自噬降低異常磷酸化tau蛋白所致的神經(jīng)細(xì)胞毒性
發(fā)布時間：2018-07-16 08:04
【摘要】：目的觀察雷帕霉素和饑餓誘導(dǎo)的自噬對表達(dá)異常磷酸化tau蛋白的神經(jīng)細(xì)胞形態(tài)、tau蛋白聚集和磷酸化tau降解的影響,探討這兩種經(jīng)典的誘導(dǎo)自噬方式抑制磷酸化tau的細(xì)胞毒性,發(fā)揮細(xì)胞保護作用的可能機制。方法體外培養(yǎng)小鼠神經(jīng)瘤母細(xì)胞株N2a并轉(zhuǎn)染tau真核表達(dá)質(zhì)粒,蛋白磷酸酯酶抑制劑岡田酸(okadaic acid,OA)誘導(dǎo)tau蛋白異常磷酸化,雷帕霉素(rapamycin,Rapa)或Earle's平衡鹽溶液(Earle's balanced salts,EBSS)誘導(dǎo)細(xì)胞自噬,巴佛洛霉素A1(Bafilomycin A1,Baf A1)抑制自噬,DAB染色觀察表達(dá)tau細(xì)胞的形態(tài)變化;激光共聚焦顯微鏡觀察細(xì)胞內(nèi)tau聚集體;TUNEL染色和caspase-3活性檢測細(xì)胞凋亡;免疫印跡(immunoblot,IB)檢測磷酸化tau和細(xì)胞自噬水平。結(jié)果過表達(dá)tau的細(xì)胞胞體變圓,突起減少;OA處理后細(xì)胞突起進一步減少甚至消失,胞質(zhì)出現(xiàn)明顯tau聚集體,凋亡細(xì)胞增加,剪切型caspase-3水平上調(diào);Rapa和EBSS處理后的細(xì)胞形態(tài)均有一定程度改善,tau聚集體明顯減少,細(xì)胞凋亡減少,剪切型caspase-3表達(dá)降低;而自噬抑制劑Baf A1處理的細(xì)胞變圓,皺縮,胞質(zhì)大量tau聚集體,凋亡細(xì)胞明顯增加。IB結(jié)果顯示Rapa明顯降低高分子量的磷酸化tau,而EBSS能明顯減少低分子量磷酸化tau的水平。結(jié)論 Rapa和EBSS誘導(dǎo)的細(xì)胞自噬均能抑制磷酸化tau蛋白的細(xì)胞毒作用,但其發(fā)揮細(xì)胞保護作用的機制不同,Rapa誘導(dǎo)自噬傾向于降解磷酸化tau的寡聚體,而EBSS更易于降解低分子量的磷酸化tau蛋白。
[Abstract]:Objective to investigate the effects of rapamycin and starvation induced autophagy on neuronal morphology tau protein aggregation and phosphorylated tau degradation in neurons expressing abnormal phosphorylated tau protein, and to explore the inhibition of cytotoxicity of phosphorylated tau by these two classical autophagy methods. The possible mechanism of cell protection. Methods Mouse neuroblastoma cell line N2a was cultured in vitro and transfected with tau eukaryotic expression plasmid. The protein phosphatase inhibitor okadaic acidine OA induced abnormal phosphorylation of tau protein, and rapamycin Rapa or Earlehs balanced saltsEBSS induced autophagy. Bafilomycin A1 (Bafilomycin A1) inhibited autophagy dab staining to observe the morphological changes of tau cells, and laser confocal microscopy was used to observe the apoptosis of tau aggregates Tunel and caspase-3 activity. Immunoblotting IB was used to detect phosphorylated tau and autophagy. Results after the over-expression of tau, the cell bodies became round, the processes decreased or even disappeared after treatment with OA, and the cytoplasm showed obvious tau aggregates, and the apoptotic cells increased. Both the up-regulation of caspase-3 level in shear type and the treatment with EBSs can improve the cell morphology, decrease apoptosis and decrease the expression of shearing type caspase-3, while the cells treated with autophagy inhibitor Baf A1 become round and shrink, and the apoptosis of the cells is decreased, and the expression of shearing type caspase-3 is decreased, while the cells treated with autophagy inhibitor Baf A1 become round and shrink. The results showed that Rapa significantly decreased the phosphorylation of tau by high molecular weight, while the level of low molecular weight phosphorylated tau was significantly decreased. Conclusion both Rapa and EBSS-induced autophagy can inhibit the cytotoxicity of phosphorylated tau protein, but the mechanisms of its protective effect are different. Rapa induced autophagy tends to degrade the oligomers of phosphorylated tau. EBSS is easier to degrade low molecular weight phosphorylated tau protein.
【作者單位】： 安徽醫(yī)科大學(xué)基礎(chǔ)醫(yī)學(xué)院;安徽醫(yī)科大學(xué)生物藥物研究所;安徽醫(yī)科大學(xué)藥學(xué)院;
【基金】：國家自然科學(xué)基金資助項目(No 81302755)
【分類號】：R96
，
本文編號：2125771
[Abstract]:Objective to investigate the effects of rapamycin and starvation induced autophagy on neuronal morphology tau protein aggregation and phosphorylated tau degradation in neurons expressing abnormal phosphorylated tau protein, and to explore the inhibition of cytotoxicity of phosphorylated tau by these two classical autophagy methods. The possible mechanism of cell protection. Methods Mouse neuroblastoma cell line N2a was cultured in vitro and transfected with tau eukaryotic expression plasmid. The protein phosphatase inhibitor okadaic acidine OA induced abnormal phosphorylation of tau protein, and rapamycin Rapa or Earlehs balanced saltsEBSS induced autophagy. Bafilomycin A1 (Bafilomycin A1) inhibited autophagy dab staining to observe the morphological changes of tau cells, and laser confocal microscopy was used to observe the apoptosis of tau aggregates Tunel and caspase-3 activity. Immunoblotting IB was used to detect phosphorylated tau and autophagy. Results after the over-expression of tau, the cell bodies became round, the processes decreased or even disappeared after treatment with OA, and the cytoplasm showed obvious tau aggregates, and the apoptotic cells increased. Both the up-regulation of caspase-3 level in shear type and the treatment with EBSs can improve the cell morphology, decrease apoptosis and decrease the expression of shearing type caspase-3, while the cells treated with autophagy inhibitor Baf A1 become round and shrink, and the apoptosis of the cells is decreased, and the expression of shearing type caspase-3 is decreased, while the cells treated with autophagy inhibitor Baf A1 become round and shrink. The results showed that Rapa significantly decreased the phosphorylation of tau by high molecular weight, while the level of low molecular weight phosphorylated tau was significantly decreased. Conclusion both Rapa and EBSS-induced autophagy can inhibit the cytotoxicity of phosphorylated tau protein, but the mechanisms of its protective effect are different. Rapa induced autophagy tends to degrade the oligomers of phosphorylated tau. EBSS is easier to degrade low molecular weight phosphorylated tau protein.
【作者單位】： 安徽醫(yī)科大學(xué)基礎(chǔ)醫(yī)學(xué)院;安徽醫(yī)科大學(xué)生物藥物研究所;安徽醫(yī)科大學(xué)藥學(xué)院;
【基金】：國家自然科學(xué)基金資助項目(No 81302755)
【分類號】：R96
，
本文編號：2125771
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