Analysis of Cytoplasmic Prion Protein Toxicity

描述（由申请人提供）：1990年代中期产生的证据表明，牛海绵状脑病（俗称疯牛病）可能传播给人类。人们普遍认为导致这些疾病的因子是形状异常的朊病毒蛋白。除了感染途径外，人类还可以偶尔或通过表达突变的朊病毒蛋白（外显率约为100%）而患上朊病毒疾病。最近的工作指出细胞质是朊病毒蛋白毒性的部位。突变的朊病毒蛋白可能是高毒性的，因为它们有更高的错误折叠的可能性，因此错误定位到细胞质。为了研究突变型朊病毒蛋白毒性增加的机制及其与胞质朊病毒毒性的关系，将对胚胎干细胞进行基因改造，使其直接在胞质中表达突变型朊病毒蛋白。这些细胞系将分化为神经元、胶质细胞或成纤维细胞，并用于研究这些突变对PrP亚细胞运输、降解和毒性的影响。基因靶向方法将允许在mRNA水平上等量表达这些结构的高概率。选择胚胎干细胞作为模型系统将允许细胞培养实验有效地过渡到小鼠。

DESCRIPTION (provided by applicant): Evidence produced in the mid 1990's suggests that bovine spongiform encephalopathy (commonly known as mad cow disease) was probably transmitted to humans. The agents widely believed to be responsible for these diseases are abnormally shapened prion proteins. Along with an infectious route, humans can also develop prion diseases either sporadically or by expressing a mutant prion protein (penetrance approximately 100%). Recent work points to the cytosol as being the site of toxicity of prion proteins. Mutant prion proteins may be highly toxic because they have an increased probability of misfolding and therefore mislocalizing to the cytosol. To study the mechanism of increased toxicity of the mutant prion proteins and how it relates to cytosolic prion toxicity, ES cells will be genetically altered to express mutant prion proteins directly into the cytosol. These cell lines will be differentiated into neurons, gila, or fibroblasts and used to study the effects these mutations have on subcellular traffciking, degradation, and toxicity of PrP. The gene-targeting approach will allow for a high probability of equivalent expression of these constructs at the mRNA level. The selection of ES cells as a model system will permit efficient transition of cell culture experiments into mice.