自噬相关基因Atg7维持成体神经干细胞稳态及其潜能的研究

Senescence and depletion of adult neural stem cells (aNSCs) are the common pathogeny of neurodegenerative diseases. Accumulative stress from hazardous stimuli plays a critical role in the aging process. Prior results of our research have revealed that the malfunctional clearance of abnormal proteins, due to the reduced catalytic ability of 20S proteasomes, contributes to replicative senescence in human bone marrow stromal cells which are similar to aNSCs in terms of their pluripotency and neural differentiation capability. As another effective regulator to sustain intracellular homostasis in addition to proteasomes, autophagy takes part in the regulation of cell development, differentiation, metabolism and renewal. Given the tight association between autophagy and maintenance of stem cell integrity suggested by multiple studies recently, we shall focus on Atg7, the crucial gene for autophagosome formation, and delve into the underlying mechanism.By virtue of the RNA interference techinque, we shall investigate the potential impact of autophagic dysfunction on the appearance of senescent markers, telomerase activity, as well as proliferation and differentiation states of aNSCs, using a combination of electromicroscopic, immunohistochemical, cellular and molecular biological methods. We shall also establish the aNSC model for oxidative stress in order to explore its influence on autophagy. The overall aim is to delineate the comprehensive effects of autophagy in maintaining the stemness of aNSCs, and the mechanism underlies the progress of aging, which raises the intriguing possibility of treating neurodegenerative diseases by potentially reviving aNSCs through modulating their autophagy.

成体神经干细胞（aNSCs）老化与干细胞库耗竭是神经退行性疾病的共同病因。应激刺激蓄积是aNSCs衰老进程中的关键事件。课题组前期结果显示20S蛋白酶体活性降低，异常蛋白清除障碍导致骨髓间充质干细胞复制性衰老；20S蛋白酶体与aNSCs衰老相关。自噬是除蛋白酶体之外维持细胞内稳态的另一有效途径，参与细胞发育、分化和代谢更新调控。近期研究提示自噬与干细胞功能维持密切相关。为此，本课题拟以自噬体形成关键基因Atg7作为切入点，借助基因沉默技术手段，通过电镜、组织化学和分子生物学等方法观察自噬功能障碍对aNSCs 衰老标志物、端粒酶活性以及增殖和分化潜能的影响；并建立aNSCs氧化应激模型，探讨氧化应激对自噬功能障碍aNSCs生存能力的影响。本课题旨在全面揭示自噬在aNSCs功能维持中的重要作用，阐明aNSCs衰老机理，寻求恢复aNSCs活力的新靶标，从而为神经退行性疾病治疗提供新的突破方向。

随年龄增加神经干细胞（neural stem cells，NSCs）的增殖和分化能力逐渐降低，这是导致成年神经系统修复能力低下，甚至出现退行性病变的重要原因，因而维持成体NSCs的活力对延缓脑老化和促进神经系统的修复再生具有重要意义。本研究中我们首先比较不同年龄阶段，神经发生的关键脑区SVZ和海马自噬相关基因的表达变化，研究结果显示成年小鼠Atg3、Atg5和Atg7的表达水平均较新生小鼠显著下调～50%。体外实验结果也证实成年NSCs Atg3、Atg5、Atg7和Beclin1 mRNA的表达水平分别较新生小鼠NSCs降低0.4、0.2、0.7和0.6倍。为进一步观察不同年龄 NSCs超微结构特点，我们制备新生和成年 NSCs电镜标本。电镜下可见成年NSCs胞质中自噬小体堆积，线粒体数量减少，且部分细胞的线粒体肿胀、嵴断裂。上述体内和体外实验结果均提示成年NSCs自噬活力降低，细胞内错误折叠蛋白和受损细胞器堆积导致NSCs代谢和细胞器更新障碍，推测自噬是影响成年NSCs活力的重要因素。应用1%血清诱导NSCs分化，比较分化前和分化后自噬相关基因表达变化。我们发现NSCs分化后Atg3、Atg5 、Atg7和Beclin1均显著增高。若应用自噬抑制剂LY294002则减少NSCs分化为神经元的比例，Tuj1阳性率由8.5±1.3%降低至3±0.5%。相反，成年NSCs应用雷帕霉素则使Tuj1阳性率较对照组提高2倍，提示在NSCs分化过程中需要自噬提供能量，改建细胞骨架和细胞形态从而促进NSCs的分化。在本研究中我们分别观察了Atg3、Atg5、Atg7和Beclin1在新生期和成年期以及NSCs分化前和分化后的表达差异，其中Atg7的表达变化最为显著。应用电穿孔介导siRNA，能够高效下调Atg7在NSCs的表达水平。研究结果显示抑制Atg7的表达水平，NSCs的自我复制能力降低，Atg7siRNA组神经球的直径和数量分别较对照组下降26%和19%。此外，Atg7siRNA组Tuj1阳性率为28.4±1.2%也较对照组47.9±1.9%显著降低，表明自噬影响NSCs分化。本课题通过电镜、组织化学和分子生物学等方法，证明年龄依赖性NSCs自噬活性下降是导致成体NSCs潜能降低的关键因素。激活自噬、提高NSCs的能量代谢可能是促进神经发生，延缓脑老化的重要途径。