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）：控制大脑中淀粉样前体蛋白（APP）衍生片段清除的机制仍然知之甚少。神经元损伤和各种神经退行性疾病后，APP 表达显着上调，导致淀粉样 (A?) 肽和具有有效生物活性的可溶性 sAPP 胞外域水平升高。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。与小胶质细胞对 Aβ 反应的广泛研究相反，这些吞噬细胞调节大脑中 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，我们采用鳞翅目曼杜卡（一种充分表征的先天免疫模型）来研究 sAPP 清除机制。与人类一样，昆虫巨噬细胞主动吞噬裂解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轻松分析。最近，我们发现曼杜卡巨噬细胞在去除APPL（APP的昆虫直系同源物）脱落的胞外域中发挥着重要作用。在发育期间和神经损伤后，巨噬细胞都位于神经元迁移、生长和修复区域，在那里它们吞噬神经元衍生的 APPL 胞外域 (sAPPL)。使用质粒表达库筛选 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。虽然联系蛋白传统上被认为是神经元特异性受体，但我们现在已经证明，小鼠小胶质细胞和巨噬细胞也表达特定的联系蛋白子集，具体取决于它们的激活状态。使用阻塞的初步试验抗体表明巨噬细胞对 sAPPL（但不是 A？）的反应需要 msContactin，这表明使用不同的机制来清除不同的 APP 片段。这些发现表明，Contactins 在介导 sAPP 的非炎症反应中发挥着之前未被认识到的作用。目标 1 将使用我们在原代细胞培养物和发育动物中的基因敲除、重新表达和成像方案来测试 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。目标 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 Contactin 依赖性对 sAPP 的反应是否代表哺乳动物吞噬细胞使用的新型信号传导途径。这些研究的成功完成将为 R01 的应用提供新的数据，目标是全面定义 Contactin 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。公共健康相关性：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有毒 sAPP 片段的积累。