Effect of RanbpM on amyloid pathology in a mouse model of Alzheimer's disease

RanbpM 对阿尔茨海默病小鼠模型淀粉样蛋白病理学的影响

基本信息

批准号：
7449214
负责人：
Madepalli Krishnappa Lakshmana
金额：
$ 6.57万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
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：
2008-09-01 至 2010-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7449214
关键词：
55-kDa Ran-binding protein Adaptor Signaling Protein Address Affect Age-Months Alzheimer&apos s Disease Americas Amino Acids Amyloid Amyloid Proteins Amyloid beta-Protein Amyloid beta-Protein Precursor Amyloid deposition Animal Model Binding Proteins Biochemical Biology Brain C-terminal Cells Cleaved cell Co-Immunoprecipitations Complex Cultured Cells Cytoplasmic Tail Data Deposition Disease Enzyme-Linked Immunosorbent Assay Foundations Future Grant Hippocampus (Brain)Hybrids Immunoblotting Intervention Intracellular Membranes Investigation LDL-Receptor Related Protein 1 Laboratories Late Onset Alzheimer Disease Length Ligand Binding Domain Ligands Link Lipoprotein Receptor Macroglobulins Mammalian Cell Membrane Membrane Protein Traffic Metabolism Mind Molecular Weight Mus Mutant Strains Mice N-terminal Nerve Degeneration Neurofibrillary Tangles Neurons Pathogenesis Pathology Patients Peptides Production Protein Binding Proteins Public Health Research Research Personnel Role SNAPIN gene Screening Result Senile Plaques Signal Transduction Site Testing Transfection Transgenic Mice Transgenic Organisms Variant Yeasts amyloid precursor protein processing apolipoprotein E-2 design extracellular in vivo insight intracellular protein transport mouse model multiple myeloma M Protein mutant novel novel therapeutics postnatal protein function protein metabolism protein transport research study secretase stable cell line therapeutic target trafficking

项目摘要

DESCRIPTION (provided by applicant): The pathological hallmarks of Alzheimer's disease (AD) are the presence of senile plaques, largely composed of extracellular deposits of amyloid beta (A2) peptide and intracellular neurofibrillary tangles. Increasing evidence suggests that the low density lipoprotein receptor-related protein (LRP) facilitates amyloidogenic processing of APP. Within LRP, we defined the last 37 C-terminal residues of LRP (LRP-C37) lacking the NPxY or YxxL domains sufficient to robustly promote A2 production. The role of LRP-C37 domain in LRP function is largely unknown. Since LRP-C37 alone was a potent inducer of A2 production, we used this domain as bait in a yeast 2-hybrid screen, resulting in the identification of Ran-binding protein M (RanbpM), implicated in intracellular membrane trafficking and signaling. RanbpM interacted with both LRP and APP in transfected cells and mouse brain homogenates. Over-expression of RanbpM also enhanced the amount of LRP/APP complex formation in mammalian cells, suggesting a role for RanbpM as an adaptor protein that facilitates the tripartite interaction among APP, RanbpM and LRP. When 90 kDa full-length RanbpM (RanbpM-FL) was over-expressed in stable cell lines, a proteolytically cleaved N-terminal 55 kDa fragment (N-55) was also generated, which interacted strongly with both APP and LRP even more than the full-length RanbpM. This proteolytically cleaved species of RanbpM was elevated by 6-fold in brains of AD patients. Indeed, over-expression of RanbpM-FL or the N55 fragment markedly increased A2 secretion. We hypothesize that RanbpM alters APP metabolism in vivo, in particular via the N55 fragment of RanbpM. The specific aims of this application are to first generate transgenic mice with neuronal expression of RanbpM-FL and RanbpM-N55 and then to cross with mice over-expressing APP to obtain RanbpM/APP double transgenic mice. Whether over-expression of RanbpM variants alter amyloidogenic processing of APP will be determined by quantifying the levels of A2 and amyloid plaques by biochemical and immunohistochemical examinations in the cortex and hippocampus of Tg RanbpM/APP double transgenic mice. The level of CHAPS-soluble and insoluble A2 will be quantified by ELISA at 2 and 10 months of age. The characterization of novel APP and LRP-interacting proteins that promote amyloidogenic processing of APP will reveal novel sites of pathogenesis and targets for anti-amyloid intervention. We believe that RanbpM represents a potential therapeutic target. In addition, the RanbpM transgenic mice generated through this R03 granting mechanism is expected to hold substantial extrinsic merit, as they will be undoubtedly be useful to many researchers who study other aspects of RanbpM. This application may also lay the foundation for a future R01 investigation on the role of RanbpM in neurodegeneration. PUBLIC HEALTH RELEVANCE: Alzheimer's disease (AD), a disease characterized by toxic amyloid (A2) accumulation in brain, affects about 5 million people in America, but despite extensive research, available treatments provide only limited symptomatic relief. Recently we found a protein called RanbpM that enhances the production of amyloid/A2 in cells cultured in the laboratory. In this application, we will express RanbpM in brains of mice to confirm the findings in animal models in the hopes of that new insights may be uncovered for therapeutically targeting amyloid production in AD, in particular with RanbpM as a therapeutic target in mind.

描述（由申请人提供）：阿尔茨海默病（AD）的病理特征是存在老年斑，主要由淀粉样β（A2）肽的细胞外沉积物和细胞内神经原纤维缠结组成。越来越多的证据表明，低密度脂蛋白受体相关蛋白 (LRP) 促进 APP 的淀粉样蛋白形成过程。在 LRP 中，我们定义了 LRP (LRP-C37) 的最后 37 个 C 端残基，缺乏足以强有力地促进 A2 产生的 NPxY 或 YxxL 结构域。 LRP-C37 结构域在 LRP 功能中的作用很大程度上未知。由于单独的 LRP-C37 是 A2 产生的有效诱导剂，我们在酵母 2 杂交筛选中使用该结构域作为诱饵，从而鉴定出与细胞内膜运输和信号传导有关的 Ran 结合蛋白 M (RanbpM)。 RanbpM 与转染细胞和小鼠脑匀浆中的 LRP 和 APP 相互作用。 RanbpM 的过表达还增加了哺乳动物细胞中 LRP/APP 复合物形成的量，表明 RanbpM 作为衔接蛋白的作用，促进 APP、RanbpM 和 LRP 之间的三方相互作用。当 90 kDa 全长 RanbpM (RanbpM-FL) 在稳定细胞系中过表达时，还会生成一个蛋白水解切割的 N 端 55 kDa 片段 (N-55)，它与 APP 和 LRP 的相互作用强烈，甚至比全长 RanbpM。这种经蛋白水解裂解的 RanbpM 在 AD 患者的大脑中升高了 6 倍。事实上，RanbpM-FL 或 N55 片段的过度表达显着增加了 A2 的分泌。我们假设 RanbpM 改变体内 APP 代谢，特别是通过 RanbpM 的 N55 片段。本申请的具体目的是首先产生神经元表达RanbpM-FL和RanbpM-N55的转基因小鼠，然后与过表达APP的小鼠杂交获得RanbpM/APP双转基因小鼠。 RanbpM 变体的过度表达是否会改变 APP 的淀粉样蛋白形成过程，将通过对 Tg RanbpM/APP 双转基因小鼠的皮层和海马进行生化和免疫组织化学检查来量化 A2 和淀粉样蛋白斑的水平来确定。 CHAPS 可溶性和不溶性 A2 的水平将在 2 月龄和 10 月龄时通过 ELISA 进行定量。促进 APP 淀粉样蛋白形成过程的新型 APP 和 LRP 相互作用蛋白的表征将揭示新的发病机制位点和抗淀粉样蛋白干预的靶点。我们相信 RanbpM 代表了一个潜在的治疗靶点。此外，通过这种 R03 授予机制产生的 RanbpM 转基因小鼠预计将具有巨大的外在优点，因为它们无疑对许多研究 RanbpM 其他方面的研究人员有用。该应用也可能为未来关于 RanbpM 在神经退行性疾病中的作用的 R01 研究奠定基础。公共健康相关性：阿尔茨海默氏病 (AD) 是一种以大脑中有毒淀粉样蛋白 (A2) 积累为特征的疾病，影响着大约 500 万人，但尽管进行了广泛的研究，现有的治疗方法只能缓解有限的症状。最近我们发现了一种名为 RanbpM 的蛋白质，它可以增强实验室培养的细胞中淀粉样蛋白/A2 的产生。在此应用中，我们将在小鼠大脑中表达 RanbpM，以证实动物模型中的发现，希望能够为治疗 AD 中淀粉样蛋白的产生找到新的见解，特别是以 RanbpM 作为治疗靶点。