Neurofilament dot blot assays: Novel means of assessing axon viability in culture
Neurofilament dot blot assays: Novel means of assessing axon viability in culture
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DOI：
10.1016/j.jneumeth.2011.03.025
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发表时间：
2011-06-15
影响因子：
3
通讯作者：
Wilkins, Alastair
中科院分区：
文献类型：
--
作者：
Hares, Kelly;Kemp, Kevin;Wilkins, Alastair
轴突的结构和完整性对整个神经元的维持和动作电位的传播至关重要。神经丝蛋白是轴突的主要细胞骨架成分之一，神经丝蛋白亚基的磷酸化调节着神经丝蛋白在轴突中的运输速度，并决定着信号传递所需的最佳轴突口径。以前对神经保护剂的许多研究都集中在神经退行性疾病模型中的神经元生存能力上，而没有特别考虑轴突功能作为神经元损伤的指标。在这项研究中，我们致力于开发新的方法，通过测量培养的皮质神经元中神经丝的磷酸化水平来确定轴突的活性。以一氧化氮供体去甲硝酸酯(NO)为炎性介质，以胶质细胞源性神经营养因子(GDNF)和超氧化物歧化酶(SOD)为潜在的轴突保护剂。使用‘斑点印迹’方法，我们发现暴露于NO介导的细胞毒性的皮质神经元中的NF磷酸化减少，并且与GDNF和SOD处理相关的NO介导的NF磷酸化的变化减弱。这些结果与免疫细胞化学计数有很好的相关性。因此，我们认为斑点印迹法是一种新的体外评价轴突完整性的方法，并可能在未来测试药物对轴突活性的影响方面发挥有用的作用，为神经退行性疾病的潜在治疗提供一种可靠和可重复性的筛选方法。(C)2011爱思唯尔B.V.保留所有权利。
Axonal structure and integrity are vital to overall neuronal maintenance and action potential propagation. Neurofilaments (NFs) are one of the main cytoskeletal components of axons and phosphorylation of NF subunits regulates speed of NF transport through axons and determines optimal axonal calibre required for signal propagation. Many previous studies of neuroprotective agents have focussed on neuronal viability in models of neurodegenerative disease, without specifically considering axon function as an indicator of neuronal damage. In this study, we have focused on developing novel assays for determining axon viability by measuring levels of neurofilament phosphorylation in cultured cortical neurons. The nitric oxide donor DETANONOate (NO) was used as an inflammatory insult and glial cell line-derived neurotrophic factor (GDNF) and superoxide dismutase (SOD) were tested as potential axonal protective agents. Using 'dot blot' methodologies, we show a decrease in NF phosphorylation in cortical neurons exposed to NO-mediated cell toxicity and an attenuation of NO-mediated changes in NF phosphorylation associated with GDNF and SOD treatment. These results correlated well with immunocytochemical counts. We propose therefore that the dot blot assay is a novel method for assessing axonal integrity in vitro and may play a useful role in the future for testing the effects of agents on axonal viability, providing a reliable and reproducible screening method for potential therapeutics for neurodegenerative diseases. (C) 2011 Elsevier B.V. All rights reserved.