苏木(SUMO)化参与AD特征性病理变化机制的研究

The level of protein SUMOylation in AD (Alzheimer's disease) patients’ brain is significantly elevated, yet it is unknown whether SUMOylation is involved in the pathogenesis of AD that is characterized by Tau hyperphosphorylation and A β toxicity. We have previously found that in cells the SUMOylation of Tau promotes its phosphorylation, inhibits its degradation that is induced by ubiquitination, and increases the insolubility of itself which leads to aggregation. Previous studies showed that the deacetylase SIRT1, as premorbid and prodromal indicators of AD in 3×Tg APP/PS1/P301L AD mouse, can make the UBC9 deacetylation, and induce substrate SUMOylation. We hypothesize that SIRT1-mediated SUMOylation plays an important role in AD pathogenesis..Based on our previous findings, we are proposing our continuing research as following:.1. We are going to investigate SIRT1 and UBC9, molecules thought to be upstream to SUMOylation, their content and acetylation level (UBC9) in the brain of APP/PS1/P301L triple transgenic mouse in different stage of AD pathogenesis and in cell line, to determine the regulation mechanism of SUMOylation via SIRT1/UBC9..2. We will overexpress SIRT1 in SD rat，and SENP1, enzyme of de-SUMOylation in APP/PS1/P301L triple transgenic mouse, respectively, to detect the SUMOylation, phosphorylation, ubiquitination and the neurofibrillary tangles formation, along with the SUMOylation of BACE1 (APP cleaving enzyme), the content of Aβand animal behavior change, to elucidate SUMOylation-involved molecular mechanism in AD pathogenesis..3. We will synthesize small peptides based on the core motifs of the SUMOylation of BACE1 and Tau protein, and will use these small peptides to intervene the triple transgenic AD mouse, in order to explore changes to the AD-like pathology brought by the small peptides..The results of above proposed research will produce new insight to AD pathogenesis, and provide molecular target for drug prevention.

AD患者脑内苏木化修饰显著升高，但其是否参与以Tau过度磷酸化和Aβ毒性为特征的AD发病机制不明。申请人发现：细胞中苏木化促进Tau磷酸化、对抗泛素化并促进Tau聚集。研究显示：去乙酰化酶SIRT1可使苏木连接分子UBC9去乙酰化，并致底物苏木化，且SIRT1上调在3×Tg-AD鼠早于Tau和Aβ病变。我们推测，SIRT1介导的苏木化在AD发病中起重要作用。本项目拟在3×Tg-AD鼠不同阶段及在细胞水平调控SIRT1和UBC9后，检测SIRT1和乙酰化UBC9水平，分析其改变与Tau和Aβ上游剪切酶BACE1苏木化的关系；通过在SD鼠过表达SIRT1和去苏木化酶干预3×Tg-AD鼠，确定苏木化参与AD特征性病理变化的具体机制；以BACE1和Tau苏木化核心模序合成小肽，探讨阻断BACE1和Tau苏木化能否改善小鼠脑损伤和认知功能。该研究将揭示AD脑中蛋白质异常聚集的新机制并提供防治新策略。

阿尔茨海默病(Alzheimer’s disease, AD)是最常见的神经退行性病变，细胞内神经纤维缠结（neurofibrillary tangles, NFTs）和细胞外老年斑（senile plaques, SPs）的沉积是其两大基本病理学特征。其中蛋白磷酸酯酶PP2A活性下调导致Tau蛋白过度磷酸化，被认为是形成NFTs的主要原因。SP由前体蛋白APP剪切而生成的β淀粉样蛋白（Aβ），引起神经毒性。AD患者脑内苏木化修饰显著升高，但其是否参与以Tau过度磷酸化和Aβ毒性为特征的AD发病机制不明。本项目研究了Aβ生成的限速酶β分泌酶（亦称BACE1）和tau关键调节分子PP2A抑制剂SET的异常苏木化修饰参与AD病变的相关机制。发现：1.在细胞和整体水平BACE1可以被苏木化修饰，其修饰位点为K275和K501；2. BACE1苏木化修饰可以提高其蛋白稳定性和酶的活性；3. BACE1苏木化修饰增加APPβ的剪切和Aβ的生成；4. BACE1苏木化修饰加剧神经元树突损伤及学习记忆障碍；5. BACE1苏木化修饰水平与年龄存在相关性；6.在AD进程中，AD关键分子Aβ促进SET苏木化修饰，引起苏木化修饰的SET滞留于胞浆，进而抑制PP2A活性，导致tau蛋白过度磷酸化、突触相关蛋白受损以及学习记忆功能障碍，加重AD发病。本研究成果为苏木化异常修饰参与AD发病机制提供了新线索，为AD药物开发提供有效特异的分子靶点。