Altered Copper Neuronal Transport and Alzheimer's Disease

项目摘要 我推测，与海马铜相关的载脂蛋白E（APOE）失调 (Cu)代谢是阿尔茨海默病（AD）发病机制中的关键步骤。该项目将 从两个具体目标探讨这一主题：1）建立铜（I） 2）研究APOE 4-改变对AD大鼠海马CA 1区的影响， 突触修剪和铜沉积在小鼠海马。这种方法是创新的， 它使用两种新开发的技术，将用于研究海马铜 调节异常、突触密度及其与毒性APOE 4等位基因的关系。CRISP-17 荧光探针是组织化学Cu定位的最新工具，具有特异性， 一价Cu远超过其他现有探针。CRISP-17将可视化铜分布后， 尸检人AD海马脑组织。将分离突触体组分， 使用原子发射光谱法测量Cu。铜转运蛋白将是 在SH-SY 5 Y神经母细胞瘤细胞中下调，分析对Cu沉积的影响 使用荧光探针和突触体部分被分离并测量Cu 程度.将在APOE 4基因敲入小鼠中研究突触密度，并与对照组进行比较。 神经保护性APOE 3基因敲入小鼠，PET扫描靶向SV 2A突触蛋白。 然后取小鼠脑组织，并使用组织化学法分析单价Cu。 CRISP-17，在动物模型中验证了这种关系。我们预计会发现， APOE 4基因敲入小鼠的突触密度与未敲入小鼠相比大大降低。 神经保护性APOE 3基因敲入小鼠，小鼠海马也表现出减少 Cu分布。这些目标旨在通过调查一个 AD的可能途径，这种疾病的发病机制尚未完全确定。

Project Summary I hypothesize that apoplioprotein E (APOE)-associated dysregulation of hippocampal copper (Cu) metabolism is a key step in Alzheimer’s disease (AD) pathogenesis. This project will approach this topic from two specific aims: 1) establish the relationship between copper (I) cation distribution and AD pathology, and 2) study the APOE4-alteration effect on hippocampal synaptic pruning and Cu deposition in the mouse hippocampus. This approach is innovative in its use of two newly developed technologies that will be used to investigate hippocampal Cu dysregulation, synaptic density and its relation to the toxic APOE4 allele. The CRISP-17 fluorescent probe is a state-of-the-art tool in histochemical Cu localization, with a specificity for monovalent Cu far beyond other existing probes. CRISP-17 will visualize Cu distribution in post- mortem human AD hippocampal brain tissue. Synaptosome fractions will be isolated and measured for Cu using atomic emissions spectroscopy. Cu transport proteins will be downregulated in SH-SY5Y neuroblastoma cells, with effects on Cu deposition being analyzed using the fluorescent probe and synaptosome fractions being isolated and measured for Cu levels. Synaptic density will be studied in APOE4 knock-in mice and compared with the neuroprotective APOE3 knock-in mice with PET scans targeting the SV2A synaptic protein. Mouse brain tissue will then be taken and histochemically analyzed for monovalent Cu using CRISP-17, verifying this relationship in the animal model. We expect to find similarly that APOE4 knock-in mice have a greatly reduced synaptic density when compared to the neuroprotective APOE3 knock-in mice, with the mouse hippocampus also exhibiting a decrease in Cu distribution. These aims are designed to further the goals of the NIH by investigating a possible pathway to AD, a disease whose pathogenesis not yet fully defined.