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

RanBP9 对阿尔茨海默病小鼠模型树突和脊柱损伤的作用

• 批准号: 8045453
• 负责人: Madepalli Krishnappa Lakshmana
• 金额: $ 28.69万
• 依托单位: TORREY PINES INST FOR MOLECULAR STUDIES
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8045453
• 关键词: 55-kDa Ran-binding protein; Address; Adhesions; Age; Age-Months; Alzheimer' s Disease; Amino Acids; Amyloid; Amyloid Proteins; Amyloid beta-Protein Precursor; Amyloid deposition; Apolipoprotein E; Appearance; Back; Behavioral; Binding; Binding Proteins; Brain; Brain region; Cell Line; Cell Nucleus; Cell Surface Receptors; Cell membrane; Cell surface; Cellular Morphology; Characteristics; Cognitive deficits; Complex; Computer software; Cytoplasm; Dendritic Spines; Formic Acids; Future; Generations; Goals; Grant; Growth; Hippocampus (Brain); Hybrids; Image; Immunoblotting; Intervention; Intracellular Membranes; Knockout Mice; LDL-Receptor Related Protein 1; Lasers; Late Onset Alzheimer Disease; Learning; Length; Ligands; Lipoprotein Receptor; Macroglobulins; Mediating; Membrane Microdomains; Membrane Protein Traffic; Memory; Memory Loss; Memory impairment; Microscope; Modeling; Molecular; Mus; Mutation; Neurites; Neurofibrillary Tangles; Neurons; Partner in relationship; Pathogenesis; Pathology; Patients; Pattern; Peptides; Production; Protein Binding; Proteins; Research; Risk Factors; Role; Scaffolding Protein; Scanning; Screening Result; Senile Plaques; Signal Transduction; Spinal Ganglia; Spinal Injuries; Staining method; Stains; Surface; Synapses; Testing; Therapeutic; Time; Transfection; Transgenic Mice; Transgenic Organisms; United States National Institutes of Health; Vertebral column; Yeasts; abstracting; amyloid precursor protein processing; base; beta-site APP cleaving enzyme 1; density; design; frontal lobe; genetic association; hippocampal pyramidal neuron; human RTN4 protein; improved; in vivo; innovation; insight; lucifer yellow; mouse model; multiple myeloma M Protein; neuronal cell body; novel; novel therapeutics; postsynaptic; presynaptic; protein B; protein metabolism; secretase; skills

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.

项目概要/摘要 阿尔茨海默病 (AD) 的特点是淀粉样 B 蛋白 (AB) 的沉积，这是一种小肽衍生的蛋白 来自淀粉样前体蛋白 (APP) 的 B 和 y 分泌酶裂解。我们最近证明了 不含 NPXY 的低密度脂蛋白受体相关蛋白 (LRP) 的最后 37 个氨基酸 (LRP-C37) 主题对于增加 AB 产量是必要且充分的。由于单独的 LRP-C37 是一种有效的诱导剂 AB 生产中，我们使用该结构域作为酵母 2 杂交筛选中的诱饵，从而鉴定出 Ranbind 蛋白 M (RanBP9)。事实上 APP 和 RanBP9-FL 或 FL 衍生的 RanBP9- 的瞬时转染 N60 显着增加多种细胞系中 AB 的分泌，表明 RanBP9 改变了 APP 代谢。 最重要的是，RanBP9 蛋白水平的免疫印迹定量表明 RanBP9-N60 和 AD 患者和 APP J20 转基因患者的大脑中 RanBP9-FL 升高了六倍和四倍以上 分别是老鼠。我们还发现，与 LRP 及其两个关键配体一样，RanBP9 与遗传相关 患有迟发性 AD。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 资助的一部分，他们培育出了在大脑中过度表达 RanBP9 的转基因小鼠。经过 将携带 APPswe、PSEN1dE9 突变的 B6C3-Tg85Dbo 小鼠 (APdE9) 与 RanBP9 转基因杂交 小鼠，产生了 RanBP9/APdE9 三重转基因小鼠，产生更多的 CHAPSO 可溶性 AB 和 早在 3 个月大时就与 APdE9 小鼠相比，发现了 c 末端片段 (CTF)，这表明 RanBP9 增加体内 APP 的淀粉样蛋白生成过程。该 R01 提案是 R03 项目的延伸。 由于突触的丧失与阿尔茨海默病患者的认知缺陷程度有更好的相关性，而且 RanBP9 大量存在于神经突中，并且是神经突生长的强抑制剂，我们接下来 希望在本提案中检查 RanBP9 诱导的 APP 加工改变是否也会导致树突 和脊柱损伤。我们已成功培育出RanBP9转基因小鼠以及杂合子无效小鼠 首次。我们建议比较树突分枝模式、树突棘密度、突触前和 海马和额叶皮层的突触后蛋白质水平，然后进行学习和记忆测试 八组小鼠在 2、5 和 10 月龄时的技能，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 单转基因、RanBP9-599/APdE9 三重 转基因、RanBP9-/- 或 RanBP9+/-、RanBP9-/- 或 RanBP9+/-/APdE9 和野生型同窝对照。 Neuron Studio，用于自动脊柱密度分析的软件，将用于分析树突分支 通过激光获得图像后，荧光黄染色的锥体神经元中的点和棘数量 扫描共焦显微镜。如果 RanBP9 在本研究中被确认为体内的真正靶标，则三重 转基因小鼠可能被证明可用作突触和行为缺陷的加速模型。