Role of the Lysosome in ER Associated Degradation of PrP

描述(由申请人提供)：Prion疾病，如疯牛病(即疯牛病)，是与正常细胞Prion蛋白(PrP-C)的错误折叠异构体(PrP-SC)有关的可传播的神经退行性疾病。最近的工作表明，错误折叠的PrP经历了内质网相关降解(ERAD)，从而被蛋白酶体反向转移到细胞质中进行降解。蛋白酶体功能障碍被认为是导致Pron病的原因。蛋白酶体抑制后PrP在胞浆中的积累(CyPrP)或直接表达到培养细胞的胞浆中会产生不溶的、抗蛋白酶K(PK)的PrP，但在表达cyPrP(Tg1D4)的转基因小鼠中不会产生PrP，也不知道这些小鼠是否产生感染性PrP。我们的初步工作提供了证据，证明内质网内错误折叠的PrP除了被传递到蛋白酶体外，还被传递到溶酶体。然而，我们发现，这条途径并没有经历有效的降解，而是促进了PrP-SC的标志物--PK抗性PrP的形成。此外，我们发现PrP-SC是从分泌型溶酶体中释放出来的，这为PrP-SC的传播提供了一种新的机制。这项工作的主要目标是更好地确定这一途径及其在Prion生成中的作用。具体来说，我们会的；1.)测试早期将PrP递送到溶酶体是ER质量控制的一个特征的假设，2.)研究PrP遵循这一途径的功能后果，以及3.)确定特定的信号和传递到溶酶体的性质。利用共聚焦免疫荧光显微镜、免疫电子显微镜、溶酶体分离、cyPrP表达(Tg1D4)小鼠、[GFP标记的轻链MAPK3表达转基因(TgGFP-Lc3)小鼠和敲除Lc3-成纤维细胞]，以及其他各种系统，我们将评估该途径与蛋白酶体途径相比，在PrP的从头产生和传播中的重要性。这些研究可能会提供关于ERAD的新概念，即错误折叠蛋白质的细胞处理，并定义治疗这些神秘且极具争议性的疾病的新途径。

DESCRIPTION (provided by applicant): Prion diseases, such as BSE (i.e. mad cow disease), are transmissible neurodegenerative diseases related to the misfolded isoform (PrP-Sc) of the normal cellular prion protein (PrP-C). Recent work suggests that misfolded PrP undergoes ER associated degradation (ERAD), whereby it is retrotranslocated to the cytosol for degradation by the proteasome. Proteasome dysfunction is hypothesized to induce prion disease. Accumulation of PrP in the cytosol (cyPrP) following proteasome inhibition or directed expression into the cytosol of cultured cells produce insoluble, Proteinase-K (PK) resistant PrP, but not in transgenic mice expressing cyPrP (Tg1D4), nor is it known if these mice produce infectious prions. Our preliminary work provides evidence that misfolded PrP within the ER is delivered to the lysosome, in addition to the proteasome. However, rather than undergoing effective degradation, we find that this pathway enhances the formation of PK-resistant PrP, a marker of PrP-Sc. In addition, we show that PrP-Sc is released from secretory lysosomes, providing a new mechanism for prion spread. The primary goal of this work is to better define this pathway and its role in prion generation. Specifically, we will; 1.) Test the hypothesis that early delivery of PrP to the lysosome is a feature of ER quality control, 2.) Study the functional consequences of PrP that follows this pathway, and 3.) Define the specific signals and nature of delivery to the lysosome. Using confocal immunohistofluorescence microscopy, immunoelectron microscopy, lysosome fractionation, cyPrP expressing (Tg1D4) mice, [GFP-tagged light chain MAP kinase 3 expressing transgenic (TgGFP-LC3) mice, and knockout LC3-fibroblasts], in addition to a variety of other systems, we will assess the importance of this pathway, in comparison with the proteasome pathway, in the de novo generation and spread of prions. These studies will likely provide new concepts about ERAD, the cellular handling of misfolded proteins, and define new avenues for treatment of these enigmatic and highly controversial diseases.