Molecular Mechanisms of Prion Protein Amyloid Formation

传染性海绵状脑病（tse或朊病毒病）是一类罕见的神经退行性疾病，包括人类克雅氏病（CJD）、绵羊痒病、牛海绵状脑病（BSE）和长尾鹿和麋鹿的慢性消耗性疾病（CWD）。TSE疾病的传染因子被称为朊病毒，主要由正常的、蛋白酶敏感的朊病毒蛋白（PrP-sen）的一种异常折叠的、蛋白酶抗性的形式（PrP-res或PrPSc）组成。PrP-res可以以弥漫性淀粉样蛋白阴性沉积或致密性淀粉样蛋白阳性沉积的形式沉积在大脑中。淀粉样形式的朊病毒疾病似乎比非淀粉样形式的传播更少。此外，目前尚不清楚PrP-res主要以淀粉样蛋白沉积的朊病毒疾病是否与PrP-res主要以非淀粉样蛋白沉积的朊病毒疾病遵循相同的致病过程。我们对了解PrP淀粉样蛋白形成的分子机制很感兴趣，并且已经开始使用体外和体内模型系统来解决这个问题。本项目的重点是：1)了解PrP淀粉样蛋白形成的途径，2)研究PrP突变如何影响家族性朊病毒疾病中PrP-res淀粉样蛋白的形成。

Transmissible spongiform encephalopathies (TSEs or prion diseases) are a group of rare neurodegenerative diseases which include Creutzfeldt-Jakob disease (CJD) in humans, scrapie in sheep, bovine spongiform encephalopathy (BSE) and chronic wasting disease (CWD) in mule deer and elk. The infectious agent of TSE diseases is called a prion and is largely composed of an abnormally refolded, protease resistant form (PrP-res or PrPSc) of the normal, protease-sensitive prion protein, PrP-sen. PrP-res can be deposited in the brain as either diffuse, amyloid negative deposits or as dense, amyloid positive deposits. Amyloid forms of prion disease appear to be less transmissible than non-amyloid forms. Furthermore, it is unclear whether or not prion diseases where PrP-res is deposited primarily as amyloid follow the same pathogenic processes as prion diseases where PrP-res is primarily deposited as non-amyloid. We are interested in understanding the molecular mechanisms underlying PrP amyloid formation and have begun to approach this issue using both in vitro and in vivo model systems. This project focuses on: 1) understanding the pathways of PrP amyloid formation and, 2) studying how mutations in PrP influence PrP-res amyloid formation in familial forms of prion disease. Using LC-MS/MS Nanospray Ion Trap Mass Spectrometry, we had previously generated the proteomes of prion infected mouse brain tissue which had accumulated PrP-res in either amyloid or non-amyloid forms. However, we discovered that non-age matched control mice had been used in the original analysis and that a new set of negative controls was required to interpret the results. In 2013, we finally completed gathering the data from a new set of appropriately age-matched negative controls. Our results suggest that cell death pathways are associated with the non-amyloid of prion disease while disruptions in lipids and cytoskeletal structures are prominent in the amyloid prion disease. Furthermore, we identified cellular pathways common to both forms of disease that appear to be related to the host response to infection. A manuscript describing our results will be submitted for publication in 2013. As another approach to delineate the mechanisms underlying amyloid and non-amyloid forms of prion disease, in 2013 we initiated in vivo work to study the pathogenesis of different forms of amyloid and non-amyloid human prion disease in transgenic mice expressing either mutant or wild-type human PrP. These studies will likely take several years to complete but will provide important information in several poorly understood areas of human prion disease including 1) the contribution of the host versus the contribution of the prion strain to different in vivo disease phenotypes and 2) the mechanisms of amyloid versus non-amyloid prion formation. Based on its ability to spread and self-replicate, it has recently been suggested that amyloid formed in non-prion protein folding diseases such as Alzheimer s disease (AD) are in fact infectious prions. In 2013, we completed studies using an in vitro system that has been shown to generate infectious prions from recombinant PrP-sen (Science 327: 1132 (2010)). Our results show that abnormal, protease-resistant forms of recombinant PrP that are self-replicating but non-infectious are generated in this system. The data show that the ability of PrP to replicate itself is not necessarily restricted to infectious prions and that self-replication is not a unique property of an infectious protein. Our results have significant implications for diseases where protein misfolding and amyloid formation are major features and suggest that it is premature to define an amyloid protein as infectious based primarily on its ability to self-replicate. This study was published in the journal PLoS One in 2013.