Quantitative analysis of BDNF/TrkB protein and mRNA in cortical and striatal neurons using ýý-tubulin as a normalization factor.
Quantitative analysis of BDNF/TrkB protein and mRNA in cortical and striatal neurons using ýý-tubulin as a normalization factor.
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使用 α-微管蛋白作为标准化因子，对皮质和纹状体神经元中的 BDNF/TrkB 蛋白和 mRNA 进行定量分析。
DOI：
10.1002/cyto.a.22073
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发表时间：
2012
期刊：
影响因子：
--
通讯作者：
Tanese,Naoko
中科院分区：
文献类型：
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作者：
Ma,Bin;Savas,JeffreyN;Chao,MosesV;Tanese,Naoko
神经营养蛋白脑源性神经营养因子（BDNF）及其受体酪氨酸激酶TrkB在神经元生物学的多个方面发挥重要的调节作用，包括细胞死亡，存活，生长，分化和可塑性。BDNF/TrkB在不同亚细胞结构域如索马、树突、轴突、生长锥、神经末梢和棘的局部可用性的调节似乎有助于其特定功能。鉴于神经元及其区室的大小和形状的变化，以前的BDNF/TrkB蛋白和mRNA的定量研究缺乏一个强大的标准化程序。为了克服这个问题，我们建立了使用α-微管蛋白的免疫荧光检测作为标准化因子的方法，用于定量分析培养的原代大鼠皮质和纹状体神经元中的蛋白质和mRNA。通过研究蛋白质和mRNA在不同生长阶段或条件下的动态分布来证明这种方法的有效性。用KCl处理培养的神经元导致TrkB蛋白水平增加，BDNF mRNA水平降低（多个转录物的复合物），皮质树突中BDNF蛋白水平略有降低。KCl处理还降低了索马中BDNF和TrkB蛋白的百分比，表明蛋白质转运。最后，对大鼠皮质和纹状体神经元的分析表明，纹状体神经元中BDNF/TrkB蛋白和BDNF mRNA的水平相当或甚至更高。因此，与以前在体内观察到的相反，纹状体神经元在体外生长培养基中培养时能够合成BDNF mRNA。本文提出的分析方法提供了对BDNF/TrkB水平响应于各种神经元活动的详细理解。我们的方法可以被广泛使用，包括在细胞和组织细胞计数的应用，以产生准确的定量数据的基因表达在细胞和亚细胞环境。© 2012国际细胞计数促进会
The neurotrophin brain‐derived neurotrophic factor (BDNF) and its receptor tyrosine kinase TrkB serve important regulatory roles for multiple aspects of the biology of neurons including cell death, survival, growth, differentiation, and plasticity. Regulation of the local availability of BDNF/TrkB at distinct subcellular domains such as soma, dendrites, axons, growth cones, nerve terminals, and spines appears to contribute to their specific functions. In view of the variance in size and shape of neurons and their compartments, previous quantitative studies of the BDNF/TrkB protein and mRNA lacked a robust normalization procedure. To overcome this problem, we have established methods that use immunofluorescence detection of α‐tubulin as a normalization factor for the quantitative analysis of protein and mRNA in primary rat cortical and striatal neurons in culture. The efficacy of this approach is demonstrated by studying the dynamic distribution of proteins and mRNA at different growth stages or conditions. Treatment of cultured neurons with KCl resulted in increased levels of TrkB protein, reduced levels of BDNF mRNA (composite of multiple transcripts) and a slight reduction in BDNF protein levels in the dendrites from the cortex. The KCl treatment also lowered the percentage of BDNF and TrkB proteins in the soma indicative of protein transport. Finally, analysis of the rat cortical and striatal neurons demonstrated comparable or even higher levels of BDNF/TrkB protein and BDNF mRNA in the neurons from the striatum. Thus, in contrast to previous observations made in vivo, striatal neurons are capable of synthesizing BDNF mRNA when cultured in growth media in vitro. The analytical approach presented here provides a detailed understanding of BDNF/TrkB levels in response to a variety of neuronal activities. Our methods could be used broadly, including applications in cell and tissue cytometry, to yield accurate quantitative data of gene expression in cellular and subcellular contexts. © 2012 International Society for Advancement of Cytometry