MECHANISM OF CELL DEATH BY PRIONS

描述(改编自申请者摘要)：Pron中的神经元死亡 疾病被认为是由一种构象变化的瘙痒病引起的 正常宿主蛋白(PrPC)的亚型(PrPSc)。高度的相关性 PrPSc沉积和神经退行性变之间存在因果关系 假设。然而，神经退行性变的存在和Pron的传递 没有检测到PrPSc的疾病表明存在替代方案 神经元死亡的机制。我们的长期目标是调查潜在的 启动正常和突变PrP代谢的神经毒性途径 神经毒性，无明显的prpSC沉积。最近，我们有 突变型PrP加工和周转的新途径 第145位密码子(PrP‘45)，与一种家族性PrP疾病有关。我们 相信PrP‘45通过细胞内途径具有神经毒性。在……里面 特别是，我们的数据表明PrPi45被蛋白酶体途径降解， 并在细胞内聚集。令人惊讶的是，相当数量的PrP‘45 也被定位到了细胞核。我们假设在这种情况下细胞毒性 是由于这些非常规生物对细胞新陈代谢的干扰造成的 PrP代谢途径。因为与PrP‘45类似片段也 由PrPC和其他突变体PrP的非典型加工产生，中央 本提案的目标是分析导致 PrPt4在细胞核内异常积聚所致的神经毒性，以及 神经细胞中产生类似PrP片段的途径。在第一个 目的，我们将识别特定的核定位信号(S)及其机制 PrP‘45向原子核的运输。第二个目标将集中在是否 细胞核中积累的PrP改变了转录活性，即 生理上相关的。在第三个目标中，我们将确定是否有目标 PRP与特定的核蛋白结合，以及这种联系是否 具有生物学意义，最后，我们将分析(S)的机制 从PrPC或其他突变体PrP产生类似于PrPt45的PrP片段， 因为它们会以类似PrP‘45的方式引起神经毒性。 设计的实验将使用各种细胞和分子生物学 技巧。体外核运输，细胞活性测定，体外 翻译，免疫共沉淀，免疫印迹，共聚焦免疫显微镜 和细胞分类将用于在具体目标1和 2.AIMS 3和AIMS 4，Far Western分析，电泳率漂移 实验、体外转录径流实验和差异mRNA展示 将会被执行。我们的研究将提供一种细胞生物学解释 与PrPSc同时运行或在PrPSc之前运行的PrP的神经毒性 沉积，并帮助制定策略来破坏这些异常 PrP代谢途径。

DESCRIPTION (Adapted from the Applicant's Abstract): Neuronal death in prion disorders is believed to result from a conformationally transformed, scrapie isoform (PrPSc) of the normal host prion protein (PrPC). The high correlation between PrPSc deposits and neurodegeneration has led to a cause and effect hypothesis. However, presence of neurodegeneration and transmission of prion diseases without detectable PrPSc suggest the presence of alternative mechanisms of neuronal death. Our long-term goal is to investigate potentially neurotoxic pathways of metabolism of normal and mutant PrP that initiate neurotoxicity without significant prpSC deposition. Recently, we have identified novel pathways of processing and turnover of mutant PrP with a stop codon at residue 145 (PrP'45), associated with a familial prion disorder. We believe that PrP'45 is neurotoxic through intracellular pathways. In particular, our data show that PrPi45 is degraded by the proteasomal pathway, and aggregates intracellularly. Surprisingly, a significant amount of PrP'45 is also rnistargeted to the nucleus. We hypothesize that cytotoxicity in this case is caused by perturbation of cellular metabolism by these unconventional pathways of PrP metabolism. Since fragments similar to PrP'45 are also generated by atypical processing of prpc and other mutant PrPs, the central goal of the present proposal is to analyze the cellular events leading to neurotoxicity by the abnormal accumulation of PrPt4s in the nucleus, and pathways of generation of similar PrP fragments in neuronal cells. In the first aim, we will identify specific nuclear localization signal(s) and the mechanism of transport of PrP'45 to the nucleus. The second aim will focus on whether accumulated PrP in the nucleus alters transcriptional activity that is physiologically relevant. In the third aim, we will determine if rnistargeted PrP is bound to specific nuclear proteins, and whether this association is biologically significant, and finally, we will analyze the mechanism(s) of generation of PrP fragments similar to PrPt45 from prpc or other mutant PrPs, since these would cause neurotoxicity in a manner similar to PrP'45. The experiments designed will use a variety of cell- and molecular biology techniques. In vitro nuclear transport, cell viability assays, in vitro translation, co-immunoprecipitation, Western blots, confocal immunomicroscopy and cell sorting will be used for the studies proposed in specific aims 1 and 2. For aims 3 and 4, Far Western analysis, electrophoretic mobility shift assay, in vitro transcriptional run-off assay, and differential mRNA display will be carried out. Our studies will provide a cell biological explanation for neurotoxicity of PrP that operates either concomitant with, or prior to PrPSc deposition, and help in developing strategies to disrupt these abnormal pathways of PrP metabolism.