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

描述（由申请人提供）：AD中神经毒性淀粉样蛋白（A）物质在脑中的蓄积加速。A？通过快速运输穿过血脑屏障（BBB）的清除需要LRP（低密度脂蛋白受体相关蛋白1），LRP是以同种型特异性方式从脑中清除A的主要受体。铜（Cu），这项研究计划的重点，与AD大脑中的淀粉样斑块有关。它与A？结合，可能促进β-淀粉样蛋白构象、聚集和毒性。铜可能会减少A从兔脑中消除，剂量与示踪剂水平的铜。我们的初步数据显示，在小鼠饮用水中给予示踪剂水平的铜：1）增加脑微血管中的铜水平和降低LRP蛋白水平，2）通过降低BBB清除率增加脑A？水平，3）APP，BACE，IDE，NEP蛋白水平无显著变化 或脑微血管中假定的受体，如PgP和β-受体。与Cu（200 nM）孵育的HBEC降低了与LRP下调相关的125 I-A 42结合，增加了LRP硝基酪氨酸化，并增强了LRP蛋白体降解。与此相反，铜没有影响HBEC介导的血管生成，细胞凋亡，NF-？B激活。Al ~（3+）、Zn ~（2+）和Fe ~（3+）在无毒浓度下不降低HBEC中的LRP蛋白水平。我们假设A由于Cu诱导的脑内皮（体内BBB部位）中LRP水平的降低，在用示踪剂水平的Cu给药的正常小鼠中，穿过BBB的清除率降低，并且这种作用随着正常衰老而增强。提出了四个目标。目标1. Cu对LRP介导的可溶性A <$单体清除正常小鼠血脑屏障的作用及衰老效应目标2.铜在体内LRP介导的可溶性A ²寡聚体穿过小鼠BBB的清除中的作用以及衰老的影响。目标3.铜对小鼠脑毛细血管中可溶性A？（单体和寡聚体）LRP结合和内化的作用。目标4。铜对人脑内皮细胞LRP水平、合成和周转的作用。我们将研究A <$40和A <$42单体和寡聚体从CNS的体内清除，并确定对照组和Cu给药组小鼠在选定年龄（给药后4、9、15和24个月）以及体外使用分离的脑毛细血管在BBB的外排。将通过ELISA测定脑、脑脊液（CSF）和血浆中的小鼠内源性A？水平。将通过石墨炉原子吸收分光光度计测定这些样品和脑微血管中的Cu水平。将在脑内皮细胞中确定Cu对LRP水平、周转和合成的影响。这项拟议的研究将首次定义Cu在调节大脑可溶性A？转运中的作用，并为如何通过控制Cu增强的神经退行性疾病中的CNS屏障转运来降低大脑A？提供新的治疗见解。项目叙述：AD患者大脑中神经毒性淀粉样蛋白（A）物质的蓄积加速。A？通过快速运输穿过血脑屏障（BBB）进行清除，需要LRP（低密度脂蛋白受体相关蛋白1），这是从大脑中清除A的主要受体。铜（Cu）与AD脑中的淀粉样斑块相关。它强烈结合A？，并可能促进β-折叠构象、聚集和毒性。我们的试验数据表明，小鼠在饮用水中给予示踪剂水平的Cu，在脑微血管中积累Cu，这种效应与这些微血管中LRP蛋白水平的下调和脑中A？保留增加有关。我们正在为Cu提出一个新的角色。我们认为，铜下调LRP在脑微血管，这种影响有助于脑A的积累。这项拟议的研究将首次将Cu的作用定义为调节可溶性A从大脑转运的环境因素。这些研究可能会提供新的治疗见解，如何降低脑A？通过控制其中枢神经系统屏障运输铜增强神经退行性疾病。

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.