MECHANISM OF CELL DEATH BY PRIONS

描述（改编自申请人的摘要）：朊病毒中的神经元死亡 疾病被认为是由于构象转化，羊瘙痒症， 正常宿主朊病毒蛋白（PrPC）的同种型（PrPSc）。的高度相关性 PrPSc沉积和神经变性之间的因果关系 假说.然而，存在神经变性和朊病毒的传播， 没有可检测到的PrPSc的疾病表明存在替代治疗。 神经元死亡的机制。我们的长期目标是调查 正常和突变型PrP代谢的神经毒性途径， 神经毒性而无显著的prpSC沉积。最近我们 确定了新的加工途径和营业额的突变PrP与停止 密码子在残基145（PrP'45），与家族性朊病毒疾病。我们 认为PrP'45通过细胞内途径具有神经毒性。在 特别地，我们的数据显示PrPi45通过蛋白酶体途径降解， 并在细胞内聚集。令人惊讶的是，大量的PrP'45 也会影响到细胞核。我们假设这种情况下的细胞毒性 是由这些非常规的细胞代谢紊乱引起的， PrP代谢途径。由于类似于PrP'45的片段也 由prpc和其他突变PrPs的非典型加工产生， 本提案的目标是分析导致以下结果的细胞事件： PrPt4s在细胞核中的异常积累导致的神经毒性，以及 在神经元细胞中产生类似PrP片段的途径。上 目的是确定特定的核定位信号及其机制 PrP'45向细胞核的转运。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， 生理相关。在第三个目标中，我们将确定rnistargeted PrP与特定的核蛋白结合，这种结合是否是 生物学意义，最后，我们将分析的机制（S） 从prpc或其它突变PrP产生类似于PrPt45的PrP片段， 因为它们会以类似于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 技术.体外核转运，细胞活力测定，体外 翻译，免疫共沉淀，蛋白质印迹，共聚焦免疫显微镜 和细胞分选将用于具体目标1中提出的研究， 2.对于目标3和4，远Western分析，电泳迁移率变化 体外转录径流测定和差异mRNA展示 将被执行。我们的研究将提供一个细胞生物学解释， 与PrPSc同时或在PrPSc之前发挥作用的PrP神经毒性 沉积，并帮助制定战略，以破坏这些异常 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.