Live-cell imaging of autophagy induction and autophagosome-lysosome fusion in primary cultured neurons.
Live-cell imaging of autophagy induction and autophagosome-lysosome fusion in primary cultured neurons.
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DOI：
10.1016/s0076-6879(08)04007-x
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发表时间：
2009
影响因子：
--
通讯作者：
Heidenreich, Kim A.
中科院分区：
文献类型：
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作者：
Bains, Mona;Heidenreich, Kim A.
自噬受损与包括癌症和神经变性在内的多种突出疾病有关，这一发现导致了该领域研究的爆炸性增长。允许研究人员观察和量化自噬过程的方法显然将促进我们对这一过程如何有助于许多临床疾病的病理生理学的了解。最近在高等真核生物中发现了必需的自噬基因，这使得分析表达带有荧光标记的自噬蛋白的哺乳动物细胞中的自噬成为可能。本章描述了使用原代培养的神经元的这种方法，当营养因子从其培养液中去除时，神经元经历了自噬的上调。持续上调的自噬反过来又导致这些神经元的死亡，从而提供了一个模型来研究增强的自噬和细胞死亡之间的关系。从出生后第7天的小鼠小脑中分离出神经元，并在营养因子和去极化浓度的钾存在下进行培养。一旦建立了神经元，就将表达MAP1-LC3的腺病毒载体与红色荧光蛋白(RFP)一起导入神经元。MAP1-Lc3是酵母自噬标记Atg8在哺乳动物中的同源物，当标记到GFP或RFP时，它是应用最广泛的自噬标记。一旦表达稳定，就可以通过去除营养因子来诱导自噬。在诱导自噬后的不同时间点，对神经元进行LysoSensor Green(一种pH依赖的溶酶体标记物)和Hoechst(一种DNA标记物)染色，并进行活细胞成像。在某些情况下，延时成像被用来检查活神经元中自噬的逐步过程。
The discovery that impaired autophagy is linked to a wide variety of prominent diseases including cancer and neurodegeneration has lead to an explosion of research in this area. Methodologies that allow investigators to observe and quantify the autophagic process will clearly advance our knowledge of how this process contributes to the pathophysiology of many clinical disorders. The recent identification of essential autophagy genes in higher eukaryotes has made it possible to analyze autophagy in mammalian cells that express autophagy proteins tagged with fluorescent markers. This chapter describes such methods using primary cultured neurons that undergo up-regulation of autophagy when trophic factors are removed from their medium. The prolonged up-regulated autophagy, in turn, contributes to the death of these neurons, thus providing a model to examine the relationship between enhanced autophagy and cell death. Neurons are isolated from the cerebellum of postnatal day 7 rat pups and cultured in the presence of trophic factors and depolarizing concentrations of potassium. Once established, the neurons are transfected with an adeno-viral vector expressing MAP1-LC3 with red fluorescent protein (RFP). MAP1-LC3 is the mammalian homologue of the yeast autophagosomal marker Atg8 and when tagged to GFP or RFP, it is the most widely used marker for autophagosomes. Once expression is stable, autophagy is induced by removing trophic factors. At various time points after inducing autophagy, the neurons are stained with LysoSensor Green (a pH-dependent lysosome marker) and Hoechst (a DNA marker) and subjected to live-cell imaging. In some cases, time-lapse imaging is used to examine the step-wise process of autophagy in live neurons.