Role of Prion Protein in Manganese Neurotoxicity

描述(由申请人提供)：环境中接触过渡金属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加剧关键的退行性变化，包括离子失衡、氧化应激和蛋白质聚集。一些金属结合蛋白调节细胞内金属动态平衡，从而维持正常的细胞功能。新出现的证据表明，Prion蛋白是一种金属结合蛋白，可以有效地与某些二价阳离子结合，包括蛋白质八肽重复区域的铜和锰。因此，金属动态平衡的失调被认为在Pron疾病的发病机制中发挥了作用。最近对普恩病毒感染的人和动物脑和血液中锰含量升高的观察表明，锰的神经毒性可能在普恩病毒疾病的病因中起作用。最近，我们证明了正常的Prion蛋白可以有效地减弱锰向神经细胞的转运，并对锰诱导的氧化应激、线粒体功能障碍、细胞抗氧化剂耗尽和细胞凋亡具有保护作用。在研究这些机制时，我们出人意料地发现，锰处理不依赖于转录而上调细胞内Prion的水平。此外，我们还发现，锰增加了稳定性，提示普恩蛋白可能促进了正常普恩蛋白(PrPC)向病理形式的普恩蛋白(PrPSc)的转化，从而导致正常普恩蛋白对锰神经毒性的保护功能丧失。因此，这一建议的中心假设是，锰与细胞蛋白的八肽(PHGGGWGQ)结构域结合，以增加蛋白质的稳定性和积累量。锰诱导的蛋白稳定化可以加速PrPC向抗蛋白酶的PrPSc聚合体的构象转化，从而导致神经毒性。将通过对下列特定目的的系统研究检验这一新的假说：i)确定慢性暴露于锰是否会增加动物模型中PrPSc的积累；ii)确定八肽重复序列在锰诱导的Prion蛋白稳定中的作用；iii)确定慢性锰暴露是否会加速PrPSc蛋白的积累和聚集，从而导致PrPSc蛋白的神经元损伤增加；iv)比较锰对PrPSc的积累和聚集以及对感染PrP的转基因动物(Tg20和TgPrPDOR转基因动物)神经元损伤的影响。总之，拟议的研究结果不仅将为Pron蛋白在锰神经毒性中的作用提供新的见解，而且将促进对金属在Pron疾病发病机制中的作用的理解。 与公共健康相关：锰是生物体正常运作所必需的一种微量元素；然而，持续接触高浓度的锰会导致不利的神经功能障碍。由于蛋白质中的八肽重复序列对包括锰在内的二价阳离子有很高的亲和力，因此该蛋白是一种可能的金属蛋白，其结合部位可能在Pron疾病的发病机制中发挥了作用。在人类和动物的Pron病的血液和脑中都观察到了锰含量的变化。此外，Pron蛋白在锰神经毒性中的作用目前尚不清楚。本研究的目的是在动物模型中确定蛋白质与锰的相互作用机制，以阐明蛋白质病的病理生理机制。这项研究的结果不仅将为了解Pron蛋白在锰神经毒性中的作用提供新的见解，而且将有助于加深对金属在Pron疾病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. PUBLIC HEALTH RELEVANCE: Manganese (Mn) is an essential trace elemental metal required by organisms for normal functioning; however, continued exposure to high concentrations of Mn results in adverse neurological deficits. The cellular prion protein is a putative metalloprotein since the octapeptide repeat sequences in the protein have high affinity for divalent cations including manganese, and the binding sites are suggested to play a role in the pathogenesis of prion diseases. Altered Mn content has been observed in the blood and brain of both human and animal prion diseases. Also, the role of prion protein in manganese neurotoxicity is currently unknown. Our proposal aims to determine the mechanisms of Mn interaction with prion protein in animal models to elucidate the pathophysiological mechanisms of prion diseases. The results of this study 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.