O-GlcNAc糖基化在神经疾病中的作用

发布时间：2018-08-06 12:19

【摘要】：第一部分O-OlcNAc糖基化对Insulin-PI3K-AKT信号通路的调节目的：在不同细胞中研究O-GlcNAc糖基化对Insulin-PI3K-AKT信号通路的影响。方法：在HEK-293FT, HepG2, N2a细胞和原代培养的小鼠海马神经元过表达或下调O-GlcNAc糖基转移酶OGT,或使用调节O-GlcNAc糖基化修饰关键酶的抑制剂处理细胞,改变细胞内O-GlcNAc糖基化水平。利用免疫共沉淀的方法研究Insulin-PI3K-AKT信号通路中可以被O-GlcNAc糖基化的组成部分。采用蛋白免疫印迹技术分析不同细胞内O-GlcNAc糖基化对AKT和GSK-3β磷酸化和活性的影响。结果： 1)在HEK-293FT细胞中,免疫共沉淀和蛋白免疫印迹分析显示,AKT和GSK-3β是Insulin-PI3K-AKT信号通路中受O-GlcNAc糖基化修饰的蛋白。 2)在不同培养条件下,改变HEK-293FT细胞的O-GlcNAc糖基化水平,影响PI3K-AKT信号通路。O-GlcNAc糖基化负性调控AKT的Thr308和Ser473磷酸化水平,改变其活性。降低细胞内O-GlcNAc糖基化,下调GSK-3β的Ser9的磷酸化,但升高细胞内的O-GlcNAc糖基化对GSK-3β的Ser9磷酸化没有影响 3)在不同培养条件下,改变HepG2细胞的O-GlcNAc糖基化水平,对AKT的GSK-3β的磷酸化没有明显的影响。 4)N2a细胞和原代培养的小鼠海马神经元,O-GlcNAc糖基化调节AKT的磷酸化,但对GSK-3β的磷酸化没有影响。结论：本研究显示O-GlcNAc糖基化对AKT和GSK-3β的调节具有蛋白特异性和细胞特异性。Insulin-PI3K-AKT在不同的器官、组织和细胞中,扮演着不同的角色,O-GlcNAc糖基化的调节也存在着不同的表现,在神经元细胞内,O-GlcNAc糖基化调节AKT的活性。第二部分O-GlcNAc糖基化在脑缺血中对细胞凋亡的影响目的：研究O-GlcNAc糖基化是否及如何引起细胞凋亡,探讨脑缺血早期升高的O-GlcNAc糖基化对细胞凋亡的影响。方法：蛋白质免疫印迹和组织免疫荧光染色分析小鼠大脑中动脉阻断(Middle Cerebral Artery Occlusion, MCAO)脑缺血模型不同时间O-GlcNAc糖基化改变和AKT的磷酸化水平。Caspase3的活性测定和Tunel测定检测小鼠脑缺血早期海马细胞发生凋亡情况。在培养的HEK-293FT细胞,过表达OGT或者shOGT下调OGT表达以改变细胞内O-GlcNAc糖基化水平,用MTT检测细胞活力,荧光染色观察细胞核的形态,AnnexinV/PI流式细胞仪检测凋亡细胞,蛋白质免疫印迹分析早期凋亡标志Caspase3的活化和PAPR的水解。利用免疫沉淀,定点突变,蛋白免疫印迹分析O-GlcNAc糖基化对AKT的影响,探讨O-GlcNAc糖基化是否通过调节AKT影响细胞凋亡。结果： 1)蛋白质免疫印迹和组织免疫荧光染色结果显示,小鼠MCAO模型脑缺血早期海马组织O-GlcNAc糖基化升高,Caspase3活性增加,Tunel染色出现凋亡细胞。 2) HEK293FT细胞内过表达OGT,细胞活力下降,细胞核浓缩,断裂,呈现凋亡样改变,流式细胞仪检测到更多的凋亡细胞,蛋白免疫印迹分析结果显示过上调细胞内O-GlcNAc糖基化,Caspase3激活,PARP蛋白被水解。 3)AKT的Thr308和Ser473受O-GlcNAc糖基化修饰。过表达OGT升高O-GlcNAc糖基化,促进AKT的糖基化,下调AKT的活性,引起下游底物Bad磷酸化下降,进而活化Caspase3而引起细胞凋亡：过表达AKT,可以改善过表达OGT引起的细胞凋亡,但过表达AKT的突变体,则不能逆转OGT引起的细胞凋亡。 4)缺血海马的O-GlcNAc糖基化水平和AKT磷酸化水平呈负相关。免疫沉淀AKT,缺血侧海马AKT的O-GlcNAc糖基化要比对照侧高。结论： AKT的Thr308和Ser473受磷酸化和糖基化双重修饰。在脑缺血的早期,由于应激反应,细胞内的O-GlcNAc糖基化增加,而使AKT的磷酸化及活性下降,磷酸化的BAD减少,经过系列反应,进而激活Caspase3,启动凋亡发生。因此,O-GlcNAc糖基化在脑缺血早期的细胞凋亡中可能起重要作用。第三部分阿尔茨海默病患者脑内F调的O-GlcNAc糖基化影响FOXO1介导的自噬和Tau病变目的：研究O-GlcNAc糖基化对转录因子FoxO1的功能进行调节,探讨阿尔茨海默病患者脑内下调的O-GlcNAc糖基化对FoxO1介导的tau病变的影响。方法：在HEK-293FT细胞过表达OGT或者shOGT下调OGT的表达,蛋白质免疫印迹技术检测O-GlcNAc糖基化对FoxO1的影响。利用蛋白合成和降解的抑制剂处理细胞,蛋白质免疫印迹和实时定量PCR研究O-GlcNAc糖基化调节FoxO1蛋白水平的途径。利用免疫沉淀技术研究FoxO1是否被O-GlcNAc糖基化修饰,及如何影响自身的降解。蛋白免疫印迹分析细胞核和细胞质中FoxO1,研究O-GlcNAc糖基化改变是否影响FoxO1的细胞分布。改变细胞内O-GlcNAc糖基化水平,检测FoxO1报告基因的荧光素酶活性,实时定量PCR检测FoxO1靶基因的表达,蛋白免疫印迹检测FoxO1对自噬的影响,研究O-GlcNAc糖基化改变是否影响FoxO1的活性。利用前脑神经元特异性敲除OGT小鼠,蛋白免疫印迹技术检测O-GlcNAc糖基化改变,AKT和foxO1的蛋白和磷酸化水平,以及自噬和蛋白酶体相关蛋白的水平,One trail物体识别,Morris水迷宫,Reversal Morris水迷宫检测OGT KO小鼠学习记忆功能变化。蛋白免疫印迹和免疫荧光染色分析AD患者脑内O-GlcNAc糖基化和FoxO1的蛋白水平,自噬相关蛋白水平,以及Tau的聚积。结果： 1)改变HEK-293FT细胞的O-GlcNAc糖基化,不仅通过调节AKT的活性,而影响FoxO1蛋白的磷酸化水平,还改变FoxO1的蛋白水平。 2)升高的O-GlcNAc糖基化抑制FoxO1经过泛素一蛋白酶体通路进行降解。 3) FoxO1本身可以被O-GlcNAc糖基化修饰,当O-GlcNAc糖基化修饰增加后,自身的泛素化减少,经泛素—蛋白酶体途径的降解受到抑制。 4) FoxO1的O-GlcNAc糖基化使得其转录活性增加,所调控的自噬功能增强,但不影响其细胞内分布。 5)前脑神经元特异性敲除OGT的小鼠,O-GlcNAc糖基化和FoxO1水平均下降,其自噬功能下调。短时程和场景记忆能力下降。 6)AD患者脑内O-GlcNAc糖基化水平、FoxO1的蛋白水平、P62蛋白水平的增加和LC3的下降提示自噬能力降低,AD脑内,FoxO1的蛋白水平与Tau聚积呈负相关。结论：转录因子FoxO1受O-GlcNAc糖基化修饰,增强其稳定性,在AD脑中,降低的O-GlcNAc糖基化水平,使FoxO1的稳定性下降而降解,受它调控的自噬功能下调,而使磷酸化和聚集的tau无法清除,形成神经纤维缠结,而导致神经退行性变。
[Abstract]:Part 1 regulation of O-OlcNAc glycosylation on Insulin-PI3K-AKT signaling pathway
Objective:
The effect of O-GlcNAc glycosylation on Insulin-PI3K-AKT signaling pathway was studied in different cells.
Method:
HEK-293FT, HepG2, N2a cells and primary cultured hippocampal neurons overexpress or downregulate O-GlcNAc glycosyltransferase OGT, or use inhibitors that regulate the key enzymes of O-GlcNAc glycosylation to treat cells and change the level of intracellular O-GlcNAc glycosylation. The method of immunoprecipitation can be used to study the Insulin-PI3K-AKT signaling pathway. The effects of O-GlcNAc glycosylation on the phosphorylation and activity of AKT and GSK-3 beta in different cells were analyzed by Western blot.
Result:
1) in HEK-293FT cells, the immunoblotting and immunoblotting analysis showed that AKT and GSK-3 beta were the O-GlcNAc glycosylated protein in the Insulin-PI3K-AKT signaling pathway.
2) under different culture conditions, the change of O-GlcNAc glycosylation level of HEK-293FT cells affects the negative regulation of.O-GlcNAc glycosylation of PI3K-AKT signaling pathway, Thr308 and Ser473 phosphorylation level, changes its activity, reduces the intracellular O-GlcNAc glycosylation and downregulates the phosphorylation of GSK-3 beta Ser9, but increases the O-GlcNAc glycosylation of the cell to GSK-3 beta. The phosphorylation of Ser9 has no effect
3) Changes of O-GlcNAc glycosylation level in HepG2 cells under different culture conditions had no significant effect on GSK-3 beta phosphorylation of AKT.
4) O-GlcNAc glycosylation regulates AKT phosphorylation in N2a cells and primary cultured mouse hippocampal neurons, but has no effect on GSK-3 beta phosphorylation.
Conclusion:
This study shows that the regulation of O-GlcNAc glycosylation on AKT and GSK-3 beta is protein specific and cell specific.Insulin-PI3K-AKT plays different roles in different organs, tissues and cells. The regulation of O-GlcNAc glycosylation also exists in different manifestations. In the neuron cell, O-GlcNAc glycosylation regulates the activity of AKT.
The second part is the effect of glycosylation of O-GlcNAc on apoptosis in cerebral ischemia.
Objective:
To study whether and how O-GlcNAc glycosylation induces apoptosis and to explore the effect of elevated O-GlcNAc glycosylation on apoptosis in the early stage of cerebral ischemia.
Method:
Protein immunoblotting and tissue immunofluorescence staining analysis of Middle Cerebral Artery Occlusion (MCAO) cerebral ischemia model in mice at different time O-GlcNAc glycosylation changes and AKT phosphorylation level.Caspase3 activity assay and Tunel determination of apoptosis in the hippocampus of mice in early cerebral ischemia. Cultured HEK-293FT cells, overexpressing OGT or shOGT down regulation of OGT expression to change the level of O-GlcNAc glycosylation, detection of cell viability by MTT, fluorescence staining to observe the morphology of nuclei, AnnexinV/PI flow cytometry to detect apoptotic cells, protein immunoblotting to analyze the activation of the early dying symbol Caspase3 and the hydrolysis of PAPR. The effects of glycosylation of O-GlcNAc on AKT were analyzed by immunoblotting and site-directed mutagenesis to explore whether O-GlcNAc glycosylation affects apoptosis by regulating AKT.
Result:
1) the results of protein immunoblotting and tissue immunofluorescence staining showed that the O-GlcNAc glycosylation of hippocampal tissue in the early cerebral ischemia of MCAO model in mice was increased, the activity of Caspase3 increased, and apoptotic cells were found in Tunel staining.
2) over expression of OGT in HEK293FT cells, cell viability decreased, nuclear concentration, fracture, and apoptotic like changes. Flow cytometry detected more apoptotic cells. Protein immunoblotting analysis showed that O-GlcNAc glycosylation, Caspase3 activation, and PARP egg white were hydrolyzed.
3) Thr308 and Ser473 of AKT are modified by O-GlcNAc glycosylation. Overexpression of OGT increases O-GlcNAc glycosylation, promotes the glycosylation of AKT, reduces the activity of AKT, causes the decrease of Bad phosphorylation in the downstream substrate, and then activates Caspase3 to induce apoptosis. It can not reverse the apoptosis induced by OGT.
4) There was a negative correlation between O-GlcNAc glycosylation and AKT phosphorylation in ischemic hippocampus.
Conclusion:
The Thr308 and Ser473 of AKT are modified by phosphorylation and glycosylation. In the early stage of cerebral ischemia, the O-GlcNAc glycosylation in the cells increased due to the stress response, and the phosphorylation and activity of AKT decreased, the BAD of phosphorylation was reduced, and the Caspase3 was activated through a series of reactions, and apoptosis occurred. Therefore, O-GlcNAc glycosylation was in the early stage of cerebral ischemia. Cell apoptosis may play an important role.
In the third part, O-GlcNAc glycosylation of F in the brain of patients with Alzheimer's disease affects FOXO1 mediated autophagy and Tau lesions.
Objective:
To investigate the function of O-GlcNAc glycosylation on the function of the transcription factor FoxO1, the effect of O-GlcNAc glycosylation on FoxO1 mediated tau lesions in Alzheimer's disease patients was investigated.
Method:
The expression of OGT or shOGT down regulated the expression of OGT in HEK-293FT cells. Protein immunoblotting was used to detect the effect of O-GlcNAc glycosylation on FoxO1. Protein synthesis and degradation inhibitors were used to treat cells. Protein immunoblotting and real-time quantitative PCR were used to study the pathway of O-GlcNAc glycosylation to regulate FoxO1 protein level. Immunoprecipitation technique was used. To study whether FoxO1 was modified by O-GlcNAc glycosylation and how to affect its own degradation. Protein immunoblotting was used to analyze FoxO1 in nuclei and cytoplasm, to investigate whether O-GlcNAc glycosylation changes affect the cell distribution of FoxO1, change the level of intracellular O-GlcNAc glycosylation, detect the luciferase activity of the FoxO1 reporter gene, and real-time quantitative PCR detection Fox O1 target gene expression, protein immunoblotting to detect the effect of FoxO1 on autophagy, and to investigate whether the O-GlcNAc glycosylation changes affect the activity of FoxO1. Using the neuron specific knockout of the OGT mice, the protein immunoblotting technique was used to detect the O-GlcNAc glycosylation changes, the protein and phosphorylation levels of AKT and foxO1, and the correlation between autophagy and proteasome. Protein level, One trail object recognition, Morris water maze, and Reversal Morris water maze test the learning and memory function of OGT KO mice. Protein immunoblotting and immunofluorescence staining were used to analyze the level of O-GlcNAc glycosylation and FoxO1 in AD patients, the level of autophagy related proteins, and the accumulation of Tau.
Result:
1) To change the glycosylation of O-GlcNAc in HEK-293FT cells not only affects the phosphorylation level of FoxO1 protein by regulating the activity of AKT, but also changes the protein level of FoxO1.
2) elevated O-GlcNAc glycosylation inhibits FoxO1 degradation through ubiquitin proteasome pathway.
3) FoxO1 itself can be modified by O-GlcNAc glycosylation, and when O-GlcNAc glycosylated modification increases, its own ubiquitination is reduced, and the degradation of ubiquitin proteasome pathway is inhibited.
4) O-GlcNAc glycosylation of FoxO1 increased its transcriptional activity and enhanced autophagy, but it did not affect its intracellular distribution.
5) The levels of O-GlcNAc glycosylation and FoxO1 in the forebrain neuron-specific OGT-knockout mice decreased, and their autophagy was down-regulated.
6) the level of O-GlcNAc glycosylation in the brain of AD patients, the level of FoxO1 protein, the increase of P62 protein level and the decrease of LC3 suggest the decrease of autophagy, and the protein level of FoxO1 is negatively correlated with the accumulation of Tau in the AD brain.
Conclusion:
The transcription factor FoxO1 is modified by O-GlcNAc glycosylation to enhance its stability. The reduced O-GlcNAc glycosylation level in the AD brain degrade and degrade the stability of FoxO1, and is regulated by its regulation of autophagic function, and the phosphorylation and aggregation of tau can not be removed, forming neurofibrillary tangles, resulting in neurodegenerative changes.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R749.16

【共引文献】