基于α-synuclein转基因大鼠帕金森病模型的内源性神经毒素致病机制的研究

发布时间：2018-04-23 08:30

本文选题：帕金森病 + α-synuclein　；参考：《北京理工大学》2015年博士论文

【摘要】：帕金森病(Parkinson’s disease,PD)是目前第二大神经退行性疾病,多于老年发病,其症状表现为静止性震颤,身心反应迟钝以及强直等,对生活质量造成严重的影响,且中国PD病人数量呈逐年增长的趋势。PD的两大病理特征为黑质区多巴胺能神经元的丧失与蛋白聚集体——路易小体(Lewy Bodies,LBs)的形成。关于PD的发病有很多假说,例如氧化应激假说,自由基假说等,然而其发病机制目前尚不清楚。遗传因素和环境因素被认为是PD的两个主要的致病原因,其中环境因素起主导作用。自从MPTP被发现以来,神经毒素作为PD的致病因子受到了广泛关注,儿茶酚四氢异喹啉类化合物(catechol tetrahydroisoquinolines,CTIQs)就是其中一类内源性的神经毒素。本课题组针对CTIQs在体内形成与代谢进行了长期研究,提出了CTIQs类神经毒素脑内形成与神经毒性的假说。当脑受到外界刺激(物理打击或环境污染等)会产生氧化应激导致脂质过氧化(lipid peroxidation,LPO),而LPO的终产物是醛类,醛类与多巴胺(dopamine,DA)反应后生成Salsolinol(Sal)等CTIQs,CTIQs经过氮甲基化和氧化代谢产生能够与线粒体复合体Ⅰ结合的离子化合物,造成细胞能量代谢障碍,进一步加剧氧化应激。另外,形成的CTIQs有可能促进脑内α-synuclein聚集的发生,而聚集进一步产生氧化应激,从而形成循环,其结果使神经元变性死亡。在本课题中,针对以上假说,通过构建α-synuclein转基因大鼠模型,重点研究内源性神经毒素N-methyl-Salsolinol(NMSal)在脑内对α-synuclein聚集的影响,阐明α-synuclein脑内聚集的机制,考察NMSal的神经毒性。通过本文的研究,对上述假说提供实验数据积累。本研究取得了以下成果:1、成功建立了脑内过表达α-synuclein的大鼠PD模型。采用了重组腺相关病毒(Recombinant Adeno-Associated Viral,rAAV)作为基因表达的载体,在研究过程中建立了一个简单,高效的纯化rAAV的方法,最终得到了滴度超过1013gc/mL的rAAV。利用脑立体定位技术将rAAV注射至大鼠大脑黑质致密部后,经过一系列时间节点的行为学观察,α-synuclein WT组和A53T组都会引发针对单侧多巴胺能神经元损伤的行为——阿扑吗啡诱导向健侧转圈,其中α-synuclein A53T组转圈速度为3.91±1.62圈/min,而WT组速度为2.68±1.74圈/min。对其脑部切片后进行免疫组化检测,外源性α-synuclein特异性地在多巴胺能神经元中表达,其中α-synuclein WT组从第3周至第12周,表达程度呈逐渐增加的趋势,而α-synucleina53t组至第9周时表达已至顶峰。同时对多巴胺能神经元标志性物质——酪氨酸羟化酶(tyrosinehydroxylase,th)的免疫组化检测表明,多巴胺能神经元的死亡与α-synuclein表达程度呈现正相关的趋势。以上结果说明α-synucleinwt与α-synucleina53t两种大鼠pd模型建立成功。2、nmsal可增强大鼠pd模型中α-synuclein产生的神经毒性。在成功建立大鼠pd模型的基础上,再次通过立体定位手术在其黑质区注射100nmolnmsal,诱导4周之后,其行为学指标显示nmsal的诱导可增加α-synucleinwt组与a53t组中阿扑吗啡诱导转圈速度。对其黑质纹状体系统进行免疫组化检测表明,nmsal能够促进大鼠pd模型中多巴胺能神经元的丧失,且α-synucleinwt组和a53t组的凋亡率分别上升了3.7%和7%。以上的结果显示nmsal能够引发大鼠pd模型中多巴胺能系统在原来基础上受到更严重的损伤。使用elisa的方法测定纹状体区丙二醛(malondialdehyde,mda)和活性氧基团(reactiveoxygenspecies,ros)的结果显示nmsal在α-synucleina53t组能够将这两个参数值提高50%,证明nmsal能够在α-synuclein过表达的鼠脑中促进氧化应激的发生。伴随着多巴胺能神经元的丧失,纹状体区多巴胺的含量也随之降低,其中α-synuclein组wt组和a53t组da含量分别约为对照组的44%与27%,nmsal的诱导可以进一步降低da含量,在wt组和a53t组分别仅为对照组的24%与15%。而内源性神经毒素的测定结果则证实了nmsal诱导之后对大鼠pd模型脑内sal和nmsal的生成具有促进作用。这些结果表明nmsal可以在大鼠pd模型中促进α-synuclein蛋白毒性作用的发挥,并且其促进效果在突变体的α-synucleina53t组表现更为显著。3、nmsal促进鼠脑中类似lbs的α-synuclein蛋白聚集。使用免疫印迹检测纹状体中不溶性的组分,结果显示经过nmsal诱导之后,α-synuclein组wt组和a53t组中α-synuclein三聚体的量分别增加为原来的2倍与3.5倍,免疫组化的结果显示nmsal的诱导会在纹状体中增加抵抗蛋白酶k消化的α-synuclein颗粒的数目。以上结果说明nmsal会促进脑内α-synuclein聚集的发生。使用s129磷酸化的α-synuclein抗体对黑质和纹状体区进行免疫组化染色的结果说明,只有在α-synucleina53t组才有显著的s129磷酸化发生,在纹状体区可以看到散布的颗粒存在,统计结果显示nmsal诱导前后颗粒数目分别为4.5±1.7和8.8±3.3,这说明nmsal可促进α-synuclein磷酸化的发生。而对纹状体进行泛素和α-synuclein免疫荧光染色的结果显示,在纹状体区出现了泛素与α-synuclein的共定位,说明出现了α-synuclein的泛素化,对荧光强度进行定量后可知,NMSal诱导可增加α-synuclein的泛素化程度。在纹状体中出现的颗粒具有磷酸化与泛素化的特性说明此α-synuclein聚集的颗粒具有与LBs相类似的性质。由此可知,NMSal可以促进α-synuclein聚集的发生,并产生类似LBs的蛋白聚集颗粒,且NMSal对α-synuclein A53T组的促进作用更为明显。4、NMSal可能抑制α-synuclein降解通路中的自噬-溶酶体途径。使用免疫印迹的方法检测了α-synuclein两大降解通路:自噬-溶酶体途径和泛素蛋白酶体系统,结果显示,在NMSal诱导之后,可导致大鼠PD模型鼠脑中自噬-溶酶体途径中Beclin-1、Atg5和Atg3的下调,而对Parkin和UCHL1的免疫印迹结果则显示NMSal的诱导对泛素蛋白酶体途径没有显著性的影响。此结果表明NMSal引发α-synuclein聚集的过程中一个可能的机制是阻断其降解通路中的自噬-溶酶体途径。本课题通过立体定位手术在大鼠PD模型中建立了α-synuclein过表达与NMSal直接定位诱导的实验,首次在动物脑内研究了过量的NMSal和过表达α-synuclein的直接相互作用,得出了NMSal可促进过表达的α-synuclein聚集并增强其对多巴胺能神经元的毒性的结论。这些结果为CTIQs类神经毒素脑内形成与神经毒性的假说的证明提供了体内的实验证据,并且为假说的进一步研究提供了非常有用的动物模型。
[Abstract]:Parkinson's disease (Parkinson 's disease, PD) is the second major neurodegenerative disease, more than the elderly, its symptoms are static tremor, mental retardation and ankylosis, which have a serious impact on the quality of life, and the number of PD patients in China is increasing year by year and the two major pathological characteristics of.PD are dopamine in the substantia nigra area. The loss of neurons and protein aggregates - the formation of the Lewy Bodies (Lewy Bodies, LBs). There are many hypotheses about the pathogenesis of PD, such as the oxidative stress hypothesis, the free radical hypothesis, etc. However, the pathogenesis is not yet clear. Genetic and environmental factors are considered to be the two main causes of PD, of which environmental factors play a leading role. Since MPTP was discovered, neurotoxin has been widely concerned as a pathogenic factor of PD. The catechol tetrahydroisoquinolines (CTIQs) of catechol (catechol tetrahydroisoquinolines, CTIQs) is a kind of endogenous neurotoxin. This research group has studied the form and metabolism of CTIQs in the body for a long time, and proposed the CTIQs class God. The hypothesis that the brain is formed and neurotoxic through the toxin. When the brain is stimulated by external stimuli (physical shock or environmental pollution), oxidative stress leads to lipid peroxidation (lipid peroxidation, LPO), and the final products of LPO are aldehydes, aldehydes and dopamine (dopamine, DA) react to Salsolinol (Sal) and other CTIQs, CTIQs through nitrogen methylation and oxidation. Metabolism produces ionic compounds that combine with mitochondrial complex I, causing cell energy metabolic disorders and further aggravating oxidative stress. In addition, the formation of CTIQs may promote the occurrence of alpha -synuclein aggregation in the brain, and the aggregation further produces oxidative stress and forms a cycle, which results in degeneration of neurons. In view of the above hypothesis, the effect of endogenous neurotoxin N-methyl-Salsolinol (NMSal) on the aggregation of alpha -synuclein in the brain was studied by constructing a -synuclein transgenic rat model. The mechanism of alpha -synuclein accumulation in the brain was elucidated and the neurotoxicity of NMSal was investigated. The experimental data were provided through this study. The following achievements have been obtained: 1, a rat model of PD was successfully established in the brain that overexpressed alpha -synuclein. The recombinant adeno-associated virus (Recombinant Adeno-Associated Viral, rAAV) was used as a carrier of gene expression. A simple, efficient method for purifying rAAV was established in the study, and the titer exceeded 1013gc/mL in the end. After injection of rAAV into the dense part of the substantia nigra of the rat brain by using rAAV. stereotaxic technique, the behavior of a series of time nodes was observed. Both the alpha -synuclein WT group and the A53T group would cause the behavior of the unilateral dopaminergic neuron injury - apomorphine induced Xiang Jian's side circle, of which the speed of the alpha -synuclein A53T group was 3.. 91 + 1.62 laps /min, while group WT was 2.68 + 1.74 laps /min. to detect the brain slices with immunohistochemical staining. Exogenous alpha -synuclein was specifically expressed in dopaminergic neurons. The expression level of alpha -synuclein WT group increased gradually from third to twelfth weeks, and the expression of alpha -synucleina53t group was at the top of ninth weeks. At the same time, the immunohistochemical detection of the Tyrosinehydroxylase (th), a marker of dopaminergic neurons, showed that the death of dopaminergic neurons was positively correlated with the expression of alpha -synuclein. The above results showed that the PD model of two rats of alpha -synucleinwt and alpha -synucleina53t was successful in establishing.2, nmsal. The neurotoxicity induced by alpha -synuclein in the rat PD model was enhanced. On the basis of the successful establishment of the rat PD model, 100nmolnmsal was injected into the substantia nigra by stereotaxic operation again. After 4 weeks of induction, its behavioral indicators showed that nmsal induction increased the speed of the alpha -synucleinwt group and the induced rotation of morphine in the a53t group. The immunohistochemical detection of the nigrostriatal system showed that nmsal could promote the loss of dopaminergic neurons in the PD model of rats, and the apoptosis rate of the alpha -synucleinwt group and the a53t group increased by 3.7% and 7%. respectively. The results showed that nmsal could cause the dopaminergic system in the rat PD model to be more severely damaged on the basis of the original basis. The results of the determination of malondialdehyde, MDA and reactive oxygen groups (reactiveoxygenspecies, ROS) in the striatum method by ELISA show that nmsal can increase these two parameters by 50% in the group of alpha -synucleina53t, proving that nmsal can promote oxidative stress in the rat brain of alpha -synuclein overexpression. The content of dopamine in the striatum decreased with the loss of the element. The Da content of group WT and a53t in group a -synuclein group was about 44% and 27% in the control group, respectively. The induction of nmsal could further reduce the content of Da. In WT group and a53t group, only 24% and 15%. in the control group and the results of endogenous neurotoxin test confirmed nmsal induction. The results showed that Sal and nmsal were promoted in the brain of rat PD model. These results showed that nmsal could promote the toxicity of alpha -synuclein protein in the rat PD model, and its promoting effect was more significant in the alpha -synucleina53t group of the mutant, and nmsal promoted the aggregation of alpha -synuclein protein in the rat brain similar to lbs. The insoluble fractions in the striatum were detected by immunoblotting. The results showed that after nmsal induction, the amount of alpha -synuclein trimer in group WT and a53t group increased to 2 times and 3.5 times, respectively. The results of immunohistochemistry showed that the induction of nmsal could be added to the alpha -synuclein particles digested by protease K in the striatum. The above results indicate that nmsal promotes the occurrence of alpha -synuclein aggregation in the brain. The results of immunohistochemical staining of substantia nigra and striatum by s129 phosphorylated alpha -synuclein antibody show that only a significant s129 phosphorylation occurs in the alpha -synucleina53t group, and the scattered particles can be found in the striate region and the statistical junction is observed. The results showed that the number of nmsal particles was 4.5 + 1.7 and 8.8 + 3.3 before and after induction, indicating that nmsal could promote the occurrence of alpha -synuclein phosphorylation. The results of ubiquitin and alpha -synuclein immunofluorescence staining in the striatum showed that ubiquitin and alpha -synuclein were found in the striatum, indicating the presence of ubiquitination of alpha -synuclein. When the fluorescence intensity is quantified, NMSal induction can increase the degree of ubiquitination of alpha -synuclein. The particles in the striatum have the properties of phosphorylation and ubiquitination indicating that the particles of this -synuclein aggregation are similar to that of LBs. Thus, NMSal can promote the occurrence of alpha -synuclein aggregation and produce a similar LBs. Protein aggregation particles, and the promotion of NMSal to the -synuclein A53T group more obviously.4, NMSal may inhibit the autophagy lysosome pathway in the alpha -synuclein degradation pathway. Using the immunoblotting method to detect the large degradation pathway of alpha -synuclein two: autophagy lysosome pathway and ubiquitin proteasome system. The results show that NMSal induced by NMSal After that, it could lead to the downregulation of Beclin-1, Atg5 and Atg3 in the autophagy lysosome pathway of rat PD model, while the immunoblotting results of Parkin and UCHL1 showed that the induction of NMSal had no significant effect on the ubiquitin proteasome pathway. The results showed that a possible mechanism in the process of NMSal induced alpha -synuclein aggregation was to block its descending. The autophagy lysosome pathway in the pathway was solved by stereotaxic surgery in the rat PD model. The experiment was established to induce the over expression of alpha -synuclein and direct localization of NMSal in the rat model. The direct interaction of excessive NMSal and over expression of alpha -synuclein was first studied in the animal brain, and the NMSal could promote the over expressed alpha -synuclein aggregation. These results provide experimental evidence for the hypothesis of the brain formation and neurotoxicity of the CTIQs neurotoxin, and provide a very useful animal model for further research on the hypothesis.

【学位授予单位】：北京理工大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R742.5

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