Targeting green fluorescent protein to dendritic membrane in central neurons
Targeting green fluorescent protein to dendritic membrane in central neurons
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DOI：
10.1016/j.neures.2008.01.014
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发表时间：
2008-05-01
影响因子：
2.9
通讯作者：
Kaneko, Takeshi
中科院分区：
文献类型：
--
作者：
Kameda, Hiroshi;Furuta, Takahiro;Kaneko, Takeshi
树突和轴突分别是神经元信息的输入和输出位点，这些过程的详细可视化对于阐明神经元回路和揭示神经元功能原理可能是必不可少的。为了建立一种完全可视化树突状突起的方法，我们首先开发了基于绿色荧光蛋白（GFP）的蛋白质，并通过使用具有神经元特异性启动子的慢病毒，检查该蛋白质是否完全可视化受感染神经元的树突状突起。当带有棕榈酰化位点（palGFP）或肉豆蔻酰化/棕榈酰化位点（myrGFP）的GFP在大鼠脑中用慢病毒表达时，myrGFP比palGFP更好地标记树突膜。随后，在纹状体黑质 GABA 能和皮质丘脑谷氨酸能神经元以及培养的皮质神经元中检查了连接到 myrGFP C 末端的三个基底外侧膜分选和三个假定的树突靶向结构域的树突靶向效率。在这六个结构域中，低密度脂蛋白受体 (LDLRCT) 的 C 端胞质结构域将蛋白质靶向树突的效率最高，在纹状体黑质神经元中的效率比 myrGFP 高 8.5-15 倍。最后，使用 Thy 1 或 Gad1 表达盒在转基因小鼠中证实了 myrGFP-LDLRCT 的树突膜靶向效力。因此，myrGFP-LDLRCT 是一种用于树突可视化的优异合成蛋白，并且可能是神经元回路形态学分析的有用工具。 (c) 2008 Elsevier Ireland Ltd 和日本神经科学学会。版权所有。
Dendritic and axonal processes are input and output sites, respectively, of neuronal information, and detailed visualization of these processes may be indispensable for elucidating the neuronal circuits and revealing the principles of neuronal functions. To establish a method for completely visualizing dendritic processes, we first developed green fluorescent protein (GFP)-based proteins and, by using lentivirus with a neuron-specific promoter, examined whether or not the protein fully visualized the dendritic processes of infected neurons. When GFP with a palmitoylation (palGFP) or myristoylation/palmitoylation site (myrGFP) was expressed in rat brain with lentiviruses, myrGFP labeled dendritic membrane better than palGFP. Subsequently, dendrite-targeting efficiencies of three basolateral membrane-sorting and three putative dendrite-targeting domains, which were attached to myrGFP C-terminus, were examined in striatonigral GABAergic and corticothalamic glutamatergic neurons, and in cultured cortical neurons. Of the six domains, C-terminal cytoplasmic domain of low density lipoprotein receptor (LDLRCT) was most efficient in targeting the protein to dendrites, showing 8.5-15-fold higher efficiency in striatonigral neurons compared with myrGFP. Finally, dendritic membrane-targeting potency of myrGFP-LDLRCT was confirmed in transgenic mice using Thy 1 or Gad1 expression cassette. Thus, myrGFP-LDLRCT is an excellent synthetic protein for dendritic visualization, and may be a useful tool for the morphological analysis of neuronal circuits. (c) 2008 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.