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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

基本信息

批准号：
8446997
负责人：
Madepalli Krishnappa Lakshmana
金额：
$ 27.11万
依托单位：
TORREY PINES INST FOR MOLECULAR STUDIES
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-04-01 至 2014-09-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8446997
关键词：
55-kDa Ran-binding protein Address Adhesions Age Age-Months Alzheimer&apos 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

项目摘要

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.

项目总结/摘要阿尔茨海默病（AD）的特征是淀粉样蛋白B蛋白（AB）的沉积，来自淀粉样前体蛋白（APP）的B-和γ-分泌酶裂解。我们最近证明，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）基序是必要的和足够的，以增加AB生产。由于单独的LRP-C37是一种有效的诱导剂， AB生产，我们使用这个结构域作为酵母双杂交筛选的诱饵，导致Ranbinding的鉴定蛋白M（RanBP 9）。事实上，APP和RanBP 9-FL或FL-衍生的RanBP 9-FL的瞬时转染是有效的。 N60在各种细胞系中强烈增加AB的分泌，表明RanBP 9改变APP代谢。最重要的是，RanBP 9蛋白水平的免疫印迹定量表明RanBP 9-N60和N60-N70蛋白表达量增加。 RanBP 9-FL在AD患者和APP J20转基因患者的脑中升高超过6倍和4倍。小鼠分别。我们还发现，像LRP和它的两个关键配体一样，RanBP 9与LRP的基因相关，晚发性AD为了更好地了解RanBP 9在AD发病机制中的体内作用，我们作为正在进行的NIH R 03资助的一部分，产生了在大脑中过度表达RanBP 9的转基因小鼠。通过将携带APPswe、PSEN 1dE 9突变的B6 C3-Tg 85 Dbo小鼠（APdE 9）与RanBP 9转基因小鼠杂交，在小鼠中，产生RanBP 9/APdE 9三重转基因小鼠，其产生更多的CHAPSO可溶性AB和早在3个月大时，与APdE 9小鼠相比，RanBP 9小鼠的c-末端片段（CTF）就已表达，这表明RanBP 9 增加体内APP的淀粉样蛋白形成过程。本R 01提案是R 03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， RanBP 9在神经突中大量存在，并且是神经突生长的强抑制剂，我们接下来我想在这个提议中检查RanBP 9诱导的APP加工改变是否也导致树突状细胞的凋亡。和脊柱损伤我们已经成功地产生了RanBP 9转基因小鼠以及杂合无效小鼠第一次我们建议比较树突分支的模式，棘密度，突触前和海马体和额叶皮层的突触后蛋白水平，随后进行学习和记忆测试技能在2，5和10个月大的八组小鼠，即，RanBP 9 -629单转基因，APdE 9双转基因转基因，RanBP 9 -629/APdE 9三转基因，RanBP 9 -599单转基因，RanBP 9 -599/APdE 9三转基因转基因、RanBP 9-/-或RanBP 9 +/-、RanBP 9-/-或RanBP 9 +/-/APdE 9和野生型同窝交配对照。 Neuron Studio是一种用于自动脊柱密度分析的软件，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和棘数扫描共聚焦显微镜如果RanBP 9在本研究中被证实为体内真正的靶点，则三重转基因小鼠可以被证明是有用的作为一个加速模型的突触和行为缺陷。