Prion Replication Elicits Cytopathic Changes in Differentiated Neurosphere Cultures
Prion Replication Elicits Cytopathic Changes in Differentiated Neurosphere Cultures
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DOI：
10.1128/jvi.00572-13
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发表时间：
2013-08-01
影响因子：
5.4
通讯作者：
Kitani, Hiroshi
中科院分区：
文献类型：
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作者：
Iwamaru, Yoshifumi;Takenouchi, Takato;Kitani, Hiroshi
朊病毒诱导的细胞毒性的分子机制在很大程度上仍不清楚。目前，只有少数细胞培养模型表现出与朊病毒感染相关的细胞病变。在这项研究中，我们介绍了一种细胞培养模型的基础上分化的神经球文化分离的新生儿朊蛋白（PrP）-空小鼠和转基因小鼠表达鼠PrP（dNP 0和dNP 20文化）的大脑。暴露于小鼠钱德勒朊病毒后，dNP 20培养物支持异常PrP的从头形成和由此产生的感染性，如通过生物测定所评估的。此外，这种培养物对各种朊病毒株敏感，包括小鼠适应性瘙痒症、牛海绵状脑病和Gerstmann-Straussler-Scheinker综合征朊病毒。重要的是，感染培养物中主要由星形胶质细胞谱系细胞组成的细胞亚群始终显示迟发性、进行性细胞毒性体征，如形态学改变、细胞活力降低和乳酸脱氢酶释放增加所证明。在感染的dNP 0培养物中未观察到这些细胞毒性迹象，表明朊病毒诱导的细胞毒性需要内源性PrP表达。胶质细胞酸性蛋白阳性的退化细胞积累异常PrP，并表现出凋亡性死亡的特征，通过活性caspase-3和末端脱氧核苷酸转移酶缺口末端染色评估。此外，半胱天冬酶抑制提供部分保护朊病毒介导的细胞死亡。这些结果表明，分化的神经球文化可以提供一个体外生物测定小鼠朊病毒，并允许朊病毒诱导的细胞毒性在细胞水平上的分子基础的研究。
The molecular mechanisms of prion-induced cytotoxicity remain largely obscure. Currently, only a few cell culture models have exhibited the cytopathic changes associated with prion infection. In this study, we introduced a cell culture model based on differentiated neurosphere cultures isolated from the brains of neonatal prion protein (PrP)-null mice and transgenic mice expressing murine PrP (dNP0 and dNP20 cultures). Upon exposure to mouse Chandler prions, dNP20 cultures supported the de novo formation of abnormal PrP and the resulting infectivity, as assessed by bioassays. Furthermore, this culture was susceptible to various prion strains, including mouse-adapted scrapie, bovine spongiform encephalopathy, and Gerstmann-Straussler-Scheinker syndrome prions. Importantly, a subset of the cells in the infected culture that was mainly composed of astrocyte lineage cells consistently displayed late-occurring, progressive signs of cytotoxicity as evidenced by morphological alterations, decreased cell viability, and increased lactate dehydrogenase release. These signs of cytotoxicity were not observed in infected dNP0 cultures, suggesting the requirement of endogenous PrP expression for prion-induced cytotoxicity. Degenerated cells positive for glial fibrillary acidic protein accumulated abnormal PrP and exhibited features of apoptotic death as assessed by active caspase-3 and terminal deoxynucleotidyltransferase nick-end staining. Furthermore, caspase inhibition provided partial protection from prion-mediated cell death. These results suggest that differentiated neurosphere cultures can provide an in vitro bio-assay for mouse prions and permit the study of the molecular basis for prion-induced cytotoxicity at the cellular level.