老年痴呆病理与SUMO-1修饰的相关研究

发布时间：2018-04-29 02:43

本文选题：老年性痴呆 + 小泛素化修饰1　；参考：《复旦大学》2012年硕士论文

【摘要】：阿尔茨海默病(Alzheimer disease, AD)是以智能衰退为主要表现的神经系统退行性疾病。AD的主要病理特征为脑内分布有大量的β-淀粉样蛋白(β-amyloid,Aβ)沉积形成的老年斑(senile plaque, SP)、申经元内高度磷酸化tau蛋白聚集形成的神经原纤维缠结(neurofibrillary tangle)、神经元营养障碍、神经元的大量丢失、突触的损害和丢失。本实验分别用SH-SY5Y细胞和APPswe/PS1△E9AD转基因鼠进行了老年痴呆病理与小泛素相关修饰物(small ubiquitin-related modifer, sumo-1)的相关研究。主要研究结果如下：第一部分：Aβ毒性作用、蛋白酶体系统抑制和SUMO-1修饰的关系 1.在Aβ对SUMO-1诱导作用的研究中,我们观察到,SH-SY5Y细胞经Aβ处理后,SUMO-1修饰没有显著变化,而在过表达SUMO-1的情况下,Aβ可促进SUMO-1的修饰；Aβ促进SUMO修饰和去修饰系统中的UBA2, RANBP2和SENP1表达,抑制UBC9表达；SUMO-1过表达可以增强Aβ对UBA2的促进作用,抑制Aβ对UBC9的抑制作用使UBC9表达明显增加,抑制Aβ对SENP1的促进作用。以上结果表明在SUMO-1过表达条件下,Aβ可能通过SUMO修饰系统诱导SUMO修饰增多。 2.在对Aβ诱导的泛素-蛋白酶体抑制与SUMO-1修饰关系的研究中,免疫印迹结果显示SH-SY5Y细胞模型经Aβ处理可诱导泛素修饰蛋白在高分子量部位的显著增加,提示Aβ可诱导蛋白酶体系统抑制,而SUMO-1过表达可增加泛素修饰蛋白的堆积。蛋白酶体抑制剂MG132可以诱导泛素化蛋白增多,对SUMO-1的修饰增加不明显；但在SUMO-1过表达条件下,蛋白酶体抑制可以促进SUMO-1的修饰,同时SUMO-1过表达可以促进MG132诱导的泛素化蛋白增加。SUMO-1过表达可以促进MG132诱导的UBC9, UBA2增加,抑制MG132诱导的SENP1增多,表明蛋白酶体抑制时,SUMO修饰系统发生改变,SUMO-1过表达可起促进作用。 3. SUMO-1抑制MG132诱导caspase3、-6的激活,SUMO-1在一定程度上对蛋白酶体抑制产生的凋亡有抑制作用,说明SUMO-1有一定的抗凋亡作用。 4.在SH-SY5Y细胞模型中,免疫印迹结果显示,SUMO-1过表达可以促进Ap诱导的高分子量tau蛋白磷酸化的增加；磷酸酯酶抑制剂可以促进SUMO-1化修饰蛋白的增加,但作用不是很强。第二部分APPswe/PS1△E9转基因鼠中SUMO-1及p-tau相关 1.同12个月龄野生型小鼠相比,12个月龄APPswe/PS1△E9AD转基因鼠脑内,免疫印迹实验检测显示多个脑区有SUMO-1蛋白表达及修饰的显著增加,同时伴有泛素修饰蛋白的增加,和tau蛋白磷酸化的增加。 2.用12个月龄的APPswe/PS1△E9转基因鼠脑可溶性蛋白组分中进行免疫沉淀研究,结果显示用SUMO-1抗体免疫沉淀的蛋白,进行免疫印迹实验,结果发现与12月龄野生型小鼠相比APP.PS422抗体标记的磷酸化tau没有明显的变化,而tau5抗体标记的总tau蛋白有所增加,而AT8抗体标记的磷酸化tau蛋白减少。 3.免疫荧光双标显示,在12个月龄的APPswe/PS1△E9转基因鼠中,Ap老年斑中存在SUMO-1,及SUMO-1与p-TAU的部分共定位。在12个月龄的APPswe/PS1△E9转基因鼠,脑可溶性蛋白组分SUMO-1化的磷酸化tau蛋白减少,可能因为这部分蛋白大多呈不容状态。这提示SUMO-1可能参与AD慢性的神经退行性变病理。总之,SUMO-1过表达可促进Ap诱导的SUMO-1修饰系统和蛋白酶体系统的变化,导致SUMO-1修饰蛋白和泛素修饰蛋白增加,Ap诱导的SUMO-1修饰系统的变化可能与Ap诱导的蛋白酶体系统的抑制有关。SUMO-1过表达可能促进蛋白的沉积和tau聚集,但是对急性的凋亡有抑制作用。
[Abstract]:Alzheimer disease (AD) is the main pathological feature of the neurodegenerative disease.AD, which is mainly manifested by the intelligence decline, which consists of a large number of beta amyloid (beta -amyloid, A beta) deposition in the brain (senile plaque, SP), and the aggregation of neurogenic fibers from the highly phosphorylated tau protein in the meta. Neurofibrillary tangle, neurotrophic disorder, massive loss of neurons, damage and loss of synapses. This experiment used SH-SY5Y cells and APPswe/PS1 Delta E9AD transgenic mice to study the pathology of Alzheimer's disease (small ubiquitin-related modifer, SUMO-1), and the main research results were as follows. Below:
Part one: the relationship between A beta toxicity, proteasome inhibition and SUMO-1 modification.
1. in the study of the induction of SUMO-1 by A beta, we observed that SUMO-1 modification did not change significantly after A beta treatment, while A beta could promote the modification of SUMO-1 in the presence of SUMO-1, and A beta promoted SUMO modification and the UBA2 in the modification of the system. RANBP2 and expression could inhibit the expression of SUMO-1. The inhibitory effect of beta on UBA2, inhibiting the inhibition of A beta on UBC9 significantly increases the expression of UBC9 and inhibits the promotion of A beta to SENP1. The above results show that A beta may induce the increase of SUMO modification by the SUMO modification system under the condition of SUMO-1 overexpression.
2. in the study of the relationship between A beta induced ubiquitin proteasome inhibition and SUMO-1 modification, the results of immunoblotting showed that the SH-SY5Y cell model could induce a significant increase in the ubiquitin modified protein in the high molecular weight site by A beta treatment, suggesting that A beta could induce the inhibition of proteasome system, and the overexpression of SUMO-1 could increase the accumulation of ubiquitin modified proteins. White enzyme inhibitor MG132 can induce the increase of ubiquitin protein and not increase the modification of SUMO-1. But under the SUMO-1 overexpression, proteasome inhibition can promote the modification of SUMO-1, while SUMO-1 overexpression can promote MG132 induced ubiquitin protein to increase.SUMO-1 overexpression can promote MG132 induced UBC9, UBA2 increase, Inhibition of MG132 induced increase in SENP1 indicates that the SUMO modification system changes when proteasome is inhibited, and over expression of SUMO-1 can play a promoting role.
3. SUMO-1 inhibited MG132 induced Caspase3 and -6 activation. SUMO-1 inhibited the apoptosis induced by proteasome inhibition to some extent, indicating that SUMO-1 had a certain anti apoptosis effect.
4. in the SH-SY5Y cell model, the results of immunoblotting showed that the overexpression of SUMO-1 could promote the increase of the phosphorylation of high molecular weight tau protein induced by Ap, and the phosphate esterase inhibitor could promote the increase of the SUMO-1 modified protein, but the effect was not very strong.
The second part is related to SUMO-1 and p-tau in APPswe/PS1 Delta E9 transgenic mice.
1. compared with 12 month old wild type mice, the immunoblotting test showed that there was a significant increase in the expression and modification of SUMO-1 protein in multiple brain regions, with the increase of ubiquitin modified protein and the increase of phosphorylation of tau protein in the brain of 12 months old APPswe/PS1 Delta E9AD transgenic mice.
2. immunoprecipitation was carried out in the APPswe/PS1 Delta E9 transgenic mouse brain soluble protein component of 12 months old. The results showed that the protein of SUMO-1 antibody immunoprecipitation was used for Western blot experiment. The results showed that the phosphorylated tau of the APP.PS422 antibody labeled with the 12 month old wild type mice had no obvious change, and the total tau5 antibody marker was total. Tau protein increased, while AT8 antibody labeled phosphorylated tau protein decreased.
3. immunofluorescence double labeling showed that in the 12 month old APPswe/PS1 Delta E9 transgenic mice, there was SUMO-1 in the Ap plaque and the co localization of SUMO-1 and p-TAU. In the APPswe/PS1 Delta E9 transgenic mice of 12 months old, the SUMO-1 phosphorylated tau protein decreased in the soluble protein component of the brain, probably because most of the proteins were incapable. SUMO-1 may be involved in AD chronic neurodegeneration.
In conclusion, overexpression of SUMO-1 can promote the changes in the Ap induced SUMO-1 modification system and proteasome system, resulting in the increase of SUMO-1 modified proteins and ubiquitin modified proteins. The changes in the SUMO-1 modification system induced by Ap may be related to the inhibition of the Ap induced proteasome system, which may promote the deposition of protein and tau aggregation. Acute apoptosis has an inhibitory effect.

【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R749.16

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