β 分泌酶在大脑衰老和退行性变化中的作用和机制研究

Aging is a slow process of becoming older and represents the accumulation of physical, psychological, and social changes over time. While the causes of aging are uncertain, studies found that dementia becomes more common with age. To better aging and prevent or delay the neurodegenerative diseases, such as AD, a better understand the normal (healthy) and abnormal (degenerative diseases) aging, as well as the transition between normal and abnormal aging process is critical. In this proposed studies, we will be focusing on the interaction between neurons and small blood vessel cells during brain aging. Our previous research showed that the β secretase (BACE1) is related with aging and could be a biomarker for AD, while Klotho also plays critical roles in aging, particularly in peripheral organs. Here, we will use our unique aging animal models which over express BACE1 in vessel endothelial or smooth muscle cells in combination with Klotho deficiency or overexpression, to investigate the interaction between BACE1 and Klotho in normal and abnormal aging. We will extend our animal mechanism investigations into human studies. We will see whether BACE1 and/or Klotho change over the transitional period from healthy aging to unhealthy aging and validate BACE1 and Klotho as potential translational targets of aging process.

衰老是一种以系统性多器官生理功能逐渐下降为特征的过程。器官衰老则是老年慢性病的基础，包括大脑就是衰老过程中受到影响最大的器官之一。衰老过程中出现的脑萎缩和脑血管退行性变化，从影响脑认知功能而演变成老年痴呆(Alzheimer's disease, AD)等疾病。到目前为止，我们对于大脑生理性衰老的机制以及如何向病理性衰老（如AD等老年退行性疾病）的转变了解甚缺。寻找衰老与相关疾病连接性靶点迫在眉睫。本研究将以脑小血管与神经系统作为研究正常大脑衰老的对象，观测与衰老相关的β蛋白分泌酶BACE1和抗衰老因子Klotho在神经元与脑血管内皮和平滑肌细胞衰老过程中的相互作用和变化规律，从动物模型到人体研究，探讨BACE1和Klotho在正常健康衰老与老年神经退行性病变之间的特异性，解析大脑衰老基础与机制，为减缓脑衰老，预防或延迟老年神经血管疾病提供新的靶点。

研究背景：本研究观测与衰老相关的β蛋白分泌酶BACE1和抗衰老因子Klotho在神经元衰老过程中的相互作用和变化规律。从动物模型到人体研究，探讨BACE1和Klotho在正常健康衰老与老年神经退行性病变（老年性抑郁症，AD）之间的特异性，解析大脑衰老基础与机制。.研究方法：144例抑郁症患者和112例健康对照者为临床研究对象。采用ELISA检测血浆BACE1和α-Klotho的蛋白水平。聚合酶链反应（PCR）/焦磷酸测序检测Klotho基因多态性和Klotho启动子甲基化水平。用汉密尔顿抑郁量表（HAMD）、汉密尔顿焦虑量表（HAMA）、威斯康星卡片分类测验（WCST）、言语流利性测试（VFT）和连线测试（MT）进行抑郁和认知功能评估。用基于时间飞行质谱（MALDI-TOF）方法鉴定BACE1酶切α-Klotho的位点，设计了荧光共振能量转移（FRET）荧光底物来检测BACE1的酶切活性。通过BACE1转基因或基因敲除小鼠验证BACE1剪切Klotho的体内生物活性。.研究结果：（1）老年首发抑郁症患者的α-Klotho水平较老年健康组显著降低，而青年抑郁症患者的α-Klotho水平与青年对照组无差异。与患者发病年龄和HAMD评分相关。血浆α-Klotho水平主要与rs9315202 T等位基因数呈正相关，而年轻人的血浆α-Klotho水平同时也受年龄和性别的影响。血浆BACE1蛋白水平也与年龄呈正相关， 而血浆BACE1蛋白水平和BACE1/APP活性与抑郁症患者临床症状没有显著相关性。（2）α-Klotho蛋白可以被BACE1切割，酶切产物测序结果与α-Klotho的氨基酸序列高度匹配，证明了α-Klotho为BACE1新的酶切底物，并鉴定出了BACE1切割α-Klotho的特异性位点F-T。BACE1酶切不同底物（α-Klotho、APP和NRG1）的活性的具有差异。（3）在BACE1基因小鼠模型中，发现BACE1与α-Klotho表达水平的相关性。.结论：老年首发抑郁症患者血浆α-Klotho水平显著降低，其与·Klotho rs9315202 次要等位基因呈正相关，与年龄呈负相关。血浆BACE1蛋白水平在抑郁症患者中发生异常改变。α-Klotho是BACE1新的酶底物，并具有特异性酶切位点。FRET荧光探针为研究衰老提供了新的工具。