Time-lapse analysis of aggregate formation in an inducible PC 12 cell model of Huntington's disease reveals time-dependent aggregate formation that transiently delays cell death
Time-lapse analysis of aggregate formation in an inducible PC 12 cell model of Huntington's disease reveals time-dependent aggregate formation that transiently delays cell death
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DOI：
10.1016/j.brainresbull.2007.08.005
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发表时间：
2008-01-31
影响因子：
3.8
通讯作者：
Morton, A. J.
中科院分区：
文献类型：
--
作者：
Gong, B.;Lim, M. C. Y.;Morton, A. J.
亨廷顿病(HD)是一种由HD基因编码区CAG重复扩增引起的进行性神经退行性疾病。翻译后的聚谷氨酰胺扩张导致HD患者和转基因小鼠模型的大脑中形成不可溶的聚集体。然而，聚集体形成和神经病理之间的关系仍不清楚。我们使用荧光蛋白标记和活细胞时间推移显微镜来研究转基因PC12细胞中可见聚集体的形成及其与细胞死亡的关系。我们使用了表达亨廷顿蛋白外显子1片段的细胞系，其中23(野生型)或74(突变型)谷氨酸在可诱导启动子的控制下与增强的绿色荧光蛋白融合。在转基因诱导96h后，我们实时观察到活细胞。我们发现在这些细胞中聚集的形成是时间依赖的，并且以核为主。我们跟踪了单个细胞的包涵体形成情况，每10分钟检查一次细胞，最多持续48小时。这揭示了包涵体形成的新细节。最初的聚集体形成很快(通常为1小时)，但需要长达18小时(48小时)才能形成最终的聚集体表型。聚集体以动态的方式形成，并在整个成熟过程中在细胞核内不断运动。存活时间越长的细胞中，大聚集体的形成越频繁。然而，聚集体的大小并不是一个很好的细胞死亡预测指标，因为细胞可以死亡，有大的(>；2微米)，小的(<；0.5微米)或没有可见的聚集体。形成大聚集体的细胞比形成小聚集体或根本没有聚集体的细胞存活时间更长。然而，细胞死亡所需的时间随着形成的最终聚集体大小的增加而减少。此外，较早形成聚集体的细胞倾向于较早死亡。总而言之，我们的数据与聚集/聚集的有毒作用是兼容的，并支持“有毒前体”假说。然而，他们也表明，在某些阶段，聚集体的形成过程是细胞保护的。(C)2007 Elsevier Inc.保留所有权利。
Huntington's disease (HD) is a progressive neurodegenerative disease caused by a CAG repeat expansion in the coding region of the HD gene. The translated polyglutamine expansion causes the formation of insoluble aggregates in the brains of HD patients and transgenic mouse models. However, the relationship between aggregate formation and neuropathology remains unknown. We used fluorescent protein tagging and live-cell time-lapse microscopy to study visible aggregate formation and its relationship to cell death in transgenic PC12 cells. We used cell lines expressing a fragment of huntingtin exon 1 with either 23 (wild type) or 74 (mutant) glutamines fused to enhanced green fluorescent protein under the control of an inducible promoter. Live cells were observed in real time after transgene induction for up to 96 h. We found that aggregate formation was time-dependent and predominantly nuclear in these cells. We followed inclusion formation in individual cells, examining the cells every 10 min for up to 48 h. This revealed new details of inclusion formation. Initial aggregate formation was rapid (often < 1 h), but many (18 -> 48) h were needed to establish a final aggregate phenotype. Aggregates formed in a dynamic manner and were in constant motion within cell nuclei throughout their maturation. The formation of large aggregates occurred more frequently in cells that survived longer. However, aggregate size was not a good predictor of cell death, since cells could die with either large (>2 mu m), small (< 0.5 mu m) or no visible aggregates. Cells that formed large aggregates survived longer than cells that formed small aggregates or no aggregates at all. However, the time taken for a cell to die decreased as a function of increasing size of final aggregate formed. Further, cells that formed aggregates earlier tended to die earlier. Together our data are compatible with a toxic role for aggregates/aggregation and support the 'toxic precursor' hypothesis. However, they also suggest that at some stages, the process of aggregate formation is cytoprotective. (C) 2007 Elsevier Inc. All rights reserved.