D-青霉胺抑制阿尔茨海默病转基因小鼠脑内β-淀粉样蛋白沉积及其机制研究

Alzheimer's disease (AD) is a neurodegenrative disorder characterized with β-amyloid protein (Aβ) deposition and plaque formation in the brain. Aβ is generated from β-amyloid precursor protein (APP) via a proteolytic process of β- and γ-secretases. Recent studies have shown that metal ions, such as copper and zinc, accumulated and combined with Aβ peptides in AD brain. Interestingly, the metal-protein attenuating compounds (MPAC) are able to compete metal ions with Aβ, reduce the activity of β- and γ-secretase, and decrease Aβ secretion/deposition in AD transgenic mouse brain. D-penicillamine (D-Pen) is a classical MPAC wich has been used for Wilson's disease therapy for decades. Recently in vitro studies have shown that D-Pen could effectively resolubilize copper-Aβ aggregates. Furthermore, D-Pen nanoparticles could release the conjugated D-Pen thorough the brain blood barrier into the brain. However, the effect and the underlying mechanism of whether D-Pen is able to inhibit Aβ deposition in AD brain has not been evaluated. In this project, we will use the D-Pen nanoparticles to treat the APP/PS1 transgenic mouse, to evaluate the effects of D-Pen on the reduction of metal ione levels and the inhibition of Aβ generation related secretase activity in APP/PS1 transgenic mouse brain. Hopefully, this project will explore the mechanisms by which D-Pen modulates the activity of Aβ generation related secretases, and provide a clue for potential use of D-Pen for the prevention and therapy of AD.

阿尔茨海默病(AD) 的主要病理特征之一是脑内出现β-淀粉样蛋白(Aβ)沉积形成的老年斑。铜、锌金属离子可通过与Aβ分子的组氨酸位点结合促进其脑内沉积，体外实验证实金属离子螯合剂D-青霉胺(D-Pen)能有效降低Aβ聚集，用纳米粒包裹的D-Pen可穿越血脑屏障进入脑组织，但D-Pen是否能抑制AD脑内Aβ沉积及其机制尚不清楚。本项目拟制备D-Pen与纳米粒共价结合的脑靶向药物传递系统，通过鼻饲处理APP/PS1转基因小鼠，结合D-Pen处理转染人APP的细胞模型，应用分子生物学和行为学等手段，检测D-Pen抑制脑内铜锌离子蓄积和Aβ的产生，改善小鼠学习记忆能力的作用，分析D-Pen对β-淀粉样蛋白前体蛋白(APP)及其剪切酶β-分泌酶、γ-分泌酶等表达的影响，明确D-Pen抑制Aβ产生与老年斑形成的可能途径，为揭示D-Pen防治AD的作用与机理，以及指导金属螯合剂类药物研发提供理论基础。

阿尔茨海默病 (AD) 的主要病理特征之一是脑内出现β-淀粉样蛋白(Aβ)沉积形成的老年斑。AD的发病过程中，APP淀粉样代谢途径被激活，产生的大量Aβ以可溶性Aβ或以寡聚肽Aβ形式存在于细胞外，具有极强的神经毒性，能导致神经元死亡以及相关的神经功能障碍或丧失。铜、锌金属离子可通过与Aβ分子的组氨酸位点结合促进其脑内沉积，体外实验证实金属离子螯合剂D-青霉胺(D-Pen)能有效降低Aβ聚集，但D-Pen是否能抑制AD脑内Aβ沉积及其机制尚不清楚。本研究以壳聚糖与甘油磷酸钠体系为载体，通过鼻饲处理APP/PS1转基因小鼠，结合D-Pen处理转染人APP的N2a细胞模型，应用分子生物学和行为学等手段，检测D-Pen抑制脑内铜锌离子蓄积和Aβ的产生，改善小鼠学习记忆能力的作用。通过体内和体外研究表明，D-Pen鼻凝胶制剂能够通过以下途径抑制Aβ的产生和沉积：① D-Pen凝胶制剂能够明显改善APP/PS1小鼠学习记忆能力，降低大脑皮层Aβ老年斑中锌离子的聚集，减少Aβ斑块的大小和数量。② D-Pen通过调节APP剪切酶ADAM10和BACE-1的表达，增加sAPPα表达降低sAPPβ的含量，阻止APP淀粉样代谢途径的激活并减少其向细胞外分泌Aβ。③ D-Pen抑制星形胶质细胞的活化，调节BAX/Bcl-2表达抑制细胞凋亡。④ D-Pen调节raf/ERK/CREB信号通路，进而影响细胞核内基因转录，减少Aβ的产生和沉积。因此本研究阐明了D-Pen抑制阿尔茨海默病学习记忆功能减退的作用机理并为研究治疗阿尔茨海默病的方法提供新思路。