Choroid Plexus as a Target in Metal-Induced Neurotoxicity

描述（由申请人提供）：特发性帕金森病（IPD）和锰（Mn）诱导的帕金森病已显示脑铁（Fe）稳态改变。目前的建议继续我们长期研究目标的中心主题，即探索脑屏障系统在金属诱导的神经毒性中的作用。二价金属转运蛋白-1 （DMT1）和金属转运蛋白-1 （MTP1）是新近发现的两种金属转运蛋白，具有跨细胞膜运输金属的功能。在进展报告中，我们已经证明DMT1和MTP1存在于血脑脊液屏障（BCB）所在的脉络丛中。我们还观察到，Mn暴露增加了DMT1的表达，并动员了BCB上皮中的亚细胞MTP1。然而，关于DMT1和MTP1在BCB中亚细胞共定位的位置，它们如何协同作用以响应BCB两侧的二价金属通量，Mn暴露通过什么机制改变这两种转运体的表达和功能，以及Mn暴露如何影响BCB中DMT1和MTP1的失调影响铁和锰的脑内稳态，这些问题仍然是个谜。因此，为了了解DMT1和MTP1在脑屏障中的结构功能及其功能障碍相关的神经元疾病，我们假设Mn暴露后脉络膜丛中DMT1和MTP1表达的改变导致了Mn诱导的脑脊液铁代谢紊乱。我们的具体目标是：(1)通过研究DMT1和MTP1在脉络膜上皮中的亚细胞位置，通过阻断或诱导DMT1和MTP1的表达来确定BCB中铁和锰的运输方向，并通过siRNA技术沉默编码DMT1和MTP1的基因来研究DMT1或MTP1敲除条件下铁和锰的摄取和运输动力学，探索DMT1和MTP1是否控制BCB中铁的运输方向；(2)利用大鼠慢性锰暴露模型和脑室-池灌注技术，探讨体内慢性锰暴露是否会扭曲BCB和特定区域血脑屏障中DMT1和MTP1的表达，并导致血液和脑脊液之间铁的通量增加；(3)探究Mn暴露是否会干扰铁调控蛋白与DMT1和MTP1 mRNA的结合，因为茎环结构分别存在于DMT1和MTP1 mRNA的3'-非翻译区（UTR）和5'-UTR。本申请中提出的研究将定义BCB中DMT1和MTP1之间的相互关系，包括它们的亚细胞位置，BCB中二价金属运输的作用，以及它们受Mn暴露影响的调节；将深入了解锰通过脑屏障影响二价铁运输的分子机制；并将最终更好地理解铁功能障碍相关的神经元疾病，如IPD。

DESCRIPTION (provided by applicant): Altered brain iron (Fe) homeostasis has been shown in idiopathic Parkinson's disease (IPD) and in manganese (Mn)-induced Parkinsonism. The current proposal continues the central theme of our long-time research goal, i.e., to explore the role of brain barrier systems in metal-induced neurotoxicities. Divalent metal transporter-1 (DMT1) and metal transport protein-1 (MTP1) are two newly discovered metal transporters and function to transport metals across the cell membrane. In the Progress Report, we have demonstrated the presence of DMT1 and MTP1 in the choroid plexus, where the blood-cerebrospinal fluid (CSF) barrier (BCB) is located. We have also observed that Mn exposure increases DMT1 expression and mobilizes subcellular MTP1 in the BCB epithelia. However, the questions as to where the DMT1 and MTP1 are subcellularly co-localized in the BCB, how they function in concert to respond to divalent-metal fluxes on both sides of the BCB, by what mechanism Mn exposure alters the expression and function of both transporters, and how the dysregulation of DMT1 and MTP1 in the BCB by Mn exposure affects brain homeostasis of Fe and Mn, remain mysterious. Thus, to understand the structural functionality of DMT1 and MTP1 in the brain barrier and their dysfunction-associated neuronal disorders, we hypothesize that the altered expression of DMT1 and MTP1 in the choroid plexus following Mn exposure contributes to Mn- induced Fe metabolism disorder in the CSF. Our specific aims are: (1) to explore whether DMT1 and MTP1 control the direction of Fe transport at the BCB by investigating the subcellular location of DMT1 and MTP1 in choroidal epithelia, by blocking or inducing DMT1 and MTP1 expression to determine the direction of Fe and Mn transport at BCB, and by using siRNA technique to silence the genes encoding DMT1 and MTP1 to investigate Fe and Mn uptake and transport kinetics under DMT1 or MTP1 knock-down conditions; (2) to explore whether in vivo chronic Mn exposure distorts the expression of DMT1 and MTP1 in the BCB and selected regional blood-brain barrier and leads to increased fluxes of Fe between the blood and CSF, by using a rat chronic Mn exposure model and by a ventriculo-cisternal perfusion technique; and (3) to explore whether Mn exposure interferes the binding of iron-regulatory proteins to mRNAs of DMT1 and MTP1, since the stem-loop structure exists in 3'-untranslated regions (UTR) and 5'-UTR in DMT1 and MTP1 mRNA, respectively. Studies proposed in this application will define the inter-relationship between DMT1 and MTP1 in the BCB with regard to their subcellular locations, roles in transport of divalent metals at the BCB, and their regulation as affected by Mn exposure; will provide insight into the molecular mechanism by which Mn affects divalent Fe transport by brain barriers; and will ultimately provide a better understanding of Fe dysfunction-related neuronal diseases such as IPD.