NCAM-140 Translocation into Lipid Rafts Mediates the Neuroprotective Effects of GDNF
NCAM-140 Translocation into Lipid Rafts Mediates the Neuroprotective Effects of GDNF
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NCAM-140 易位至脂筏介导 GDNF 的神经保护作用
DOI：
10.1007/s12035-016-9749-x
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发表时间：
2017-05-01
影响因子：
5.1
通讯作者：
Gao, Dianshuai
中科院分区：
文献类型：
--
作者：
Li, Li;Chen, Huizhen;Gao, Dianshuai
胶质细胞源性神经营养因子（GDNF）是一种对黑质多巴胺（DA）能神经元细胞有重要作用的神经营养因子。最近的研究表明，神经细胞粘附分子作为GDNF的信号转导受体发挥作用。本研究旨在揭示神经细胞粘附分子（NCAM）是否介导GDNF对DA神经细胞的保护作用，并进一步探讨其作用机制。我们利用SH-SY 5 Y细胞系建立了6-羟基多巴胺（6-OHDA）损伤的DA神经元细胞模型。构建慢病毒载体来敲减或过表达NCAM-140，并采用密度梯度离心法分离膜脂筏。采用MTT比色法、流式细胞术和Western blotting法检测GDNF的保护作用。结果表明，GDNF对6-OHDA损伤的SH-SY 5 Y细胞具有保护作用，其作用机制可能与GDNF提高细胞存活率、降低细胞死亡率和降低caspase-3表达有关。NCAM-140敲低降低了细胞活力，增加了细胞死亡率和切割的caspase-3表达，而其过表达具有相反的效果。值得注意的是，GDNF处理后，位于脂筏中的NCAM-140的量增加。用蛋白质棕榈酰化的特异性抑制剂2-溴棕榈酸酯预处理，抑制NCAM-140向脂筏的移位，并降低NCAM介导的GDNF对损伤的DA神经元细胞的保护作用。GDNF可能通过影响NCAM-140向脂筏的转位而对损伤的DA细胞起保护作用。
Glial cell line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor for substantia nigra dopaminergic (DA) neuronal cells. Recent studies have demonstrated that neural cell adhesion molecule functions as a signal transduction receptor for GDNF. The purpose of this study is to reveal whether neural cell adhesion molecule (NCAM) mediates the protective effects of GDNF on DA neuronal cells and further explore the mechanisms involved. We utilized SH-SY5Y cell line to establish a model of 6-hydroxydopamine (6-OHDA)-injured DA neuronal cells. Lentiviral vectors were constructed to knockdown or overexpress NCAM-140, and a density gradient centrifugation method was employed to separate membrane lipid rafts. 3-(4,5-Dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), flow cytometric analysis, and western blotting were used to evaluate the protective effects of GDNF. The results showed that GDNF could protect 6-OHDA-injured SH-SY5Y cells via improving cell viability and decreasing the cell death rate and cleaved caspase-3 expression. NCAM-140 knockdown decreased cell viability and increased the cell death rate and cleaved caspase-3 expression, while its overexpression had the opposite effects. Notably, the amount of NCAM-140 located in lipid rafts increased after GDNF treatment. Pretreatment with 2-bromopalmitate, a specific inhibitor of protein palmitoylation, suppressed NCAM-140 translocation to lipid rafts and reduced the NCAM-mediated protective effects of GDNF on injured DA neuronal cells. Our results suggest that GDNF have the protective effects on injured DA cells by influencing NCAM-140 translocation into lipid rafts.