摘要
阿尔茨海默病(Alzheimer’s disease, AD)的两大重要的病理学特征是神经细胞内的大量神经原纤维缠结(neurofibrillary tangles,NFT)和神经细胞间的大量老年斑(senile plaques,SP),临床表现为进行性记忆减退。神经原纤维缠结主要由异常过度磷酸化的骨架蛋白tau构成,而老年斑的核心成分是由β-淀粉样前体蛋白(β-amyloid precursor protein,APP)降解产生的β-淀粉样多肽(β-amyloid, Aβ)。目前AD的发病机制仍不清楚,解释其发病机制的主要学说有“tau异常学说”和“Aβ毒性学说”,但孤立地用一种学说不能全面地解释AD脑中的病理改变。虽然存在很多发病过程,但其确切的影响因素和发病机制至今仍未完全明了。
同型半胱氨酸(Homocysteine,Hcy)是甲基化循环的一个中间产物,它在细胞蛋白质、DNA和脂类的甲基化中起重要作用。流行病学调查和临床研究发现,AD的发生与血浆的Hcy关系密切,高同型半胱氨酸血症(hyperhomocysteinemia,HHcy)是AD强而独立的危险因子。我们在本研究中,利用大鼠经尾静脉注射Hcy14天,或辅饲叶酸和维生素B12进行治疗,来探讨HHcy是否引起大鼠出现AD样改变及其可能的机制。
第一部分
同型半胱氨酸诱导大鼠海马tau蛋白过度磷酸化及其机制研究
在本实验中,我们利用青年健康的雄性SD大鼠,经尾静脉注射Hcy复制HHcy模型,来研究大鼠是否出现AD样tau蛋白磷酸化并探讨其机制。具体的方法和结果如下:
一、Hcy升高对大鼠海马tau蛋白磷酸化的影响
为了研究Hcy对tau蛋白磷酸的影响,大鼠尾静脉注射Hcy 400μg/kg/day 3、7、14、21天,结果显示:tau蛋白在Ser396和Ser199/Ser202位点出现过度磷酸化,并且磷酸化的程度随着时间的延长而加重。然后我们用两个不同的Hcy剂量(400μg /kg/day或1600μg /kg/day)对大鼠注射14天,或同时辅以叶酸和维生素B12作治疗组,用相关抗体进行Western-blotting检测,结果发现tau蛋白出现多位点的磷酸化;免疫组化和银染的结果与上述一致,叶酸和B12能减轻Hcy的此种效应。Hcy的剂量升高到1600μg/kg/day时,除Thr205和Ser214两个位点外,其余位点的磷酸化没有进一步增加。因此,我们就选择Hcy 400μg/kg/day的量注射大鼠14天,作为机制探讨的模型,来进行更进一步的研究。
二、HPLC测定的血液中Hcy浓度
为了证实大鼠tau蛋白出现的过度磷酸化是Hcy浓度升高所致,我们采用高压液相色谱检测大鼠血浆中的Hcy的浓度。经过14天的Hcy注射,血浆中的Hcy浓度显著的升高,当增补叶酸和B12后明显的降低Hcy的浓度。这些结果表明提高血液中的Hcy能诱导大鼠出现AD样多位点的磷酸化。
三、血浆中Hcy的升高对PP-2A和GSK-3活性的影响及与活性相关的修饰我们用32P-ATP检测了PP-2A的活性,发现注射Hcy后,海马神经元PP-2A的活性降低40%。Western-blotting检测发现PP-2Ac总的表达水平没有变化,PP-2Ac的Leu309的甲基化(活性形式)水平显著降低,而Leu309去甲基化水平(失活形式)显著提高。我们又检测了PP-2Ac的磷酸化,发现PP-2Ac Tyr307位点的磷酸化显著增加,与PP-2Ac Leu309位点的去甲基呈正相关。当增补叶酸和B12时,PP-2A的活性可以恢复至对照组的85%,并增加Leu309位点的甲基化和降低Tyr307位点磷酸化。这些说明Hcy通过阻断PP-2Ac的甲基化和增加PP-2Ac的磷酸化来抑制PP-2A的活性。
GSK-3是AD中一个关键的激酶,我们利用32P-ATP检测它的活性,发现各组间没有显著性差异;Western-blotting显示,GSK-3总的表达水平和Ser-9位点的磷酸化水平无明显变化。这就可以排除GSK-3参与了Hcy诱导大鼠tau蛋白的过度磷酸化。
我们又检测了调节PP-2Ac甲基化的两个酶:甲酯酶(PME)和S-腺苷甲硫氨酸依赖的甲基转移酶(PPMT)表达情况。结果发现PPMT的表达水平各组间无差异,但在Hcy组PME的表达水平显著增加,这就表明Hcy注射引起PP-2Ac的去甲基化是由PME导致的,增补叶酸和维生素B12可以部分恢复Hcy导致的PME的升高。
四、PP-2Ac去甲基化对PP-2A生物学功能的影响
为了探讨Hcy诱导的PP-2Ac去甲基化对PP-2Ac生物学功能的影响,我们用总PP-2A抗体进行免疫沉淀,用Western-blotting检测发现,PP-2Ac的总量各组间无变化,但Hcy组的去甲基化水平和磷酸化水平比对照组显著增加,且沉淀物中PP-2AB显著减少,说明PP-2Ac的去甲基化降低了PP-2Ac结合调节B亚基的能力。接着用去甲基化的PP-2Ac抗体免疫沉淀,发现沉淀中去甲基化的PP-2Ac同时被磷酸化,PP-2Ac的去甲基化和磷酸化间有高度的正相关;增补叶酸和维生素B12可以减轻Hcy引起的这些变化。这些结果表明PP-2Ac的去甲基化可以促进它的磷酸化,并共同抑制PP-2A的活性。
五、甲基化修饰的PP-2Ac和磷酸化tau蛋白在Hcy诱导的大鼠海马和AD脑中的相互关系和分布
为了探讨去甲基化的PP-2Ac和磷酸化的tau蛋白共定位关系,我们用甲基化的PP-2Ac、去甲基化的PP-2Ac和PS396抗体进行免疫荧光双标。结果发现Hcy组大鼠海马CA1区磷酸化显色明显加强,同时去甲基化PP-2Ac的免疫活性也相应增加;但是甲基化PP-2Ac的显色显著减弱。同时还发现只有去甲基化的PP-2Ac与磷酸化的tau蛋白共定位,而甲基化的PP-2Ac与磷酸化的tau蛋白完全不共定位。上述这种定位关系在AD病人脑海马中也同样显著。这些结果强烈说明了在Hcy诱导的大鼠脑和AD病人脑中,PP-2Ac的去甲基化导致了tau蛋白的磷酸化。
结论
在本实验中,我们发现提高血液中的Hcy能通过失活PP-2A来导致tau的磷酸化。致使PP-2A失活的机制主要是PP-2Ac的去甲基化,并由此导致PP-2Ac的磷酸化和PP-2Ac结合PP-2AB亚基的能力降低。PME表达增加可能是Hcy升高时引起PP-2Ac去甲基化机制。同时增补叶酸和维生素B12可以部分的恢复血浆Hcy的水平,并减轻PP-2Ac的异常修饰和降低tau蛋白的磷酸化。
第二部分同型半胱氨酸诱导大鼠空间记忆障碍和Aβ增多并聚集的机制
我们在本实验中,用同型半胱氨酸进行大鼠尾静脉注射14天,复制高同型半胱氨酸血症模型,来探讨Hcy引起大鼠空间记忆障碍和Aβ产生增加的机制。实验方法和结果如下:
一、Hcy升高对大鼠海马APP代谢的影响
为了研究Hcy升高对大鼠海马内APP的代谢影响,我们采用尾静脉注射Hcy 400μg/kg/day,分为3、7、14、21天。结果显示:4G8和6E10的显色加深,说明APP在β-位点的切割增加,并随着时间的延长而增加。
因为14天的Hcy注射能显著的诱导APP蛋白分解代谢变化,然后我们用两个不同的Hcy剂量(400μg /kg/day或1600μg /kg/day)注射大鼠14天,或同时辅以叶酸和维生素B12治疗,结果发现两个剂量都能导致APP在β-位点的切割增加,叶酸和维生素B12对上述有拮抗作用。当Hcy的剂量升高到1600μg/kg/day时,Aβ的产生没有进一步增加。因此,我们就选择Hcy 400μg/kg/day的量注射大鼠14天,作为机制探讨的模型,
我们利用大鼠脑切片,用Aβ的抗体免疫组化和用Thioflavin-S染色,结果进一步证明,Hcy升高可以导致大鼠皮质和海马Aβ的产生增加,并且叶酸和B12能减轻Hcy的此种作用。说明了Hcy导致AD样毒性部分是通过Aβ的产生增加而致。
二、Hcy对大鼠空间记忆的影响
为了证明Hcy引起了大鼠行为学的损伤,我们采用Morris水迷宫的方法检测大鼠的空间记忆情况。结果发现HHcy可以明显的损伤大鼠的空间记忆能力,其中以低剂量的Hcy结果最明显。叶酸和维生素B12可以拮抗Hcy的作用,提高大鼠的记忆能力。
三、ELISA测定大鼠皮质和海马Aβ40的含量
用ELISA的方法测定大鼠皮质和海马Aβ40的实际含量。两个不同剂量的Hcy注射14天后,大鼠皮质和海马Aβ产生的量显著升高,以低剂量的Hcy最明显,并且皮质的变化最显著,当辅饲叶酸和维生素B12后,能明显的降低Aβ的产生。
四、RT-PCR检测APP及其代谢分泌酶基因水平的变化
用相应引物进行RT-PCR反应。结果发现除γ-分泌酶的PS1基因在mRNA水平表达升高外,其余酶和蛋白在RNA水平均无显著性变化。当加饲叶酸和维生素B12后,能显著降低γ-分泌酶PS1在RNA水平上的变化。这说明Aβ增加不是因为APP表达水平的改变引起,而是受其代谢变化或其它变化影响所致。
五、翻译后蛋白水平的检测
因为APP和α-、β-分泌酶在基因水平上没有改变,接着我们用Western-blotting来检测β-和γ-分泌酶PS2的表达,结果发现它们在翻译水平上也无明显的改变。因为用4G8和6E10两抗体检测,β切割产生的片段呈强阳性反应,且Aβ的产生量显著增加,我们就用p-Thr668-APP抗体(识别磷酸化的APP Thr668位点)检测APP磷酸化的修饰情况,发现APP在Thr668位点的磷酸化加强,与对照组相比有显著的差异性,叶酸和维生素B12能显著降低这种变化。免疫组化结果与Western-blotting结果一致。说明Hcy导致APP出现了异常磷酸化修饰.
六、磷酸化APP对自身代谢的影响
为了进一步探讨磷酸化修饰的APP对自身代谢的影响,我们又用磷酸化p-Thr668-APP的抗体去免疫沉淀,结果发现磷酸化的APP阳性显色增加,还可以见切割的磷酸化APP片段;并且沉淀中BACE1和PS2数量显著升高;叶酸和维生素B12对上述Hcy的影响有拮抗作用。这说明APP磷酸化修饰后,增加了与β-和γ-分泌酶的亲和力,使APP便于β、γ-分泌酶切割,因此导致了Aβ的产生增加。
结论
总之在本实验中, HHcy一方面通过增加PS1的表达,提高γ-分泌酶的活性,另一方面通过增加APP Thr668的磷酸化修饰,增加APP与分泌酶的亲和性,致使Aβ产生增多。增补叶酸和维生素B12可以部分的恢复血浆Hcy的水平的同时,并减轻PS1的表达和APP的异常修饰,降低Aβ的产生和聚集。
Alzheimer’s disease is characterized by intracellular neurofibrillary tangles and extracellular senile plaques, and clinical manifestation is progressive memory impair. Neurofibrillary tangles are composed of aberrantly hyperphosphorylated tau protein, andβ-amyloid (Aβ), a peptide derived from cleavage ofβ-amyloid precursor protein (APP), is the major component of senile plaques. The pathogenesis of AD is still elusive, there are two dominant theories to underscore the pathogenesis of Alzheimer’s disease: tau hypothesis and Aβhypothesis. However, neither of the two theories alone can fully explain the neuropathology in AD brain. Although many pathologic processes have been investigated in this disease, but the concrete influence factors and mechanisms remain unclear.
Hcy is central to the methyl cycle and plays an important role in cellular methylation for protein,DNA and lipid, and elevated plasma Hcy levels precedes the onset of AD and is an independent risk factor for the disease. Epidemiology and clinical data have demonstrated that there present a positive correlation between elevated plasma homocysteine (Hcy) and the occurrence of AD, and thus hyperhomocysteinemia(HHcy) has been proposed to be an independent risk factor of AD. In this study, we injected Hcy into rats by vena caudalis, or supplement of folate and vit-B12 to investigate whether HHcy could induce AD-like alteration in rats and the potential mechanisms.
PartⅠ
The study of the hyperphosphorylation of tau and the underlying mechanisms induced by homocysteine in rats
In this study, in order to reproduce the HHcy model, we selected the health young male SD rats, injected the rats with Hcy by vena caudalis, to explore whether there would be AD-like hyperphosphorylation of tau and the potential mechanisms. And the concrete methods and results are shown as follows:
1.Effect of high plasma Hcy on tau phosphorylation in rat hippocampus
To investigate the effect of Hcy on tau phosphorylation, we injected the rats with Hcy (400μg/kg/day) by vena caudalis for 3, 7, 14 and 21 days, respectively. It was shown that tau was hyperphosphorylated at ser 396 and ser199/ser202 site, and the immunoreactivity of hyperphosphorylation increased time-dependently. Then, we injected the rats for 14 days with two different dosages of Hcy (400μg/kg/day or 1600μg/kg/day), and the rats were treated simultaneously with or without supplement of folate and vit-B12. We observed that a significantly increased tau phosphorylation at many sites by western-blotting. Increased phosphorylation of tau was demonstrated by immunohistochemistry and silver staining, it was attenuated by folate/vit-B12. No further elevation of tau phosphorylation except at PT205 and PS214 was observed when the concentration of Hcy was raised from 400μg/kg/day to 1600μg/kg/day. With these results, we selected 400μg/kg/day of Hcy and injected for 14 days in our further studies of the mechanism.
2. The level of plasma Hcy was detected by HPLC
To confirm the role of Hcy , we measured the plasma level of Hcy by HPLC. A significant elevation of Hcy was shown after the injection, and the supplement of folate/vit-B12 distinctly attenuated the elevation of plasma Hcy. These data strongly suggest that elevated plasma Hcy can induce tau hyperphosphorylation at multiple AD-sites in the hippocampus of the rat brains.
3. Effect of Hcy on the activity and the activity-related modifications of PP2A and GSK-3
We measured the activity of PP2A by 32p-ATP and found that the activity of PP2A decreased to ~60% of the control level in Hcy-injected rats . With basically unchanged total protein level of PP2AC, the level of the Leu309-methylated PP2AC (activated form) decreased significantly and the level of the Leu309-demethylated PP2AC (inactivated form) increased after the injection of Hcy. We measured the phosphorylation of PP2AC, and observed that the level of the Tyr307-phosphorylated PP2AC increased and this change was positively correlated with the alteration of the Leu309-demethylated PP2AC. A simultaneous supplement of folate/vit-B12 restored the activity of PP2A to ~85% of the control level with a simultaneous restoration of the methylation and phosphorylation of PP2AC. These data together suggest that Hcy inhibits PP2A through interrupting the methylation of PP2AC and phosphorylation of PP2AC may also contribute to the inactivation of the phosphatase.
We also measured the activity of GSK-3 with 32p-ATP , GSK3 is a crucial kinase in AD-like tau hyperphosphorylation, but no obvious change was observed in the activity of GSK-3, as well as the levels of the total and the Ser9-phosphorylated GSK-3 by western-blotting. These results ruled out the involvement of GSK-3 in Hcy-induced tau hyperphosphorylation in the rats.
The methylation of PP2AC is regulated by a specific methylesterase (PME) and S-adenosylmethionine-dependent methyltransferase (PPMT), therefore, we detected the expression of these two enzymes. It was shown that the level of PME increased significantly and the level of PPMT was not changed in Hcy-injected rats.This suggested the role of PME in Hcy-induced demethylation of PP2AC. Simultaneous supplement of folate/vit-B12 partially restored the Hcy-induced elevation of PME.
4. Effect of Hcy-induced demethylation on the biological function PP2AC To explore the influence of Hcy-induced demethylation on the biological function of PP2AC, we immunoprecipitated with an anti-total PP2AC antibody. With a significantly elevated demethylation/phosphorylation of PP2AC and an unchanged total PP2AC, we observed a remarkable decrease of PP2AB in the precipitates of the Hcy-injected rats, suggesting a decreased binding capacity of PP2AC with PP2AB.
Then, we analyzed the phosphorylation status of PP2AC with immunoprecipitation by demethylated-PP2AC antibody. It was shown that the demethylated PP2AC was phosphorylated, and a highly positive correlation between demethylation and phosphorylation was observed. Simultaneous supplement of folate/vit-B12 partly attenuated the Hcy-induced alterations. These results indicate that demethylation may promote phosphorylation of PP2AC and thus inhibits its activity
5. The in situ relationship between modified PP2AC and hyperphosphorylated tau in the hippocampal neurons of the Hcy-injected rats and the AD patients
To explore the in situ relevance of the methylation of PP2AC and the phosphorylation of tau, we did co-labeling studies using the antibody of methylated PP2AC, demethylated PP2AC and Ser-396-phosphorylated tau. Compared with control subjects, the immunoreactivity of Ser-396-phosphorylated tau was significantly increased in hippocampal CA1 neurons of the Hcy-injected rats; concomitantly, the immunoreactivity of the demethylated PP2AC was increased but that of methylated PP2AC was decreased dramatically, and only the demethylated PP2AC but not the methylated PP2AC was co-localized with the hyperphosphorylated tau. The co-localization was also remarkable in AD brains. These data together strongly suggest that demethylation of PP2AC contributes to tau hyperphosphorylation in rats induced by Hcy and in the AD brains.
Conclusion
Taken together, we have found in the present study that elevation of plasma Hcy can induce tau hyperphosphorylation through inactivating PP-2A. The underlying mechanism for the inactivation of PP-2A involves demethylation of PP2AC, which favors phosphorylation of PP2AC and disrupts the binding of PP2Ac to PP2AB. The elevation of PME may also contribute to the Hcy-induced demethylation of PP2AC. Simultaneous supplement of folate and vit-B12 restores partially the plasma Hcy level and thus attenuates the abnormal modification/inactivation of PP2AC and tau hyperphosphorylation.
PartⅡ
The mechanisms of impairment of spatial memory and increase and accumulation of Aβby Hcy in rats
In this study, in order to reproduce the HHcy model, we injected the rats with Hcy by vena caudalis, to explore memory impairment of rats and Aβaccumulation and the potential mechanisms. The concrete methods and results are shown as follows:
1. The effect of APP catabolism by HHcy in rats hippocampal
To investigate the effect of Hcy on APP catabolism, we injected the rats with Hcy (400μg/kg/day) by vena caudalis for 3, 7, 14 and 21 days, respectively. It was demonstrated that injection of Hcy could induce APP catabolic time dependently at both 4G8 and 4E10 epitopes. Then, we injected the rats for 14 days with two different dosages of Hcy (400μg/kg/day or 1600μg/kg/day), and the rats were treated simultaneously with or without supplement of folate and vit-B12. We observed that a significantly increased APP dissection atβ-sites by western-blotting. It was attenuated by folate/vit-B12. Without further elevation of Aβproduction and accumulation was observed when the concentration of Hcy was raised from 400μg/kg/day to 1600μg/kg/day. With these results, we selected 400μg/kg/day of Hcy and injected for 14 days in our further studies of the mechanism.
APP dissection was further demonstrated by immunohistochemistry with the antibody of Aβand Thioflavin-S staining, the results was similar to above,and folate/vit-B12 attenuated the alteration.These results manifested that one of toxicity of Hcy for AD is through on Aβaccumulation.
2. The impairment of spatial memory of rats by Hcy
To investigate the impairment of Hcy on spatial memory,we detected the rats spatial memory by Morris Water Maze.The ability of spatial memory was impaired significantly by Hcy,especially in lower dosage of Hcy.Folate and vit-B12 could prevent the spatial memory from impairment.
3. The level of Aβwas detected by ELISA in rats cortex and hippocampal
ELISA detects the actually level of Aβ40 in rats cortex and hippocampal. After 14 days Hcy injection, the level of Aβ40 in cortex and hippocampal increased significantly when compared to the control, especially in contex and lower dosage Hcy. While folate and vitB12 were supplement, the level of Aβ40 decreased.
4. The alterations of mRNA of APP and its metabolic secretase by RT-PCR
With RT-PCR, we found that besides the PS1 mRNA, other mRNA were not changed by Hcy. Folate and vit-B12 could decrease the PS1 mRNA.This results demonstrated that the increasing of Aβwasn’t induced by the expression of APP, but there were other mechanisms involving in it.
5. Detection of proteins expression and the modification
Since there was no change on the gene level of APP and its secretase, we detected the expression of BACE andγ-secretase by western-blotting, and no change was observed on the level. Now that the peptide of APP dissection byβ-secretase was strong positive reaction,with the 4G8 and 6E10 detection, and the level of Aβincreased significantly, nextly we explored the phosphorylation modification of APP with the p-Thr668-APP antibody. Comparation to the control, phosphorylation of APP increased significantly, and folate/vit-B12 decreased partially the alteration, The same alterations were visible in rats brain slice by immunohistochemistry staining. The results demonstrated that APP was abnormally phosphorylated by Hcy..
6. The influence of APP catabolism by its phosphorylation
In order to further explore the influence of phosphorylation modified APP on itself catabolism, we immunoprecipitated with an anti-p-Thr668-APP antibody, with a significantly elevated phosphorylation of APP, and the dissected phosphorylated peptide of APP could be visible. We observed a remarkable increase of BACE1 and PS2 in the precipitates of the Hcy-injected rats. Simultaneous supplement of folate/vit-B12 partly attenuated the Hcy-induced alterations. The results explained that phosphorylation modificated APP increased the ability and affinity for APP to bind its secretase, and this induced APP processing and dissection byβ- andγ-secretase, and increased Aβlevel.
Conclusion
Taken together, we have found in the present study that elevation of plasma Hcy can increase the expression of PS1, enhance theγ-secretase activity; and induce the APP phosphorylation, increase the ability and affinity for APP to bind its secretase and increase the Aβproduction. Simultaneous supplement of folate and vit-B12 restores partially the plasma Hcy level, and thus attenuates the abnormal hyperphosphorylation of APP, and decrease the Aβproduction and accumulation.
引文
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