CHOROID PLEXUS AS A TARGET IN METAL-INDUCED NEUROTOXICITY

描述（由申请人提供）：脑铜（Cu）稳态改变与特发性帕金森病（IPD）、阿尔茨海默病（AD）、肌萎缩侧索硬化症（ALS）和朊病毒病有关。我们最近发现，暴露于锰的人类工人的血液和唾液中的铜水平显著升高，暴露于锰的动物的脑脊液和脑组织中的铜水平也显著升高。虽然已经证实在脑屏障中存在可能负责铜转运的转运体，如Cu转运蛋白-1 （Ctr1）、二价金属转运蛋白-1 （DMT1）和ATP7A，但这些转运体如何在脑屏障中转运铜，以及暴露于锰（Mn）改变这些转运体表达和功能的机制尚不清楚。该研究项目旨在验证以下假设：脉络膜丛是形成血液和脑脊液（CSF）之间屏障的脑组织，通过关键转运蛋白调节血液和CSF之间的Cu运输；暴露于锰会改变这些转运体的功能，导致脑脊液中铜稳态的扭曲。为了验证这一假设，我们设计了4个具体目标。在Aim 1中，我们将确定在大鼠模型中亚慢性暴露于Mn是否会改变血脑屏障（BBB）和血-CSF屏障（BCB）中Ctr1、DMT1和ATP7A的表达，从而导致从血液到脑实质的铜流入增加和从CSF到血液的铜流出减少。在Aim 2中，我们将揭示Ctr1和DMT1是否协调血脑屏障和血脑屏障表面的Cu摄取，以及Mn暴露是否破坏这些过程，导致脑屏障细胞对Cu的过载。Aim 3旨在研究ATP7A的细胞内转运是否决定BCB的铜转运方向，以及Mn暴露是否通过作用于ATP7A改变血液和CSF之间的铜转运方向。最后，在Aim 4中，我们将与普度大学物理系教授合作，使用同步加速器快速扫描x射线荧光（RS-XRF）技术，建立三维模型，同时定位和量化Mn暴露大鼠大脑中的Cu， Fe， Mn和Zn。本申请中提出的研究将确定脑屏障中Ctr1、DMT1和ATP7A之间的相互关系，包括它们在亚细胞位置、Cu运输中的作用以及它们受Mn暴露影响的调节；将提供对锰通过脑屏障影响铜转运的分子机制的见解；并将最终更好地理解铜调节失调相关的神经退行性疾病。

DESCRIPTION (provided by applicant): Altered brain copper (Cu) homeostasis has been associated with idiopathic Parkinson's disease (IPD), Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), and Prion disease. We have recently discovered that Cu levels in blood and saliva of Mn-exposed workers from human cohorts are significantly increased, and so are the Cu levels in the CSF and brain tissues of Mn- exposed animals. While the presence of transporters possibly responsible for Cu transport in brain barriers such as Cu transport protein-1 (Ctr1), divalent metal transporter-1 (DMT1) and ATP7A has been demonstrated, how Cu is transported by these transporters in brain barriers and by what mechanism exposure to manganese (Mn) alters the expression and function of these transporters are unknown. This research project is designed to test the hypothesis that the choroid plexus, a brain tissue forming a barrier between the blood and cerebrospinal fluid (CSF), regulates Cu transport between the blood and CSF through the critical transporters; exposure to Mn alters the functions of these transporters, leading to a distorted Cu homeostasis in the CSF. To test this hypothesis, we have designed 4 specific aims. In Aim 1, we will determine if subchronic exposure to Mn in a rat model alters the expression of Ctr1, DMT1 and ATP7A in blood-brain barrier (BBB) and blood-CSF barrier (BCB), leading to an increased influx of Cu from the blood to brain parenchyma and a decreased Cu efflux from the CSF to blood. In Aim 2, we will reveal if Ctr1 and DMT1 coordinate the Cu uptake on the surface of the BBB and BCB and if Mn exposure disrupts these processes, leading to cellular overload of Cu by the brain barrier cells. Aim 3 is designed to investigate if the intracellular trafficking of ATP7A determines the direction of Cu transport by the BCB and if Mn exposure, by acting on ATP7A, may alter the direction of Cu transport between the blood and the CSF. Finally, in Aim 4, we will use the synchrotron rapid scanning X-ray fluorescence (RS-XRF) technique, by collaboration with the professor in Purdue's Physics department, to establish 3D model to simultaneously localize and quantify Cu, Fe, Mn and Zn in brains of Mn-exposed rats. The studies proposed in this application will define the inter-relationship between Ctr1, DMT1 and ATP7A in brain barriers with regard to their subcellular locations, roles in transport of Cu, and their regulation as affected by Mn exposure; will provide the insight into the molecular mechanism by which Mn affects Cu transport by brain barriers; and will ultimately provide a better understanding of Cu dysregulation-related neurodegenerative diseases. PUBLIC HEALTH RELEVANCE: Altered brain copper (Cu) homeostasis has been associated with idiopathic Parkinson's disease (IPD), Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS) and Prion disease. This project will uncover how Cu is transferred in and out of the brain and how exposure to environmental toxicant manganese (Mn) interferes with the Cu balance in brain, which in turn causes the degenerative manganese parkinsonism.