Waguri Satoshi, Komatsu Masaaki
Department of Anatomy and Histology, Fukushima Medical University School of Medicine, Fukushima, Japan.
Methods Enzymol. 2009;453:181-96. doi: 10.1016/S0076-6879(08)04009-3.
Autophagy-deficient mice exhibit the formation of ubiquitin-inclusions in the liver and brain, which is not attributed to the dysfunction of the ubiquitin-proteasome system. Moreover, it is also clear that a multifunctional protein p62/A170/SQSTM1 (hereafter referred to as p62) links autophagy and inclusion formation, being one of the key components of the ubiquitin inclusions. The ubiquitin/p62 inclusions can be detected in the detergent-insoluble fraction by western blot analysis, while morphological information can be obtained by immunohistochemistry at both the light and electron microscopy levels. Importantly, p62 has become a reliable marker, with which we can identify inclusions and estimate autophagic activity in diseased tissues or cells. In this chapter, we describe the methods used for biochemical and morphological detection of ubiquitin/p62-inclusions in autophagy-suppressed Atg7-deficient mice. These methods are suitable for examination of cells and tissues with conditions associated with reduced autophagy (e.g., aging and mice models of intractable diseases such as Alzheimer's disease), and their applications should enhance our understanding of the pathophysiological mechanisms involved in the formation of intracellular inclusions.
自噬缺陷小鼠在肝脏和大脑中会出现泛素包涵体的形成，这并非归因于泛素 - 蛋白酶体系统功能障碍。此外，多功能蛋白p62/A170/SQSTM1（以下简称p62）连接自噬与包涵体形成这一点也很明确，它是泛素包涵体的关键组成部分之一。通过蛋白质印迹分析可在去污剂不溶性组分中检测到泛素/p62包涵体，而通过免疫组织化学在光学显微镜和电子显微镜水平均可获得形态学信息。重要的是，p62已成为一种可靠的标志物，利用它我们可以在患病组织或细胞中识别包涵体并评估自噬活性。在本章中，我们描述了用于在自噬受抑制的Atg7缺陷小鼠中对泛素/p62包涵体进行生化和形态学检测的方法。这些方法适用于对与自噬减少相关的条件下（如衰老以及阿尔茨海默病等难治性疾病的小鼠模型）的细胞和组织进行检测，其应用应能增进我们对细胞内包涵体形成所涉及的病理生理机制的理解。
