STEP61 negatively regulates amyloid beta-mediated ERK signaling pathway in Alzheimer’s disease cell model

doi:10.3969/j.issn.2095-4344.0796

Abstract

Abstract:

BACKGROUND: Striatal-enriched phosphatase 61 (STEP61) has been shown to play an important role in
synaptic plasticity. STEP is located at the postsynaptic terminal, and affects the formation of synaptic enhancement probably through the dephosphorylation and inactivation of key signal proteins such as MAPK kinase ERK1/2.
OBJECTIVE: To explore the mechanism of STEP61 negatively regulating amyloid β-protein (Aβ)-mediated ERK signaling pathway in Alzheimer’s disease cell model.
METHODS: There were three groups. Normal group was primary cortical neuron cells of C57BL/6 mice. In Alzheimer’s disease cell model group, 1 μmol/L Aβ1-42 (condensed state) was added into the cortical neuron cells for 1 hour culture to prepare Alzheimer’s disease cell model. In Aβ1-42 + RNAi group, STEP61 gene silencing was performed in Alzheimer’s disease cell model by sRNAi technique. The effect of STEP61 RNAi on ERK signaling pathway was detected by western blot assay.
RESULTS AND CONCLUSION: The expression of STEP61 protein in the Alzheimer’s disease cell model group was increased by 223% compared with the normal group, and the ratio of phosphorylated STEP61 was reduced to (75.6 ± 4.6)% (P < 0.05). There was no significant difference in the ratio of STEP61 phosphorylation between Aβ1-42 + RNAi and Alzheimer’s disease cell model groups. Western blot analysis showed an evident decrease of pERK1/2 levels in the Alzheimer’s disease cell model group compared with the control group (P < 0.05). The expression of pERK1/2 protein in the Aβ1-42 + RNAi group was significantly higher than that in the Alzheimer’s disease cell model group (P < 0.05). These results suggest that STEP61 negatively regulates Aβ mediated ERK signaling pathway in the Alzheimer’s disease cell model, possibly by dephosphorylation.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: Tissue Engineering, Alzheimer Disease, RNA Interference

CLC Number:

R318

Zhang Lin1, Yang Jing2, Liu Zan-hua1 . STEP61 negatively regulates amyloid beta-mediated ERK signaling pathway in Alzheimer’s disease cell model [J]. Chinese Journal of Tissue Engineering Research, 2018, 22(28): 4507-4512.

Figures/Tables 2

2.1 Aβ对皮质神经元细胞中STEP61的影响 STEP61在外源性加入Aβ1-42的皮质神经元细胞中的表达变化：Western Blot结果显示，外源性加入Aβ1-42使STEP61蛋白表达水平增加了223%(P < 0.05；图1A，B)。使磷酸化STEP61的比例减少到(75.6±4.6)% (P < 0.05；图1C)。作为对照实验使用β-淀粉样肽42-1(1 μmol/L，1 h)，发现其对STEP61的蛋白表达和磷酸化比例无影响(STEP61蛋白水平：(105.0±4.2)%；磷酸化STEP61比例：(98.0±1.6)%；图1D-F)，提示外源性加入Aβ1-42有助于STEP61在皮质神经元细胞突触小体中的蛋白表达和活性形式的增加。 2.2 STEP61 RNAi鉴定结果实验接下来对STEP61的干扰效率进行检测，分为正常对照组，转染STEP siRNA，non-silencing siRNA和转染试剂组。Western Blot结果显示，转染STEP siRNA组可明显抑制STEP61蛋白表达，干扰后48 h为抑制效果最佳时间点(瞬转法)。与正常组相比，干扰后48 h STEP61表达量减少了80.7%(P < 0.01，图2A，B)；转染STEP siRNA组与non-silencing siRNA 组相比，STEP61表达量减少了69%(P < 0.01，图2C，D)。 2.3 STEP61 RNAi对ERK信号通路的影响实验首先检测谷氨酸刺激后的ERK1/2活化的时程。免疫印迹结果显示，在谷氨酸刺激原代皮质神经元细胞5 min后ERK1/2出现了快速的激活，15-30 min则出现了ERK1/2的延迟失活(图3A，B)。接下来检测STEP61脱磷酸化时程，谷氨酸刺激神经元0，5，10，15和30 min，实验使用抗-STEP抗体来分析细胞裂解产物。谷氨酸刺激15-30 min STEP61显示了明显的上面条带的蛋白表达减少和下面条带的蛋白表达增加，而5-10 min STEP61上面条带的蛋白表达轻微减少，表明STEP61在谷氨酸刺激15-30 min发生了脱磷酸化(图3C，D)。 Western blot 结果显示，Aβ1-42处理组STEP61水平 (1 μmol/L，1 h)较对照组明显增加(P < 0.05；图4A，B)，而STEP61磷酸化的比例减少(P < 0.05，图4C)。尽管转染STEP61 siRNA明显减少了STEP61水平(P < 0.05；图4C)，而Aβ1-42+RNAi组STEP61磷酸化的比例与 Aβ1-42组相比无显著差异(P > 0.05，图4C)。 ERK1/2是STEP的底物，可以使突触加强和记忆巩固。为了检测阿尔茨海默中STEP61是否调控pERK1/2 (Thr202 and Tyr204，STEP61去磷酸化位点)。Western blot结果显示1 μmol/L Aβ1-42处理皮质神经元细胞1 h后，pERK1/2的蛋白表达量较对照组减少(P < 0.05)，而Aβ1-42+RNAi组pERK1/2的蛋白表达量较Aβ1-42组有显著增加(P < 0.05；图4D，E)。"

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