The Cell Biology of Neurodegeneration Caused by the Prion Protein

朊病毒蛋白（PrP）是一种广泛表达的高度保守的细胞表面糖蛋白，其功能尚不清楚。PrP的异常代谢是人和动物中多种神经退行性疾病的原因。这些疾病包括传染性“朊病毒病”，如牛海绵状脑病，以及由PrP基因突变引起的遗传性神经退行性疾病。在这两种情况下，导致细胞死亡和神经元损伤的途径都不清楚。该项目的总体目标是确定PrP介导的神经变性的途径。为了实现这一目标，我们正在研究PrP生物合成，细胞内运输，代谢和降解的分子途径。对这些事件的定量分析预计将揭示PrP的各种遗传突变如何以导致细胞功能障碍的方式影响其生物合成或代谢。 我们的分析表明，至少有两种细胞毒性形式的PrP（称为CtmPrP和cyPrP）在初始易位PrP进入内质网（ER）。现在已经建立了转基因小鼠，以确定CtmPrP介导的神经变性和cyPrP介导的神经变性是否可以在体内通过调节PrP生物发生过程中新发现的这一步骤来避免。我们也在研究各种形式的PrP被细胞正常代谢的途径，以确定这些事件的调节是否参与神经变性的进展。预期某些形式的PrP的生物合成和/或清除中的缺陷的组合协作最终导致神经元功能障碍和死亡。相反，对这些事件的操纵可能能够减缓或逆转这些疾病中的神经退行性过程。 与此同时，我们也进行了系统的生物合成，贩运和疾病相关的PrP突变体的代谢分析。这些研究的目的是精确地确定PrP错误折叠启动疾病过程的细胞位置和机制。我们目前的分析发现，对于大量的突变体，错误折叠的PrP被发现在ER后的位置，从那里它被路由到溶酶体降解。这一观察结果是值得注意的，因为它表明错误折叠的PrP种类以某种方式逃脱了ER中的正常细胞质量控制机制，而是在高尔基体中使用尚未鉴定的质量控制途径。目前正在研究这些新的质量控制途径。 在平行研究中，PrP错误折叠和聚集的下游后果正在研究中，以确定这些事件导致细胞功能障碍的机制。我们现在已经发现，这些聚集体募集各种细胞因子，从而耗尽它们的功能可用性。一个这样的因素是特别重要的，因为它在小鼠中的破坏直接导致神经退行性表型，让人想起由PrP引起的疾病。 最后，我们正在研究蛋白质聚集体的一般特性，这些特性与多种疾病有关。我们已经发现胞质聚集体的存在可以显著影响正常细胞质量控制的途径。在一个具体的例子中，我们已经发现，聚集体影响的命运错误定位的分泌和膜蛋白。聚集体的存在导致这些蛋白质在胞质溶胶中保持不降解，而不是被快速降解。这导致它们的共聚集，促进现有聚集体的繁殖并导致细胞死亡。目前正在研究这种现象的分子基础。

The prion protein (PrP) is a widely expressed and highly conserved cell surface glycoprotein of uncertain function. Aberrant metabolism of PrP is responsible for a variety of neurodegenerative diseases in both people and animals. These diseases include the transmissible 'prion diseases' such as bovine spongiform encephalopathy, as well as inheritable neurodegenerative diseases caused by mutations in the PrP gene. In neither case is the pathway(s) leading to cell death and neuronal damage understood. The overall goal of this project is to define the pathways of PrP-mediated neurodegeneration. To achieve this goal, we are studying the molecular pathways of PrP biosynthesis, intracellular trafficking, metabolism, and degradation. A quantitative analyses of these events are expected to shed light on how the various inherited mutations in PrP influence its biosynthesis or metabolism in a manner that leads the cellular dysfunction. Our analysis suggests that at least two cytotoxic forms of PrP (termed CtmPrP and cyPrP) are during the initial translocation of PrP into the endoplasmic reticulum (ER). Transgenic mice have now been created to determine whether CtmPrP-mediated neurodegeneration and cyPrP-mediated neurodegeneration can be averted in vivo by modulating this newly discovered step during PrP biogenesis. We are also investigating the pathways by which the various forms of PrP are normally metabolized by the cell to determine whether modulation of these events are involved in the progression of neurodegeneration. It is anticipated that a combination of defects in biosynthesis and/or clearance of certain forms of PrP collaborate to eventually cause neuronal dysfunction and death. Conversely, manipulation of these events may be able to slow or reverse the neurodegenerative process in these diseases. In parallel, we are also performing a systematic analysis of the biosynthesis, trafficking, and metabolism of disease-associated PrP mutants. The aim of these studies is to identify precisely the cellular locale and mechanism of PrP misfolding that initiates the disease process. Our current analyses have found that for a large number of mutants, misfolded PrP is found in a post-ER location, from where it is routed to lysosomes for degradation. This observation is notable because it suggests that the misfolded PrP species have somehow escaped the normal cellular quality control mechanisms in the ER, and instead use yet unidentified quality control pathways in the Golgi. These new pathways of quality control are now being investigated. In parallel studies, the downstream consequences of PrP misfolding and aggregation are being studied to identify the mechanism by which these events lead to cellular dysfunction. We have now found that these aggregates recruit various cellular factors, therby depleting their functional availability. One such factor is of particular importance because its disruption in mice leads directly to a neurodegenerative phenotype reminiscent of diseases caused by PrP. And finally, we are investigating the general properties of protein aggregates, which are associated with a wide range of diseases. We have discovered the the presence of cytosolic aggregates can significantly influence the pathways of normal cellular quality control. In one specific example, we have found that aggregates influence the fate of mislocalized secretory and membrane proteins. Rather than being rapidly degraded, the presence of aggregates causes these proteins to remain undegraded in the cytosol. This leads to their co-aggregation, facilitating the propogation of the existing aggregates and causing cell death. The molecular basis of this phenomenon is now being investigated.