CHOROID PLEXUS AS A TARGET IN METAL-INDUCED NEUROTOXICITY

描述(申请人提供)：脑铜(铜)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Prion病有关。我们最近发现，人类队列中接触锰的工人血液和唾液中的铜水平显著升高，接触锰的动物的脑脊液和脑组织中的铜水平也显著升高。虽然铜转运蛋白-1(CTR1)、二价金属转运蛋白-1(DMT1)和ATP7A等转运体的存在可能与铜在脑屏障中的转运有关，但这些转运体是如何在脑屏障中转运的，以及暴露于锰(Mn)后这些转运体的表达和功能发生变化的机制尚不清楚。本研究旨在验证脉络丛通过关键转运蛋白调节血和脑脊液(CSF)之间铜转运的假说；暴露于锰会改变这些转运蛋白的功能，导致脑脊液中铜的稳态被扭曲。为了验证这一假设，我们设计了4个具体目标。在目标1中，我们将在一个大鼠模型中确定亚慢性暴露是否改变了血脑屏障(BBB)和血脑脊液屏障(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，从脑脊液到血液的铜外流减少。在目标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铜的摄取，以及锰暴露是否扰乱了这些过程，导致脑屏障细胞对铜的超载。AIM 3旨在研究ATP7A的细胞内转运是否决定了BCB转运铜的方向，以及锰暴露是否通过作用于ATP7A而改变铜在血液和脑脊液之间的转运方向。最后，在目标4中，我们将使用同步辐射快速扫描X射线荧光(RS-XRF)技术，与普渡大学物理系教授合作，建立三维模型，同时定位和定量暴露在锰暴露大鼠脑内的铜、铁、锰和锌。本申请中提出的研究将确定CTR1、DMT1和ATP7A在脑屏障中的亚细胞位置、在铜的运输中的作用以及它们在锰暴露影响下的调控等方面的相互关系；将有助于深入了解锰通过脑屏障影响铜运输的分子机制；并最终将更好地理解铜调节失调相关的神经退行性疾病。 公共卫生相关性：脑铜(铜)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Prion病有关。该项目将揭示铜是如何进出大脑的，以及暴露在环境毒物锰(Mn)中如何干扰大脑中的铜平衡，从而导致退行性锰帕金森症。

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.