Mechanisms of Jedi-mediated engulfment of apoptotic cells

绝地介导的凋亡细胞吞噬机制

• 批准号: 8525795
• 负责人: Chelsea Suzanne Sullivan
• 金额: $ 2.69万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-03-01 至 2014-08-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8525795
• 关键词: Actins; Adaptor Protein Complex 2; Apoptosis; Apoptotic; Autoimmune Diseases; Autoimmune Diseases of the Nervous System; Autoimmunity; Bacteria; Binding; Biological Assay; Caenorhabditis elegans; Cell Count; Cell membrane; Cells; Clathrin; Clathrin Adaptors; Clathrin Heavy Chains; Confocal Microscopy; Cryoultramicrotomy; Data; Development; Disease; Dominant-Negative Mutation; Drosophila genus; Embryo; Endocytosis; Etiology; Excision; Failure; GTP Binding; Guanosine Triphosphate Phosphohydrolases; Immune system; In Situ Nick-End Labeling; In Vitro; Inflammation; Knockout Mice; Lupus; Measures; Mediating; Molecular; Mus; Nervous system structure; Neuroglia; Neurons; PTB Domain; Patients; Peripheral Nervous System; Peripheral Nervous System Diseases; Phagocytosis; Process; Proteins; Role; Signal Transduction; Site; Small Interfering RNA; Sorting - Cell Movement; Spinal Ganglia; Staining method; Stains; Symptoms; TFAP2A gene; Tyrosine; Vesicle; adapter protein; base; cell type; egg; in vivo; inhibitor/antagonist; insight; knock-down; macrophage; mouse model; mutant; nervous system development; neuron development; novel; public health relevance; receptor; receptor internalization; response; scaffold; small hairpin RNA; trafficking; uptake

DESCRIPTION (provided by applicant): Programmed cell death is a natural part of the development of the mammalian nervous system, necessary for establishing proper cell numbers and connections. The neuronal corpses must be efficiently removed in order to avoid an immune system response, which can induce inflammation and autoimmunity (Krysko et al., 2006). The cells responsible for neuron corpse clearance in the peripheral nervous system (PNS) were recently identified as satellite glial cell precursors (Wu et al., 2009). Furthermore, a novel engulfment receptor, Jedi, was identified that was required for engulfment of apoptotic neurons in vitro. Jedi is homologous to the Drosophila and C. elegans engulfment receptors, Draper and CED-1, which signal at least partly through the engulfment adapter protein GULP, but the mechanism of Jedi signaling is unknown. GULP binds to the NPXY motif of CED-1 and Draper via its phosphotyrosine binding (PTB) domain. We have determined that Jedi and GULP interact via the NPXY motif of Jedi, and we have determined that GULP is important for Jedi-mediated engulfment signaling by knocking down GULP using siRNA and also by evaluating non-binding mutants of Jedi in an engulfment assay. While GULP is known to be essential for engulfment signaling downstream of Draper and CED-1, its function in this process has not been determined. Since GULP binds to NPXY motifs, which are typically involved in receptor trafficking and internalization through clathrin mediated endocytosis, this adapter may have a role in Jedi sorting and/or endocytosis. GULP binds to clathrin (Martins-Silva, 2006), and it also regulates Arf6 (Ma et al., 2007), a GTPase known to promote recruitment of the clathrin adapter AP- 2 to the cell membrane (Paleotti et al., 2005). Based on preliminary data that the NPXY motif of Jedi is important for internalization of the receptor and engulfment, we hypothesize that Jedi mediates phagocytosis through a clathrin dependent mechanism involving GULP and Arf6. The importance of clathrin-dependent mechanisms for engulfment will be determined using pharmacological inhibitors or shRNA knock-down of clathrin heavy chain. We have also generated a Jedi knock-out mouse which will allow us to investigate the in vivo role of Jedi, and we will also be able to examine the potential effects of failure to clear dead neurons and the development of autoimmune disease. Some mouse models of autoimmunity are associated with impaired cell corpse removal by macrophages (e.g. Hanayama et al., 2004); however, the effects of impaired neuronal corpse engulfment have not been studied despite the fact that most patients with rheumatoid disease and lupus develop peripheral neuropathy (Spirin et al., 2007). Determining the mechanisms of apoptotic neuron clearance will benefit our understanding of mammalian nervous system development and possibly provide vital insights into the etiology of autoimmune disorders of the nervous system.

描述（由申请方提供）：程序性细胞死亡是哺乳动物神经系统发育的自然部分，是建立适当细胞数量和连接所必需的。必须有效地去除神经元尸体以避免免疫系统反应，其可诱导炎症和自身免疫（Krysko等人，2006年）。负责周围神经系统（PNS）中神经元尸体清除的细胞最近被鉴定为卫星神经胶质细胞前体（Wu et al.，2009年）。而且有 鉴定了一种新的吞噬受体Jedi，它是体外吞噬凋亡神经元所必需的。Jedi与果蝇和C. elegans吞噬受体，德雷珀和CED-1，其至少部分通过吞噬衔接蛋白GULP发出信号，但Jedi信号的机制尚不清楚。GULP通过其磷酸酪氨酸结合（PTB）结构域与CED-1和德雷珀的NPXY基序结合。我们已经确定Jedi和GULP通过Jedi的NPXY基序相互作用，并且我们已经确定GULP对于Jedi介导的吞噬信号传导是重要的，通过使用siRNA敲低GULP，并且还通过在吞噬测定中评估Jedi的非结合突变体。虽然已知GULP对于德雷珀和CED-1下游的吞噬信号传导是必需的，但其在该过程中的功能尚未确定。由于GULP与NPXY基序结合，NPXY基序通常通过网格蛋白介导的内吞作用参与受体运输和内化，因此该衔接子可能在Jedi分选和/或内吞作用中起作用。GULP与网格蛋白结合（Martins-Silva，2006），并且它还调节Arf 6（Ma等人，2007），一种已知促进网格蛋白衔接子AP- 2向细胞膜募集的GT酶（Paleotti等人，2005年）。基于初步的数据，绝地的NPXY基序是重要的受体和吞噬的内化，我们假设，绝地介导的吞噬作用，通过网格蛋白依赖的机制，涉及GULP和Arf 6。将使用网格蛋白重链的药理学抑制剂或shRNA敲低来确定网格蛋白依赖性机制对于吞噬的重要性。我们还产生了一只Jedi基因敲除小鼠，这将使我们能够研究Jedi在体内的作用，我们也将能够研究未能清除死亡神经元和自身免疫性疾病发展的潜在影响。一些自身免疫的小鼠模型与巨噬细胞对受损细胞尸体的清除有关（例如Hanayama et al.，2004年）;然而，尽管大多数患有类风湿性疾病和狼疮的患者发展为周围神经病变，但是受损的神经元尸体吞噬的作用还没有被研究（Spirin等，2007年）。确定凋亡神经元清除的机制将有助于我们了解哺乳动物神经系统的发展，并可能提供重要的见解神经系统的自身免疫性疾病的病因。