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

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。APP的表达在神经元损伤和各种神经退行性疾病中显著上调，导致淀粉样蛋白（a ?）肽和具有强大生物活性的可溶性sAPP外结构域水平升高。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。与对小胶质细胞对A？然而，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，我们采用鳞翅目物种Manduca（一种具有良好特征的先天免疫模型）来研究sAPP清除的机制。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，这一过程可以很容易地在体外和体内进行分析。最近，我们发现Manduca巨噬细胞在去除APPL （APP的昆虫同源物）的脱落外畴中起着重要作用。无论是在发育过程中还是在神经损伤后，巨噬细胞都进入神经元迁移、生长和修复的区域，在那里它们吞噬神经元来源的APPL外畴（sAPPLs）。利用质粒表达文库筛选appll结合伙伴，我们发现Manduca Contactin （msContactin）是昆虫巨噬细胞表达的sAPPL候选受体。虽然Contactins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Contactins亚群，这取决于它们的激活状态。使用阻塞的初步试验

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.