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Copper's Role in Brain LRP-mediated Abeta Efflux and Aging

铜在大脑 LRP 介导的 Abeta 流出和衰老中的作用

基本信息

批准号：
7800884
负责人：
RASHID DEANE
金额：
$ 25万
依托单位：
UNIVERSITY OF ROCHESTER
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-04-01 至 2013-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7800884
关键词：
Affect Affinity Age Alzheimer&apos s Disease Amyloid Amyloid beta-Protein Precursor Apoptosis Binding Blood - brain barrier anatomy Blood capillaries Brain Cerebrospinal Fluid Cerebrum Copper Data Dose Down-Regulation Endothelial Cells Endothelium Environmental Risk Factor Enzyme-Linked Immunosorbent Assay Human In Vitro Incubated Insulinase Intercellular Fluid Ions LDL-Receptor Related Protein 1 Lipoprotein Receptor Low Density Lipoprotein Receptor Mediating Metabolic Clearance Rate Metals Molecular Conformation Mus Natural graphite Neprilysin Neurodegenerative Disorders Oryctolagus cuniculus Peptide Hydrolases Peptides Plasma Production Protein Isoforms Proteins Research Proposals Role Sampling Senile Plaques Site Testing Time Toxic effect Tracer absorption age effect angiogenesis beta pleated sheet beta-site APP cleaving enzyme 1 capillary drinking water in vivo insight monomer neurotoxic normal aging novel therapeutics protein aggregate receptor

项目摘要

DESCRIPTION (provided by applicant): Accumulation of neurotoxic amyloid-¿ (A¿) species in brain is accelerated in AD. A¿ clearance by rapid transport across the blood-brain-barrier (BBB), requires LRP (low-density lipoprotein receptor-related protein1), the main receptor that clears A¿ from brain in an isoform specific manner. Copper (Cu), the focus of this research proposal, is associated with amyloid plaques in AD brains. It avidly binds A¿ and may promote betasheet conformation, aggregation and toxicity. Cu may reduce A¿ elimination from brain in rabbits, dosed with tracer levels of Cu. Our preliminary data using mice dosed with tracer levels of Cu in their drinking water showed: 1) increased Cu levels and decreased LRP protein levels in brain microvessels, 2) increased brain A¿ levels by reducing its BBB clearance and 3) no significant changes in protein levels of APP, BACE, IDE, NEP or A¿ putative receptors such as PgP and RAGE in brain microvessels. HBEC incubated with Cu (200 nM) had decreased 125I-A¿42 binding associated with LRP down-regulation, increased LRP nitrotyrosination, and enhanced LRP proteosomal degradation. In contrast, Cu did not affect HBEC-mediated angiogenesis, apoptosis, NF-?B activation. Al3+, Zn2+ and Fe3+, did not reduce LRP protein levels in HBEC at non-toxic concentrations. We hypothesize that A¿ clearance across the BBB is reduced in normal mice dosed with tracer levels of Cu due to Cu-induced decrease in LRP levels in cerebral endothelium, the BBB site in vivo, and that this effect is potentiated with normal aging. Four aims are proposed. Aim 1. The role of Cu on LRPmediated soluble A¿ monomers clearance across the normal mouse BBB in vivo and effect of aging. Aim 2. The role of Cu on LRP-mediated soluble A¿ oligomers clearance across the mouse BBB in vivo and effect of aging. Aim 3. The role of Cu on soluble A¿ (monomers and oligomers) LRP binding and internalization in mouse brain capillaries. Aim 4. The role of Cu on LRP levels, synthesis and turnover in human brain endothelial cells. We will study clearance of A¿40 and A¿42 monomers and oligomers from the CNS in vivo and determine efflux at the BBB in control and Cu-dosed mice, at selected ages (4,9,15 and 24 months, after dosing), as well as in vitro using isolated brain capillaries. Mouse endogenous A¿ levels in brain, cerebrospinal fluid (CSF) and plasma will be determined by ELISA. Cu levels in these samples and in brain microvessels will be determined by graphite furnace atomic absorption spectrophotometer. The effects of Cu on LRP levels, turnover and synthesis will be determined in brain endothelial cells. The proposed study will, for the first time, define Cu's role in the modulation of soluble A¿ transport from brain, and provide new therapeutic insights on how to lower brain A¿ by controlling its CNS barriers transport in Cu-potentiated neurodegenerative disorders. Project narrative: Accumulation of neurotoxic amyloid-¿ (A¿) species in brain is accelerated in AD. A¿ clearance by rapid transport across the blood-brain-barrier (BBB), requires LRP (low-density lipoprotein receptor-related protein1), the main receptor that clears A¿ from brain. Copper (Cu) is associated with amyloid plaques in AD brains. It avidly binds A¿ and may promote beta-sheet conformation, aggregation and toxicity. Our pilot data showed that mice, dosed with tracer levels of Cu in their drinking water, accumulates Cu in brain microvessels, and this effect is associated with down-regulation of LRP protein levels in these microvessels and increased A¿ retention in brain. We are proposing a new role for Cu. We suggest that Cu down-regulates LRP in brain microvessels, and that this effect contributes to brain A¿ accumulation. The proposed study will, for the first time, define Cu's role as an environmental factor in the modulation of soluble A¿ transport from brain. These studies may provide new therapeutic insights on how to lower brain A¿ by controlling its CNS barriers transport in Cu-potentiated neurodegenerative disorders.

描述(申请人提供)：神经毒性淀粉样蛋白(A)在大脑中的积累在AD时加速。通过血脑屏障(BBB)的快速转运清除需要LRP(低密度脂蛋白受体相关蛋白1)，LRP是以异构体特异性方式从大脑中清除A的主要受体。铜(铜)，这项研究建议的重点，与AD大脑中的淀粉样斑块有关。它能强烈地结合A？，并可能促进Betasheet的构象、聚集和毒性。在示踪水平的铜剂量下，铜可减少兔脑中的A？排泄。我们在饮水中加入示踪剂铜的小鼠的初步数据显示：1)增加了脑微血管中铜的水平，降低了LRP蛋白的水平，2)通过降低血脑屏障的清除率提高了脑A的水平，3)APP、BACE、IDE、NEP的蛋白水平没有显著变化或脑微血管中可能的受体，如Pgp和RAGE。与铜(200 NM)孵育的HBEC减少了与LRP下调相关的125I-A？42结合，增加了LRP亚硝基酪氨酸化，并促进了LRP蛋白酶体的降解。相反，铜不影响HBEC介导的血管生成、细胞凋亡和核因子？B的激活。Al~(3+)、Zn~(2+)和Fe~(3+)在无毒浓度下对HBEC LRP蛋白水平无明显影响。我们假设，由于铜诱导体内血脑屏障部位--脑内皮细胞LRP水平的降低，服用示踪剂铜的正常小鼠血脑屏障上的A清除量减少，并且这种影响随着正常年龄的增加而增强。提出了四个目标。目的1.铜对LRP介导的可溶性A单体体内清除正常小鼠血脑屏障的作用及衰老的影响。目的2.铜对LRP介导的小鼠血脑屏障清除可溶性A？寡聚体的作用及衰老的影响。目的3.铜对小鼠脑毛细血管中可溶性单体和寡聚体LRP结合和内化的作用。目的4.铜对人脑内皮细胞LRP水平、合成和转运的影响。我们将在体内研究A？40和A？42单体和低聚物从中枢神经系统中的清除，并在选定的年龄(给药后4、9、15和24个月)测定对照组和铜剂量组小鼠血脑屏障的外流，以及使用分离的脑毛细血管进行体外测定。用双抗体夹心法测定小鼠脑、脑脊液和血浆中内源性Aβ水平。这些样品和脑微血管中的铜含量将用石墨炉原子吸收光谱仪测定。铜对脑血管内皮细胞LRP水平、周转和合成的影响将被确定。这项拟议的研究将首次明确铜在调节脑内可溶性Aβ运输中的作用，并为如何在铜强化的神经退行性疾病中通过控制其中枢神经系统屏障运输来降低脑Aβ提供新的治疗见解。项目简介：神经毒性淀粉样蛋白在大脑中的积累在阿尔茨海默病中加速。通过血脑屏障(BBB)的快速转运清除需要LRP(低密度脂蛋白受体相关蛋白1)，LRP是从大脑中清除A的主要受体。铜与AD大脑中的淀粉样斑块有关。它热切地结合A？，并可能促进β-折叠构象、聚集和毒性。我们的实验数据显示，在饮水中加入示踪剂水平的铜的小鼠，在脑微血管中积累了铜，这种效应与这些微血管中LRP蛋白水平的下调和大脑中A的滞留增加有关。我们正在为铜提出一个新的角色。我们认为，铜可下调脑微血管中LRP的表达，这一作用有助于脑内Aβ的蓄积。这项拟议的研究将首次确定铜作为环境因素在调节脑中可溶性A的运输中所起的作用。这些研究可能为如何在铜强化的神经退行性疾病中通过控制其中枢神经系统屏障运输来降低脑Aé提供新的治疗见解。