Mechanisms of Jedi-mediated engulfment of apoptotic cells

Vanderbilt University, Nashville TN

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Abstract

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.