Role of RanBP9 on dendritic and spine injury in an Alzheimer's mouse model

项目摘要/摘要 阿尔茨海默病S病(AD)的特征是淀粉样蛋白(AB)的沉积，这是一种源于 从淀粉样前体蛋白(APP)的B-和Y-分泌酶裂解。我们最近展示了 不含NPXY的低密度脂蛋白受体相关蛋白的最后37个氨基酸(LRP-C37) 主题是必要的和足够的，以增加AB&61472；产量。因为LRP-C37本身就是一种有效的诱导剂 AB&#61472；生产中，我们将该结构域作为诱饵在酵母双杂交筛选中进行，从而鉴定了Ranbinding. 蛋白M(RanBP9)。事实上，APP和RanBP9-FL或FL衍生的RanBP9-FL的瞬时转染- N60在不同的细胞系中显著增加AB&61472；的分泌，表明RanBP9改变了APP的新陈代谢。 最重要的是，对RanBP9蛋白水平的免疫印迹定量表明，RanBP9-N60和 AD患者和APP J20转基因患者脑内RanBP9-FL分别升高6倍和4倍以上 分别为小鼠。我们还发现，与LRP及其两个关键配体一样，RanBP9也与基因相关 患有晚发性阿尔茨海默病。为了更好地了解RanBP9在AD发病机制中的体内作用，我们 作为正在进行的NIH R03拨款的一部分，产生了在大脑中过度表达RanBP9的转基因小鼠。通过 携带APPsWE、PSEN1dE9突变的B6C3-Tg85Dbo小鼠(APdE9)与RanBP9转基因小鼠的杂交 小鼠，RanBP9/APdE9三转基因小鼠产生了更多的CHAPSO可溶性AB&#61472； C末端片段(CTF)与3个月龄的APdE9小鼠进行比较，表明RanBP9 增加体内APP的淀粉样蛋白形成过程。本R01方案是R03项目的延伸。 由于突触丢失与阿尔茨海默病患者认知障碍的程度有更好的相关性，S自 RanBP9在神经突起中大量存在，是神经突起生长的强有力的抑制物，我们接下来 我想在这个建议中检查RanBP9诱导的APP变化处理是否也会导致树突状细胞 和脊椎受伤。我们已成功培育出RanBP9转基因小鼠和杂合子缺失小鼠 这是第一次。我们建议比较树突分枝的模式，脊椎密度，突触前和 海马区和额叶皮质突触后蛋白水平以及学习和记忆测试 2、5和10月龄八组小鼠的技能，即RanBP9-629单转基因、APdE9双转基因 转基因，RanBP9-629/APdE9三重转基因，RanBP9-599单一转基因，RanBP9-599/APdE9三重 转基因、RanBP9-/-或RanBP9/-、RanBP9-/-或RanBP9/-/APdE9和野生型产仔对照。 用于自动脊椎密度分析的软件Neuron Studio将用于分析树突分支 激光成像后荧光黄染色锥体神经元的点数和棘数 扫描共聚焦显微镜。如果在这项研究中RanBP9被确认为体内的一个真正的靶点，那么三重 转基因小鼠作为突触和行为缺陷的加速模型可能被证明是有用的。

Project Summary/Abstract Alzheimer¿s disease (AD) is characterized by the deposition of amyloid Bprotein (AB), a small peptide derived from B- and y-secretase cleavages of the amyloid precursor protein (APP). We recently demonstrated that the last 37 amino acids (LRP-C37) of low-density lipoprotein receptor-related protein (LRP) without the NPXY motifs to be necessary and sufficient to increase ABproduction. Since LRP-C37 alone was a potent inducer of ABproduction, we used this domain as bait in a yeast 2-hybrid screen, resulting in the identification of Ranbinding protein M (RanBP9). Indeed transient transfections of APP and RanBP9-FL or FL-derived RanBP9- N60 robustly increased secretion of ABin varieties of cell lines, indicating that RanBP9 alters APP metabolism. Most importantly, immunoblot quantification of RanBP9 protein levels demonstrated that RanBP9-N60 and RanBP9-FL were elevated more than six and four-folds in the brains of AD patients and APP J20 transgenic mice respectively. We also found that like LRP and two of its key ligands, RanBP9 is genetically associated with late-onset AD. To gain a better insight on the in vivo role of RanBP9 in the pathogenesis of AD, we generated transgenic mice over expressing RanBP9 in the brain as a part of an ongoing NIH R03 grant. By crossing B6C3-Tg85Dbo mice (APdE9) carrying APPswe, PSEN1dE9 mutations with RanBP9 transgenic mice, RanBP9/APdE9 triple transgenic mice were generated, which produced more CHAPSO-soluble ABand c-terminal fragments (CTFs) compared to APdE9 mice as early as 3 months of age, suggesting that RanBP9 increases amyloidogenic processing of APP in vivo. This R01 proposal is an extension of the R03 project. As loss of synapses is a better correlate of the extent of cognitive deficits in Alzheimer¿s patients and since RanBP9 is present in substantial amounts in neurites and is a strong inhibitor of neurite outgrowth, we next want to examine in this proposal, whether RanBP9-induced altered processing of APP also leads to dendritic and spine injury. We have successfully produced RanBP9 transgenic mice as well as heterozygous null mice for the first time. We propose to compare the pattern of dendritic arborization, spine density, presynaptic and postsynaptic protein levels in the hippocampus and frontal cortex followed by tests for learning and memory skills at 2, 5 and 10 months of age in eight groups of mice, i.e., RanBP9-629 single transgenic, APdE9 double transgenic, RanBP9-629/APdE9 triple transgenic, RanBP9-599 single transgenic, RanBP9-599/APdE9 triple transgenic, RanBP9-/- or RanBP9+/-, RanBP9-/- or RanBP9+/-/APdE9 and wild type litter-mate controls. Neuron Studio, software for automated spine density analysis, will be used to analyze dendritic branching points and spine numbers in Lucifer-yellow-stained pyramidal neurons after obtaining images by laser scanning confocal microscope. If RanBP9 is confirmed as a bona fide target in vivo in this study, the triple transgenic mice may prove to be useful as an accelerated model for synaptic and behavioral deficits.