Formation, Stability, and Structure of Hirano Bodies Using a Live Cell System

随着人口老龄化，神经退行性疾病成为一个主要的健康问题。最少的之一 所研究的神经退行性疾病的结构表现为平野小体。平野小体是肌动蛋白 基于包裹体的，已在具有广泛范围的 包括阿尔茨海默病在内的神经退行性疾病。虽然超微结构和蛋白质 众所周知，平野体的组成是导致平野体形成的一系列事件 仍然不为人知。确定导致集合的事件对于理解 病理学上的平野小体。我们的假设是，平野小体的形成是不受控制的肌动蛋白的结果 交联剂的活性和这种不受控制的交联物，与细胞的自然交联物相一致 能力，产生稳定的平野身体结构。平野天体形成的主要原因之一是 没有被研究的是缺乏活细胞模型系统。我们已经证明了一个被截断的 具有不受控制的肌动蛋白交联活性的肌动蛋白结合蛋白可诱导大鼠体内平野小体的形成 真核模型系统盘基盘基菌(Maselli，2002；Maselli，2003)。模特平野 网柄菌体与人脑中发现的平野小体具有相似的特征 (Maselli，2002)。表达截短的肌动蛋白结合蛋白(t-ABP)绿色荧光蛋白(GFP) 与可诱导表达系统融合后，我们将能够观察细胞中平野小体的形成。通过 结合t-ABP探针和丝状肌动蛋白探针，我们可以观察到在 平野体形成的最早阶段。扩展我们的假设，t-ABP在人类细胞中的一个可能来源 是细胞自身肌动蛋白结合蛋白的蛋白水解性裂解片段。我们建议检验一下我们的假设 通过在细胞中表达可能的切割片段，并观察细胞是否形成包涵体。这个 平野小体的超微结构是了解其形成和稳定性的关键。我们将决定 最佳固定方法及免疫电子显微镜和FIAsH标记技术 从光学显微镜到超微结构的观察。更好地了解事件的级联和 平野身体形成的可能触发因素将进一步加深我们对平野潜在作用的理解 神经退行性疾病中的身体。

Neurodegenerative diseases become a major health concern as the population ages. One of the least studied structural manifestations of neurodegenerative disease are Hirano bodies. Hirano bodies are actin based inclusions, which have been identified in the brains of individuals with a broad range of neurodegenerative disorders including Alzheimer's disease. Although the ultrastructure and protein composition of Hirano bodies is well known the cascade of events that leads to Hirano body formation remains unknown. Determining the events that lead to assembly is important to understanding the role of Hirano bodies in pathology. Our hypothesis is that Hirano bodies form as the result of uncontrolled actin cross-linker activity and that this uncontrolled cross-linking, in concert with the cell's natural cross-linking ability, generate a stable Hirano body structure. One of the major reasons that Hirano body formation has not been studied is the lack of a live cell model system. We have shown that the expression of a truncated actin binding protein with uncontrolled actin cross-linking activity can induce the formation of Hirano bodies in the eukaryotic model system Dictyostelium discoideum (Maselli, 2002 ;Maselli, 2003). The model Hirano bodies formed in Dictyostelium have similar characteristics to the Hirano bodies found in the human brain (Maselli, 2002). By expressing a truncated actin binding protein (t-abp) Green Fluorescent Protein (GFP) fusion with an inducible expression system we will be able to observe Hirano body formation in cells. By combining the t-abp probe with a probe for filamentous actin we can observe changes that take place at the earliest stages of Hirano Body formation. Extending our hypothesis, a likely source of t-abp in human cells are proteolytic cleavage fragments of the cells own actin biding proteins. We propose to test our hypothesis by expressing the likely cleavage fragments in cells, and observing the cells for inclusion formation. The ultrastructure of Hirano Bodies is key to understanding both their formation and stability. We will determine the optimal fixation method and used immuno electron microscopy and FIAsH tag technology to correlate our observations from light microscopy to the ultrastructure. Better understanding the cascade of events and the possible triggers for Hirano Body formation will further our understanding of the potential roles of Hirano Bodies in neurodegenerative disease.