硫胺素缺乏对β-淀粉样多肽前体蛋白加工和Aβ生成的作用及机制研究

【作者】 张骑鹏；

【导师】 孙安阳；

【作者基本信息】 复旦大学 ， 神经生物学， 2008， 博士

【摘要】 阿尔茨海默病（Alzheimer disease,AD）是老年痴呆的最常见类型,其神经病理学特征主要表现为沉积在细胞外的老年斑（Senile plaques,SPs）和细胞内聚集的神经纤维缠结（Neurofibrillary tangles,NFTs）以及区域性的神经元丧失。目前,对于AD的发病原因及其病理机制了解甚少,尚没有有效的治疗方法。β-淀粉样多肽（β-Amyloid peptide,Aβ）是SPs的主要成分,来源于β-淀粉样多肽前体蛋白（Amyloid precursor protein,APP）的蛋白剪切。大量研究表明Aβ的代谢异常可能是AD起始性的病理环节。研究Aβ的代谢对于探讨AD的发病机理较为重要。硫胺素（Thiamine）即维生素B₁,是人体必需的营养成分。硫胺素在体内主要以焦磷酸硫胺素（Thiamine pyrophosphate,TPP）的形式存在,TPP是糖代谢途径中的丙酮酸脱氢酶复合体,α-酮戊二酸脱氢酶复合体以及磷酸戊糖途径中的转酮醇的辅酶因子。硫胺素在维持脑内氧化代谢平衡方面也发挥了重要作用。硫胺素缺乏（Thiamine deficiency,TD）会导致以上三种酶类的活性下降、三羧酸循环发生障碍、三磷酸腺苷（adenosine-triphosphate,ATP）产生受阻。此外,硫胺素缺乏过程中产生了大量的活性氧（reactive oxygen species,ROS）,导致了氧化应激的发生。主要依靠糖代谢提供能量的神经组织对硫胺素缺乏高度敏感,硫胺素缺乏的中枢神经表现称为脑型维生素B₁缺乏症,也称韦尼克—科尔萨科夫综合症（Wernicke-Korsakoff’s syndrome,WKS）,病人除了有明显的认知丧失、记忆力减退外,晚期WKS病人出现脑室扩大,神经元死亡。其脑内的KGDHC活性也明显降低。在硫胺素缺乏动物模型上,硫胺素缺乏处理的小鼠脑内出现了神经元死亡,小胶质细胞、星型胶质细胞肥大、增生等神经退行性疾病的共同病理表现。此外,研究显示,在AD病人中,TPP含量和TPP依赖型的酶类的活性都有明显的下降,表明硫胺素缺乏可能在AD病理中起了一定的作用。但硫胺素缺乏在AD中的具体作用尚未得到系统的研究。我们在体外培养的稳定转染了瑞典型突变APP基因（APPsw）的人源神经母细胞瘤细胞株（SH-SY5Y）上和C57BL/6J小鼠上成功诱导了TD模型,并通过酶连免疫吸附实验、免疫印迹实验、免疫组织化学等技术手段系统地研究了TD对APP加工（APP processing）的影响以及其可能的机制。首先,在APPsw SH-SY5Y细胞上,TD处理5天导致了大约13.5%的细胞死亡,发生凋亡的细胞增加了2倍。MTT法检测表明TD处理5天导致细胞活力下降了大约50%。第二,酶连免疫吸附实验测定Aβ的结果显示:TD处理显著增加细胞外和细胞内的Aβ的含量。与对照组(胞外的Aβ₄₀和Aβ₄₂分别是5.52±0.12和0.66±0.23 pg/ml·mg)相比,TD5天组胞外的Aβ₄₀和Aβ₄₂分别上升到18.57±2.31和3.85±0.52pg/ml·mg;细胞内的Aβ₄₀和Aβ₄₂分别从对照组的23.4±5.1 7和7.06±1.03 pg/mg分别上升到86.03±8.19和23.52±1.02 pg/mg。第三,免疫印迹实验显示在TD过程中,细胞中成熟形式的APP蛋白水平降低,不成熟形式的APP蛋白水平没有明显变化。说明在TD过程中APP的表达水平没有增加,应该不是导致Aβ积累的原因。第四,酶活力测定的结果表明负责APP加工的β-分泌酶的活性在TD过程中明显增高,而γ-分泌酶的活性在这一过程中没有显著性变化。前人研究结果证实β-site APP cleaving enzyme 1（BACEl）具有β-分泌酶的活性,我们分别从转录以及翻译水平研究了BACE1基因的表达情况,发现在TD处理过程中,BACE1基因的mRNA水平没有明显变化,BACE1的蛋白总量也没有明显改变。然而,与对照组相比,BACE1蛋白的成熟形式增加了大约2倍,而不成熟形式的BACE1降低了大约50%。表明β-分泌酶的活性调节不是通过增加BACE1基因的转录水平或翻译水平,而是通过促进BACE1蛋白的翻译后修饰（BACE1蛋白的成熟过程）来实现的。为了进一步确定β-分泌酶的活性在TD过程中明显增高,我们检测了TD过程中β-分泌酶产物:β-C terminalfragment（β-CTF）的变化,免疫印迹结果显示β-CTF含量在TD过程中明显增高。第五,TD处理5天后,补充硫胺素24小时明显地减弱了BACE1蛋白的成熟过程,下调β-分泌酶的活性,降低了Aβ的积累。表明补充硫胺素对TD引起的Aβ的积累具有一定的补救作用。第六,在TD处理5天时,向培养液中添加β-分泌酶抑制剂24小时可以抑制Aβ和β-CTF的积累。第七,TD增加了细胞内活性氧的积累,抗氧化剂Trolox不仅可以降低TD导致的细胞内活性氧的积累,也抑制了BACE1蛋白的成熟过程和Aβ的积累。表明氧化应激可能是TD引起Aβ积累的内在机制。最后,在TD处理过程中,添加低浓度（250pmol/L）的化学合成的Aβ到培养液里明显加剧了TD导致的氧化应激。表明Aβ的积累增强了TD引发的氧化应激。以上的离体实验结果在TD小鼠模型上得到了验证:（1）TD处理9天或更长时间会导致丘脑区域的丘脑中线旁丘脑核（Submedial thalamic nucleus,SmTN）出现明显的神经元死亡;（2）在TD小鼠丘脑区域,Aβ₄₀和Aβ₄₂含量显著增加:对照组和TD10天组的Aβ₄₀分别是0.97±0.15和3.34±0.33 pmol/g;Aβ₄₂分别是0.16±0.01和0.49±0.02 pmol/g;（3）TD处理8天后补充硫胺素可以减少丘脑区域Aβ₄₂的积累。综上,我们的实验结果表明:（1）由TD引起的氧化应激导致了APP蛋白加工过程中产生了更多的Aβ;（2）氧化应激的作用靶点是BACE1蛋白。进一步,TD对β-分泌酶的活性调节是通过促进BACE1蛋白的翻译后修饰（BACE1蛋白的成熟过程）来实现的;（3）Aβ的积累也加剧了TD导致的氧化应激。提示氧化应激和Aβ的积累之间存在着正反馈的效应。本工作发现硫胺素缺乏引起的氧化应激促进了APP加工中产生更多的Aβ,Aβ的积累反过来加剧了硫胺素缺乏导致的氧化应激。氧化应激和Aβ的积累之间存在着正反馈的效应。这一效应很可能参与了AD的发生、发展。最后,体外、体内补充硫胺素实验都表明及时补充硫胺素可以缓解硫胺素缺乏对APP加工的影响,表明对于出现硫胺素缺乏相关症状的AD病人,考虑及时补充硫胺素是有益的。更多还原

【Abstract】 Alzheimer’s disease（AD）is the most common cause of dementia in the elderly worldwide.Neuropathological hallmarks of AD are extracellular senile plaques（SPs）, intracellular neurofibrillary tangles（NFTs）and massive neuronal loss in vulnerable brain regions.The pathogenesis of AD is still poorly understood and current available treatments for AD are unsatisfactory.β-amyloid peptide（Aβ）is the major component of SPs,which is derived from proteolytic processing ofβ-amyloid precursor protein （APP）.Mounting evidence suggests that Aβplays an initial role in AD pathogenesis, Therefore,unraveling the molecular mechanism of Aβmetabolism is important to AD pathogenesis research.Thiamine（Vitamin B₁）is an essential nutrition factor for human beings.The active form of thiamine in vivo is thiamine pyrophosphate（TPP）.TPP acts as a co-enzyme of thiamine-dependent enzymes,which include the pyruvate dehydrogenase complex（PDHC）,α-ketoglutarate dehydrogenase complex（KGDHC） and transketolase（TK）.Thiamine is also essential to keep the cellular redox state and glutathione reductase activity.Thiamine deficiency（TD）impairs the activities of thiamine-dependent enzymes which result in dysfunction of tricarboxylic acid cycle and shortage of adenosine-triphosphate（ATP）.Simultaneously,the accumulation of reactive oxygen species（ROS）leads to severe oxidative impairment during TD process.The nervous system is highly sensitive to TD,which may cause a serious neurological disorder called Wernicke-Korsakoff’s syndrome（WKS）.The clinical features of WKS include confusion,cognitive deficits,memory loss,and,at late stage of WKS,enlarged ventricle and neuronal loss are also found.The activity of KGDHC decreases in WKS patient brain.In brains of TD animal models,common features of neurodegenerative diseases can be found,such as selective neuronal loss,activation and proliferation of microglia and astrocytes cells.Furthermore,it is reported that TPP and the activities of thiamine-dependent enzymes both decline in AD patients, which suggests that TD may contribute to the AD pathology.However,the exact effect（s）of TD in AD pathogenesis is still unclear.In present work,we investigated the effect（s）of TD on APP processing using cultured Swedish mutant APP（APPsw）transfected SH-SY5Y cells and C57BL/6J mouse with a series of biochemical and morphological techniques.Firstly,in APPsw stably transfected SH-SY5Y cells,TD caused moderate cell death（about 20%）after 5-days treatment,while the numbers of apoptotic cells elevated by 2-fold and the cell viability decreased by 50%.Secondly,the amounts of Aβ,both secreted and intracellular forms were elevated during TD:After 5 days TD treatment,the basic secreted（5.52±0.12 pg/mg）and intracellular（23.4±5.17 pg/mg） Aβ₄₀were elevated to 18.57±2.31 pg/mg and 86.03±8.19 pg/mg,respectively.The basic secreted（0.66±0.23 pg/mg）and intracellular（7.06±1.03 pg/mg）Aβ₄₂were elevated to 3.85±0.52 pg/mg and 23.52±1.02 pg/mg,respectively.Thirdly,during the TD process,the mature from of APP is decreased and the immature from is unchanged.Therefore,Aβaccumulation should not be derived from increased expression of its precursor protein.Fourthly,the activity ofβ-secretase was up regulated and the activity ofγ-secretase is unchanged during TD.Real-time polymerase chain reaction（Real time-PCR）and immunoblotting assay showed that the expression ofβ-site APP cleaving enzyme 1（BACE1）gene is unaltered,and, however,the maturation of BACE1 increased,the mature BACE1 increased by about 2-fold,and immature BACE1 decreased by about 50%.This change may result in an elevatedβ-secretase activity.To further confirm the changes ofβ-secretase activity, we examined the level ofβ-C terminal fragment（β-CTF）of APP,the product ofβ-secretase cleavage.The level ofβ-CTF was elevated during TD.Fifthly,the above abnormal alteration induced by TD can be restored with thiamine re-supplement for 24 hours after 5 days TD treatment,implying that the effectiveness of thiamine re-supplement after TD.Sixthly,addingβ-secretase inhibitor to the 5-day TD treated culture medium could efficiently inhibit the accumulation of Aβandβ-CTF. Seventhly,5-day TD treatment increased ROS by nearly 3-fold compared with the control groups,which can be significantly attenuated by antioxidant Troiox.Trolox also can inhibit the TD induced Aβaccumulation and maturation of BACE1.Finally, synthetic Aβ_1-40exposure（250pmol/L）during TD treatment can elevate the ROS level significantly compared with TD treatment only,indicating that Aβaccumulation enhanced the oxidative stress caused by TD.These findings on TD cell model were also confirmed in vivo in TD mouse model: （1）neuronal loss was found in submedial thamalic nucleus（SmTN）after 9-days TD treatment;（2）dramatic Aβaccumulation was detected in thalamus region:the Aβ₄₀ level of control group and TD 10-days group is 0.97±0.15 and 3.34±0.33 pmol/g, respectively,and the Aβ₄₂level of control group and TD 10-days group is 0.16±0.01 and 0.49±0.02 pmol/g;（3）restoration with thiamine after 8-days TD treatment can reduce the Aβ₄₂accumulation caused by TD.Taken together,our results imply that:（1）the oxidative stress caused by TD promotes the amyloidogenic APP processing and resulting in Aβaccumulation;（2） the oxidative stress up-regulates theβ-secretase activity though enhancing the BACE1 maturation;（3）conversely,Aβaccumulation exacerbates the oxidative stress during TD treatment,suggesting that there may be a positive feedback cycle between Aβaccumulation and oxidative stress.We have found that TD can cause oxidative stress and promote the amyloidogenic APP processing,and resulting in Aβaccumulation.Furthermore,a positive feedback cycle exists between Aβaccumulation and oxidative stress.These effects may play important roles in AD pathogenesis and/or progression.Finally,our thiamine re-supplement experiment results showed that it may be effective and important to supply thiamine for treatment of the AD patients who were proved to be TD.更多还原