Role of Prion Protein in Manganese Neurotoxicity

描述（由申请人提供）：过渡金属的环境暴露与各种神经退行性疾病的病理过程有关，因为金属神经毒性通常会增强关键的退行性变化，包括离子失衡、氧化应激和蛋白质聚集。几种金属结合蛋白调节细胞内金属稳态，从而维持正常的细胞功能。新出现的证据表明，朊病毒蛋白是金属结合蛋白，可以有效地结合到某些二价阳离子，包括铜和锰的八肽重复区的蛋白质。因此，金属稳态失调已被认为在朊病毒疾病的发病机制中发挥作用。最近观察到患有朊病毒疾病的人类和动物的脑和血液中锰（Mn）水平升高，表明锰神经毒性可能在朊病毒疾病的病因学中起作用。最近，我们证明，正常朊病毒蛋白有效地减弱锰转运到神经元细胞，并保护对锰诱导的氧化应激，线粒体功能障碍，细胞抗氧化剂消耗和细胞凋亡。在研究这些机制时，我们意外地发现锰处理上调细胞朊病毒水平，而不依赖于转录。此外，我们发现锰增加了朊蛋白的稳定性，这表明朊蛋白可能促进正常朊蛋白（PrPC）转化为朊蛋白（PrPSc）的病理形式，这导致正常朊蛋白对锰神经毒性的保护功能丧失。因此，该提议的中心假设是锰与细胞朊病毒蛋白的八肽（PHGGGWGQ）结构域结合以增加蛋白的稳定性和积累。锰诱导的朊病毒蛋白的稳定化加速了PrPC向蛋白酶抗性朊病毒蛋白（PrPSc）聚集体的构象转化，从而诱导神经毒性。这一新的假设将通过对以下具体目标的系统研究进行检验：i）确定慢性暴露于锰是否增加动物模型中朊病毒蛋白的积累，ii）确定八肽重复序列在锰诱导的朊病毒蛋白稳定化中的作用，iii）确定慢性锰暴露是否加速羊瘙痒症形式的朊病毒蛋白（PrPSc）的积累和聚集并在朊病毒病的小鼠模型中引起增加的神经元损伤，iv）比较锰对PrPSc的积累和聚集以及对小鼠羊瘙痒病感染的朊病毒过表达和八肽缺失转基因动物（Tg20和TgPrPDOR转基因小鼠）中神经元损伤的影响。总之，从拟议的研究结果不仅将提供新的见解朊病毒蛋白在锰神经毒性的作用，但也将推进金属的朊病毒疾病的发病机制中的作用的理解。

DESCRIPTION (provided by applicant): Environmental exposure to transition metals is linked to pathological processes of various neurodegenerative conditions since metal neurotoxicity often augments key degenerative changes including ionic imbalance, oxidative stress and protein aggregation. Several metal binding proteins regulate intracellular metal homeostasis and thereby maintain normal cellular function. Emerging evidence indicates that prion proteins are metal binding proteins that can efficiently bind to certain divalent cations including copper and manganese at the octapeptide repeat regions of the protein. Therefore, dysregulation of metal homeostasis has been suggested to play a role in the pathogenesis of prion diseases. Recent observations of elevated manganese (Mn) levels in the brain and blood of humans and animals afflicted with prion diseases suggest that manganese neurotoxicity may play a role in the etiology of prion diseases. Recently, we demonstrated that normal prion protein effectively attenuates manganese transport into neuronal cells and protects against manganese-induced oxidative stress, mitochondrial dysfunction, cellular antioxidant depletion, and apoptosis. While investigating these mechanisms, we unexpectedly found that manganese treatment upregulates cellular prion levels independent of transcription. Furthermore, we found manganese increases stability, suggesting that prion protein may promote the conversion of normal prion protein (PrPC) to the pathological form of prion (PrPSc), which results in the loss of normal prion protein's protective function against manganese neurotoxicity. Thus, the central hypothesis of this proposal is that manganese binds to the octapeptide (PHGGGWGQ) domain of cellular prion protein to increase the stability and accumulation of the protein. Manganese-induced stabilization of prion protein accelerates conformational conversion of PrPC to proteinase-resistant prion protein (PrPSc) aggregates and thereby induces neurotoxicity. This novel hypothesis will be tested through a systematic investigation of the following specific aims: i) to determine whether chronic exposure to manganese increases prion protein accumulation in animal models, ii) to determine the role of octapeptide repeat sequences in the manganese-induced stabilization of prion protein, iii) to determine whether chronic manganese exposure accelerates the accumulation and aggregation of the scrapie form of prion protein (PrPSc) and causes increased neuronal damage in a mouse model of prion disease, iv) to compare the effect of manganese on the accumulation and aggregation of PrPSc and on neuronal damage in mouse scrapie-infected prion overexpressing and octapeptide deletion transgenic animals (Tg20 and TgPrPDOR transgenic mice). Together, results from the proposed studies will not only provide new insights into the role of prion protein in manganese neurotoxicity but also will advance understanding of the role of metals in the pathogenesis of prion diseases.