Role of Ninjurin1 during Leukocyte Trafficking in the Inflamed Central Nervous System

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약학대학 약학과
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서울대학교 대학원
Experimental autoimmune encephalomyelitis (EAE)Leukocyte traffickingNinjurin1Membrane protrusion formationPosttranslational modifications
학위논문 (박사)-- 서울대학교 대학원 : 약학과, 2013. 2. 김규원.
Ninjurin1 (nerve injury-induced protein) is an adhesion molecule that is essential for cell-to-cell interactions. However, the pathophysiological relevance of Ninjurin1 in vivo and its precise regulatory mechanisms in inflamed Central Nervous System (CNS) remain largely undefined. Here, it is demonstrated that Ninjurin1 is involved in leukocyte trafficking through promigratory activity and its posttranslational modifications by using in vivo animal model and in vitro cell culture system. Ninjurin1 was dominantly expressed in myeloid cells (macrophages/monocytes and neutrophils) and endothelial cells (ECs) in the brain of experimental autoimmune encephalomyelitis (EAE), the animal model of Multiple sclerosis (MS). Both Ninjurin1 KO and neutralized mice alleviated the severity of EAE by reducing the recruitment of leukocyte into inflamed lesions, suggesting the contribution of Ninjurin1 in leukocyte trafficking in vivo. With in vitro experiments on gain or loss of Ninjurin1 activity, we proved the dual functions of Ninjurin1, adhesive and protrusive activity, depending on the steps of leukocyte trafficking. Ninjurin1 contributes to the later crawling and transmigration stages via formation of membrane protrusion as well as to the initial rolling and adhesion stages via homophilic binding.
Next, we investigated the contribution of posttranslational modifications of Ninjurin1, proteolytic cleavage and N-glycosylation, on the leukocyte trafficking. The fragmentations of Ninjurin1 are occurred not only in a vector system in vitro but also in mouse tissues in vivo. MMP9 is responsible for the cleavage of mouse Ninjurin1 between Leu56 and Leu57 in N-terminal ectodomain. Intriguingly, the liberated ectodomain of Ninjurin1 seems to have a chemotactic activity which is supported by its secondary structure similar to well-known chemokines. We also found that Ninjurin1 is glycosylated on Asn60 residue of N-terminal ectodomain. Mutagenesis of Asn60 to Ala60 (N60A) decreased the formation of membrane protrusions and showed impaired localization to plasma membrane and loss of homophilic binding activity. Additionally, leukocyte-endothelial adhesion and transendothelial migration (TEM) activity were reduced in N60A mutant transfectants. Altogether, the in vitro and in vivo studies clearly demonstrated that Ninjurin1 enhances leukocyte trafficking through adhesive and protrusive activities that are regulated via its posttranslational modifications, proteolytic cleavage or N-glycosylation.
Therefore, we strongly suggest that Ninjurin1 is a beneficial therapeutic target for modulating pathogenesis of inflamed CNS including MS.
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College of Pharmacy (약학대학)Dept. of Pharmacy (약학과)Theses (Ph.D. / Sc.D._약학과)
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