测量生长锥中 GTP 结合的 Rac1、Cdc42 和 RhoA 的活性 Rho GTPase 水平。通过数字形态计量学和免疫细胞化学评估轴突长度、生长锥面积和生长锥表面酪氨酸激酶 B（TrkB）的表达，TrkB 是 BDNF 的受体。
尽管乙醇增加了生长锥的表面积，但在没有外源性 BDNF 的情况下，轴突生长锥中活性 Rho GTPase 的水平没有受到影响。相反，乙醇暴露以剂量依赖性方式抑制 BDNF 诱导的 Rac1/Cdc42 激活，并在测试的最高浓度下增加 RhoA 激活。在对照和乙醇处理神经元的轴突生长锥的表面观察到相似的 TrkB 表达。
这些结果揭示了乙醇在体外海马发育的早期阶段通过小 Rho GTPases 对生长锥信号的抑制作用，并提出了一种机制，即乙醇可能破坏神经营养因子对轴突生长和导向的调节。
