Biology of Prion Protein and the TSE Diseases

Pron病或传染性海绵状脑病是人和动物的传染性神经退行性疾病。Prion病的一个主要特征是将正常的宿主蛋白Prion蛋白(PrP)折叠和聚集成疾病相关的蛋白酶抵抗形式(PrPres)，这可能会导致脑损伤。 有一种很强的物种屏障可以阻止或延缓跨物种的病毒感染，而这种屏障主要取决于不同物种PrP的氨基酸序列差异。例如，尽管有广泛的流行病学证据表明人类接触了羊痒病感染的绵羊组织，但人类似乎对羊痒病具有抵抗力。同样，小鼠适应的绵羊瘙痒病菌株尚不能感染表达人PrP的转基因小鼠，而这些小鼠对来自患有散发性克雅氏病的人类的普恩病毒株敏感。 在2015财年，我们测试了Pron蛋白C末端糖磷脂酰肌醇(GPI)膜锚定基团对鼠与人之间Pron物种屏障的影响。小鼠适应的绵羊瘙痒病(22L株)感染表达PrP的小鼠，在有或没有锚定GPI部分的情况下产生PrP库。然后对表达人PrP的tg66转基因小鼠进行不同稀释度的脑内注射，检测这些股票的相似小鼠感染滴度，并跟踪小鼠的临床瘙痒症状。在tg66小鼠中，没有观察到来自表达正常锚定PrP的小鼠的病毒。然而，tg66小鼠对表达无锚定PrP的小鼠的普恩敏感。因此，缺乏PrP上的GPI锚基似乎减少了人-鼠物种屏障的影响。这一结果依赖于人PrP在tg66小鼠中的高表达，而在低水平表达人PrP的小鼠中则看不到。 在2015财年，我们还研究了两种不同品系的小鼠(22L和RML)感染后临床前各时间点小鼠脑内的神经炎性反应。基因调控的改变通过使用SuperArray试剂盒使用实时定量RT-PCR分析脑RNA来定量，重点关注与炎症反应有关的基因，如细胞因子、细胞因子受体、JAK/STAT和IL6/STAT3通路。这些实验检测到这些阵列中15个以前未报道的基因上调，其中许多也被定量蛋白质分析证实。结果表明，22L株和RML株在感染后70d开始出现明显的神经炎性反应。尽管已知这两种菌株感染不同的大脑区域，但两种菌株上调了相似的基因。这一发现表明，在临床症状和空泡病理之前就可以检测到这种反应，这表明神经炎症可能参与了致病损害过程，并可能成为治疗干预的目标。

Prion diseases or transmissible spongiform encephalopathies are infectious neurodegenerative diseases of humans and animals. A major feature of prion diseases is the refolding and aggregation of a normal host protein, prion protein (PrP), into a disease-associated protease-resistant form (PrPres) which may contribute to brain damage. There is a strong species barrier which prevents or delays cross-species prion infection, and this barrier mostly depends on amino acid sequence differences in the PrP of various species. For example, humans appear to be resistant to sheep scrapie in spite of extensive epidemiological evidence of exposure of humans to scrapie-infected sheep tissues. Similarly mouse-adapted sheep scrapie strains are not known to infect transgenic mice expressing human PrP, whereas these mice are susceptible to prion strains from humans with sporadic Creutzfeldt-Jakob disease. In FY15 we tested the effect of the presence of the prion protein C-terminal glycophosphatidylinositol (GPI) membrane anchoring group on the prion species barrier between mice and humans. Prion stocks of mouse-adapted sheep scrapie (strain 22L) were generated by infection of mice expressing PrP with or without the anchoring GPI moiety. Similar mouse infectious titers of these stocks were then tested by intracerebral injection of various dilutions in tg66 transgenic mice expressing human PrP, and mice were followed for signs of clinical scrapie. No disease was observed in tg66 mice given prions from mice expressing normal anchored PrP. However, tg66 mice were susceptible to prions from mice expressing anchorless PrP. Thus the lack of the GPI anchor group on PrP appeared to reduce the effect of the mouse-human species barrier. This result was dependent on high expression of human PrP in tg66 mice and was not seen in mice expressing human PrP at lower levels. In FY15 we also studied the neuroinflammatory response in mouse brain at preclinical time-points after infection with 2 different strains of mouse prions (22L and RML). Alterations in gene regulation were quantitated by analysis of brain RNA using quantitative real-time RT-PCR with SuperArray kits focusing on genes involved in inflammatory responses, such as cytokines, cytokine receptors, JAK/STAT and IL6/STAT3 pathways. These experiments detected upregulation of 15 previously unreported genes in these arrays, and many of which were also confirmed by quantitative protein analysis. The results indicated that a prominent neuroinflammatory response occurred starting at 70 days post-infection for scrapie strains 22L and RML. Similar genes were upregulated with both strains even though these strains are known to infect different brain regions. The finding that this response was detectable prior to clinical signs and vacuolar pathology suggested that neuroinflammation might contribute to the pathogenic damage process, and might be a possible target for therapeutic intervention.