Mechanisms of Jedi-mediated engulfment of apoptotic cells

描述(由申请人提供)：细胞程序性死亡是哺乳动物神经系统发育的一个自然部分，对于建立适当的细胞数量和连接是必要的。必须有效地移走神经元身体，以避免免疫系统反应，这可能会引发炎症和自身免疫(Krysko等人，2006年)。负责清除周围神经系统(PNS)中神经元身体的细胞最近被确认为卫星神经胶质细胞前体(Wu等人，2009年)。此外，a 一种新的吞噬受体JEDI被鉴定出来，它是在体外吞噬凋亡神经元所必需的。绝地与果蝇和线虫的吞噬受体Draper和CED-1同源，后者至少部分通过吞噬适配器蛋白GULP发出信号，但绝地信号的机制尚不清楚。GUP通过其磷酸酪氨酸结合(PTB)结构域与CED-1和Draper的NPXY基序结合。我们已经确定绝地和GULP是通过绝地的NPXY基序相互作用的，我们已经通过使用siRNA击倒GULP和在吞噬实验中评估绝地的非结合突变体来确定GULP对于绝地介导的吞噬信号是重要的。虽然已知GUPP在Draper和CED-1下游的吞噬信号中是必不可少的，但它在这一过程中的功能尚未确定。由于GULP与NPXY基序结合，而NPXY基序通常通过网状蛋白介导的内吞作用参与受体的运输和内化，因此这种适配器可能在绝地分选和/或内吞作用中发挥作用。GUP与clathrin结合(Martins-Silva，2006年)，它还调节Arf6(Ma等人，2007年)，这是一种已知能促进clathrin适配器AP-2募集到细胞膜的GTPase(Paleotti等人，2005年)。根据初步数据，绝地的NPXY基序对于受体的内化和吞噬很重要，我们假设绝地通过涉及GULP和Arf6的胞内蛋白依赖机制来介导吞噬作用。依赖于笼蛋白的吞噬机制的重要性将通过使用药物抑制剂或敲除笼蛋白重链的shRNA来确定。我们还培育了一只绝地基因敲除小鼠，这将使我们能够研究绝地在体内的作用，我们还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清除受损的细胞身体有关(例如，Hanayama等人，2004年)；然而，尽管大多数类风湿疾病和狼疮患者都会发生周围神经病变，但受损的神经元身体吞噬的影响尚未被研究(Spirin等人，2007年)。确定神经元凋亡清除的机制将有助于我们了解哺乳动物神经系统的发育，并可能为神经系统自身免疫性疾病的病因学提供重要的见解。

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.