神经丝的O-糖基化失调对其磷酸化和组装的影响
发布时间：2019-02-23 20:03
【摘要】：目的：阿尔茨海默病(Alzheimer's Disease, AD)是一种最常见的神经退行性疾病,主要临床表现为认知和记忆功能进行性减退。细胞骨架蛋白神经丝(neurofilaments, NFs)是神经元特异的中间丝成分,由三种不同分子量的亚单位组装成的多聚体。异常过度磷酸化的神经丝聚集在细胞轴突,是AD脑中神经元纤维缠结(NFT)的一种重要成分和早期的病理改变,但其发病机制目前尚不清楚。除了磷酸化,NFs蛋白还受细胞内葡萄糖代谢产生的O-糖基化(O-GlcNAcylation)修饰,我们的最新研究发现：在细胞水平NFs的糖基化和磷酸化之间存在相互调节,AD病人的脑神经细胞内聚集的异常磷酸化的NFs其O-糖基化明显减少。本课题是在上述研究结果的基础上,主要研究神经丝的O-糖基化失调对其磷酸化和组装的影响,探讨神经丝过度磷酸化的发病机制。 方法：我们主要采用体内和体外实验观察神经丝的O-糖基化的改变对其磷酸化和组装的影响。4-6月龄清洁级雄性SD大鼠35只大鼠平均分5组,每组7只,脑室内分别注射Alloxan (OGT抑制剂,2mg/kg)、选择性葡萄糖胺酶抑制剂NAG-AE (NAG-thiazoline,5mg/kg)组、Don (HBP途径抑制剂,2mg/kg)、 OA (PP2A抑制剂,50ng/kg)、实验对照组(生理盐水),空白对照鼠3只。24小时后处死大鼠取脑,采用蛋白免疫印迹检测脑组织NFs蛋白的糖基化和磷酸化水平；采用脑匀浆液高速离心和神经丝体外组装技术,结合透射电镜和免疫印迹方法,定性和定量对比不同糖基化水平的鼠脑NFs的体外组装的情况和形态。进一步我们采用体外酶化学技术,用β-乙酰糖胺酶去除脑组织匀浆液蛋白质的糖基化,用电镜技术观察神经丝组装的结构,探讨是NFs糖基化本身还是通过继发的磷酸化影响鼠脑NFs组装。 结果：我们的预试验表明神经丝提取效率高,纯度较高,电镜观察组装后神经丝由直径10nm左右的纤维主干以及由主干周期性发出的侧臂组成,侧臂一般较短,大小及结构与其它方法提取的神经丝相同。在体实验中,与对照组相比Alloxan、DON、OA组神经丝蛋白O-糖基化水平降低而磷酸化增加,神经丝较短且组装的比例明显减低；而NAG-Ae处理与对照组相比,O-糖基化水平明显增加而磷酸化水平是明显降低,神经丝纤维呈长丝状,组装的比例较高。提示神经丝的O-糖基化的改变对神经丝的组装有一定的影响。进一步在体外实验,我们只改变NFs的糖基化但不改变其磷酸化水平,电镜负染神经丝与对照组相比,神经丝的看起来较粗糙,仍然组装成较长的丝,提示单纯去糖基化对组装影响不明显。 结论：体内O-糖基化的改变影响NFs的磷酸化,减少的O-糖基化可能通过影响其磷酸化和神经丝的组装而影响其结构和功能,导致AD神经细胞的退行性变的发生。该研究结果为AD发病机制理论研究和药物的研发提供新的思路。
[Abstract]:Objective: Alzheimer's disease (Alzheimer's Disease, AD) is one of the most common neurodegenerative diseases. The cytoskeletal protein neurofilament (neurofilaments, NFs) is a neuron-specific intermediate filament composed of three different molecular weight subunits. Abnormal hyperphosphorylation of neurofilament is an important component and early pathological change of neurofibrillary tangles (NFT) in AD brain, but its pathogenesis is still unclear. In addition to phosphorylation, NFs proteins are also modified by Oglycosylation (O-GlcNAcylation) produced by glucose metabolism in cells. Our latest study has found that there is a mutual regulation between glycosylation and phosphorylation of NFs at the cellular level. Abnormal phosphorylation of NFs in brain nerve cells of AD patients decreased significantly in O-glycosylation. On the basis of the above research results, this paper mainly studies the effect of the disorder of O-glycosylation on the phosphorylation and assembly of neurofilament, and probes into the pathogenesis of neurofilament hyperphosphorylation. Methods: the effects of changes of O-glycosylation of neurofilament on phosphorylation and assembly of neurofilament were observed in vivo and in vitro. 35 male SD rats aged 4-6 months were divided into 5 groups with 7 rats in each group. Intraventricular injection of Alloxan (OGT inhibitor (2mg/kg), selective glucosaminase inhibitor NAG-AE (NAG-thiazoline,5mg/kg), Don (HBP pathway inhibitor, 2mg/kg), OA (PP2A inhibitor, 50ng/kg, respectively. The control group (normal saline) and the blank control group (3 rats) were killed 24 hours later. The glycosylation and phosphorylation of NFs protein in brain tissue were detected by Western blot. High speed centrifugation of brain homogenate, in vitro assembly of neurofilament, transmission electron microscopy and Western blotting were used to qualitatively and quantitatively compare the in vitro assembly and morphology of brain NFs with different glycosylation levels. Furthermore, we used in vitro enzyme chemistry to remove the glycosylation of protein from brain homogenate by 尾 -acetylglycosaminase, and observed the structure of neurofilament assembly by electron microscopy. To investigate whether NFs glycosylation itself or secondary phosphorylation affects brain NFs assembly. Results: our pre-test showed that the extraction efficiency of neurofilament was high and the purity was high. Electron microscopic observation showed that the nerve filament was composed of the fiber trunk about diameter 10nm and the lateral arm which was issued periodically by the trunk, and the lateral arm was generally short. The size and structure were the same as those obtained by other methods. In vivo, compared with the control group, the level of O-glycosylation of neurofilament protein in Alloxan,DON,OA group was decreased, the phosphorylation was increased, and the proportion of neurofilament was shorter and the proportion of assembly was significantly lower than that in control group. Compared with the control group, the level of O-glycosylation was significantly increased and the phosphorylation level was significantly decreased in NAG-Ae treatment. The neurofilament fibers were long filamentous and the proportion of assembly was higher. These results suggest that the changes of O-glycosylation of neurofilament have a certain effect on the assembly of neurofilament. Further in vitro, we only changed the glycosylation of NFs but did not change its phosphorylation level. The results suggest that the effect of deglycosylation alone on assembly is not obvious. Conclusion: the changes of Oglycosylation in vivo affect the phosphorylation of NFs, and the decrease of Oglycosylation may affect the structure and function of NFs by affecting its phosphorylation and neurofilament assembly, resulting in the degeneration of AD nerve cells. The results provide new ideas for the theoretical study of pathogenesis of AD and the development of drugs.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R749.16
本文编号：2429147
[Abstract]:Objective: Alzheimer's disease (Alzheimer's Disease, AD) is one of the most common neurodegenerative diseases. The cytoskeletal protein neurofilament (neurofilaments, NFs) is a neuron-specific intermediate filament composed of three different molecular weight subunits. Abnormal hyperphosphorylation of neurofilament is an important component and early pathological change of neurofibrillary tangles (NFT) in AD brain, but its pathogenesis is still unclear. In addition to phosphorylation, NFs proteins are also modified by Oglycosylation (O-GlcNAcylation) produced by glucose metabolism in cells. Our latest study has found that there is a mutual regulation between glycosylation and phosphorylation of NFs at the cellular level. Abnormal phosphorylation of NFs in brain nerve cells of AD patients decreased significantly in O-glycosylation. On the basis of the above research results, this paper mainly studies the effect of the disorder of O-glycosylation on the phosphorylation and assembly of neurofilament, and probes into the pathogenesis of neurofilament hyperphosphorylation. Methods: the effects of changes of O-glycosylation of neurofilament on phosphorylation and assembly of neurofilament were observed in vivo and in vitro. 35 male SD rats aged 4-6 months were divided into 5 groups with 7 rats in each group. Intraventricular injection of Alloxan (OGT inhibitor (2mg/kg), selective glucosaminase inhibitor NAG-AE (NAG-thiazoline,5mg/kg), Don (HBP pathway inhibitor, 2mg/kg), OA (PP2A inhibitor, 50ng/kg, respectively. The control group (normal saline) and the blank control group (3 rats) were killed 24 hours later. The glycosylation and phosphorylation of NFs protein in brain tissue were detected by Western blot. High speed centrifugation of brain homogenate, in vitro assembly of neurofilament, transmission electron microscopy and Western blotting were used to qualitatively and quantitatively compare the in vitro assembly and morphology of brain NFs with different glycosylation levels. Furthermore, we used in vitro enzyme chemistry to remove the glycosylation of protein from brain homogenate by 尾 -acetylglycosaminase, and observed the structure of neurofilament assembly by electron microscopy. To investigate whether NFs glycosylation itself or secondary phosphorylation affects brain NFs assembly. Results: our pre-test showed that the extraction efficiency of neurofilament was high and the purity was high. Electron microscopic observation showed that the nerve filament was composed of the fiber trunk about diameter 10nm and the lateral arm which was issued periodically by the trunk, and the lateral arm was generally short. The size and structure were the same as those obtained by other methods. In vivo, compared with the control group, the level of O-glycosylation of neurofilament protein in Alloxan,DON,OA group was decreased, the phosphorylation was increased, and the proportion of neurofilament was shorter and the proportion of assembly was significantly lower than that in control group. Compared with the control group, the level of O-glycosylation was significantly increased and the phosphorylation level was significantly decreased in NAG-Ae treatment. The neurofilament fibers were long filamentous and the proportion of assembly was higher. These results suggest that the changes of O-glycosylation of neurofilament have a certain effect on the assembly of neurofilament. Further in vitro, we only changed the glycosylation of NFs but did not change its phosphorylation level. The results suggest that the effect of deglycosylation alone on assembly is not obvious. Conclusion: the changes of Oglycosylation in vivo affect the phosphorylation of NFs, and the decrease of Oglycosylation may affect the structure and function of NFs by affecting its phosphorylation and neurofilament assembly, resulting in the degeneration of AD nerve cells. The results provide new ideas for the theoretical study of pathogenesis of AD and the development of drugs.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R749.16
【参考文献】
相关期刊论文 前2条
1 崔冉亮;胡海燕;吕朴;戎凯;陈宁;邓艳秋;;神经丝蛋白质糖基化与磷酸化的相互调节和神经退行性疾病[J];生命的化学;2009年06期
2 佟向军,陈建国,翟中和;低分子量神经丝蛋白(NF-L)的体外组装[J];实验生物学报;1998年03期
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