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

描述(申请人提供)：阿尔茨海默病(AD)的病理特征是存在老年斑，主要由细胞外沉积的淀粉样β(A2)多肽和细胞内的神经纤维缠结组成。越来越多的证据表明，低密度脂蛋白受体相关蛋白(LRP)促进了APP的淀粉样变过程。在LRP中，我们定义了LRP的最后37个C末端残基(LRP-C37)，缺少足以有力地促进A2产生的NPxY或YxxL结构域。LRP-C37结构域在LRP功能中的作用在很大程度上还不清楚。由于LRP-C37单独是A2产生的有效诱导者，我们在酵母双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转导有关的Ran结合蛋白M(RanbpM)。在转基因细胞和小鼠脑匀浆中，RanbpM与LRP和APP均有相互作用。RanbpM的过表达也增加了哺乳动物细胞中LRP/APP复合体的形成，表明RanbpM作为一种接头蛋白促进了APP、RanbpM和LRP之间的三方相互作用。当90 kDa全长RanbpM(RanbpM-FL)在稳定的细胞系中过表达时，还会产生一个N末端55 kDa的蛋白水解性片段(N-55)，它与APP和LRP的相互作用甚至比全长RanbpM更强。这种蛋白分解的RanbpM物种在AD患者的大脑中增加了6倍。事实上，RanbpM-FL或N55片段的过度表达显著增加了A2的分泌。我们假设RanbpM改变了体内的APP代谢，特别是通过RanbpM的N55片段。本应用的具体目的是首先建立神经元表达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。通过生化和免疫组织化学方法检测TG RanbpM/APP双转基因小鼠大脑皮质和海马区A2和淀粉样斑块的水平，以确定RanbpM变异体的过度表达是否会改变APP的淀粉样蛋白形成过程。在2月龄和10月龄时，用酶联免疫吸附试验测定CHAPS可溶性和不溶性A2的水平。新的APP和LRP相互作用促进APP淀粉样蛋白形成的蛋白的特征将揭示新的发病机制和抗淀粉样蛋白干预的靶点。我们认为RanbpM代表了一个潜在的治疗靶点。此外，通过R03授予机制产生的RanbpM转基因小鼠有望具有实质性的外在优点，因为它们无疑将对许多研究RanbpM其他方面的研究人员有用。这一应用也可能为未来R01研究RanbpM在神经退行性变中的作用奠定基础。与公共卫生相关：阿尔茨海默病(AD)是一种以有毒淀粉样蛋白(A2)在大脑中堆积为特征的疾病，在美国约有500万人受到影响，但尽管进行了广泛的研究，现有的治疗方法只能起到有限的症状缓解作用。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。在这项应用中，我们将在小鼠的大脑中表达RanbpM，以证实动物模型中的发现，希望能为治疗AD的淀粉样蛋白产生发现新的见解，特别是将RanbpM作为治疗靶点。

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.