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Macrophage-dependent regulation of neurotoxic APP fragments in a model system

模型系统中神经毒性 APP 片段的巨噬细胞依赖性调节

基本信息

批准号：
8701023
负责人：
PHILIP F COPENHAVER
金额：
$ 23.1万
依托单位：
OREGON HEALTH & SCIENCE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-02-15 至 2016-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8701023
关键词：
Address Affect Alzheimer&apos s Disease Amyloid Amyloid beta-Protein Precursor Animals Behavior Binding Biochemical Biological Biological Assay Biological Availability Biological Markers Biological Models Blocking Antibodies Blood - brain barrier anatomy Brain CD36 gene Cell Death Cerebrospinal Fluid Cleaved cell Clinical Data Development Disease Embryo Expression Library Family member Future Genes Genome Goals Growth Home environment Homing Human Image Immune response In Vitro Inflammatory Inflammatory Response Injury Insecta Lesion Manduca Manduca sexta Mediating Membrane Proteins Methods Microglia Modeling Mononuclear Mus Natural Immunity Nerve Degeneration Nervous system structure Neurodegenerative Disorders Neuronal Injury Neurons Operative Surgical Procedures Orthologous Gene Pathway interactions Patients Peripheral Phagocytes Phagocytosis Physiological Plasmids Play Primary Cell Cultures Process Protein Family Protein Fragment Proteins Protocols documentation Recruitment Activity Regulation Research Role Senile Plaques Signal Pathway Signal Transduction Source Synapses System Testing Traumatic Brain Injury Work amyloid peptide beta-site APP cleaving enzyme 1 cell motility contactin embryo culture in vivo inhibitor/antagonist injured macrophage migration monocyte nerve injury neurogenesis neuronal growth neurotoxic new therapeutic target novel novel therapeutics peptide 32 peptide A prevent protein expression public health relevance receptor repaired response response to injury scavenger receptor secretase synaptogenesis tool uptake

项目摘要

DESCRIPTION (provided by applicant): The mechanisms that control the clearance of Amyloid Precursor Protein (APP)-derived fragments in the brain remain poorly understood. APP expression is dramatically upregulated following neuronal injury and in a variety of neurodegenerative diseases, resulting in elevated levels of both amyloid (A?) peptides and soluble sAPP ectodomains that have potent biological activities. Growing evidence suggests that the misregulation of sAPP levels can be harmful to the nervous system, while abnormal sAPP levels may provide useful biomarkers for a variety of neurodegenerative conditions and injury responses. In contrast to extensive work on microglial responses to A?, the mechanisms by which these phagocytic cells regulate sAPP levels in the brain has remained largely unexplored. To address this issue, we have adapted the Lepidopteran species Manduca (a well-characterized model of innate immunity) to investigate the mechanisms of sAPP scavenging. As in humans, insect macrophages actively phagocytose cleaved membrane proteins and cellular debris to protect the nervous system, a process that can be readily analyzed both in vitro and in vivo. Recently, we discovered that Manduca macrophages play a prominent role in removing the shed ectodomains of APPL (the insect ortholog of APP). Both during development and following nerve injury, macrophages home to regions of neuronal migration, growth, and repair, where they phagocytose neuronally derived APPL ectodomains (sAPPLs). Using a plasmid expression library to screen for APPL-binding partners, we identified Manduca Contactin (msContactin) as a candidate sAPPL receptor that is expressed by insect macrophages. Whereas Contactins have traditionally been considered neuronal-specific receptors, we have now shown that both mouse microglia and macrophages also express specific subsets of Contactins, depending on their activation state. Initial trials using blocking antibodies indicate that msContactin is required for macrophage responses to sAPPLs (but not to A?), suggesting that distinct mechanisms are used to clear different APP fragments. These discoveries suggest a previously unrecognized role for Contactins in mediating non-inflammatory responses to sAPP. Aim 1 will test the hypothesis that msContactin specifically regulates the homing and phagocytic responses of insect macrophages to sAPPL fragments, using our gene knockdown, re-expression, and imaging protocols in primary cell cultures and developing animals. Aim 2 will use primary cultures of mouse microglia and macrophages to test whether Contactin-dependent responses to sAPP represents a novel signaling pathway used by mammalian phagocytic cells. Successful completion of these studies will provide new data for an R01 application, with the goal of comprehensively defining the mechanisms by which Contactin-dependent signaling regulates the microglial control of sAPPs in the nervous system. Public Heath Relevance: Understanding these mechanisms will support new therapeutic strategies for preventing the accumulation of toxic sAPP fragments associated with neurodegenerative disease and traumatic brain injury.

描述（由申请人提供）：控制淀粉样前体蛋白（APP）衍生片段在脑中清除的机制仍知之甚少。APP的表达显着上调神经元损伤后，在各种神经退行性疾病，导致淀粉样蛋白（A？）肽和可溶性sAPP胞外域，具有有效的生物活性。越来越多的证据表明，sAPP水平的失调可能对神经系统有害，而异常的sAPP水平可能为各种神经退行性疾病和损伤反应提供有用的生物标志物。与关于小胶质细胞对A？反应的大量研究相反，这些吞噬细胞调节脑中sAPP水平的机制在很大程度上仍未被探索。为了解决这个问题，我们已经适应了鳞翅目物种Manduca（先天免疫的良好表征模型），以研究sAPP清除的机制。与人类一样，昆虫巨噬细胞积极吞噬切割的膜蛋白和细胞碎片以保护神经系统，这一过程可以在体外和体内进行分析。最近，我们发现，曼陀罗巨噬细胞在去除APPL（APP的昆虫直系同源物）的脱落胞外结构域中起着突出的作用。在发育过程中和神经损伤后，巨噬细胞归巢到神经元迁移、生长和修复的区域，在那里它们吞噬神经源性APPL胞外域（sAPPs）。使用质粒表达文库来筛选APPL结合伴侣，我们将Manduca Contactin（msContactin）鉴定为由昆虫巨噬细胞表达的候选sAPPL受体。虽然接触素传统上被认为是神经元特异性受体，我们现在已经表明，小鼠小胶质细胞和巨噬细胞也表达接触素的特定子集，这取决于它们的激活状态。使用区组的初始试验抗体表明msContactin是巨噬细胞对sAPLs（但不是对A？）的应答所必需的，表明不同的机制用于清除不同的APP片段。这些发现表明接触蛋白在介导对sAPP的非炎症反应中的先前未被认识的作用。目的1将测试msContactin特异性调节昆虫巨噬细胞对sAPPL片段的归巢和吞噬反应的假设，在原代细胞培养物和发育中的动物中使用我们的基因敲除、再表达和成像方案。目的2将使用小鼠小胶质细胞和巨噬细胞的原代培养物来测试对sAPP的接触蛋白依赖性反应是否代表哺乳动物吞噬细胞使用的新信号传导途径。这些研究的成功完成将为R01的应用提供新的数据，其目标是全面定义接触素依赖性信号传导调节神经系统中sAPP的小胶质细胞控制的机制。公共卫生相关性：了解这些机制将支持新的治疗策略，以防止与神经退行性疾病和创伤性脑损伤相关的毒性sAPP片段的积累。