遗传编码的NAD+探针在器官衰老研究中的应用

With the increasing accumulation of damages, living organisms elicit the compensatory responses by metabolic reprogramming together with cellular senescence, which is the critical step toward organismal aging. NAD+ is one of the most important metabolites, whose level decreases with aging. The diminishing NAD+ accelerates the aging process of organs which is significantly delayed by repletion of NAD+. Hence it is of great interest to examine the potential of cellular NAD+ levels as a predictor of aging process as well as seek the compounds raising the NAD+ level. However, traditional methods including enzymatic assays, chromatography and mass spectrometry suffer from some defects in NAD+ assay, and are not suitable for high-throughput screen. The current project aims to develop a novel genetically encoded fluorescent sensor for NAD+ with high spatio-temporal resolution, establishing the state-of-art imaging tool for NAD+ in living organism, single-cell as well as sub-cellular settings. The sensor is also used to evaluate the ability of cellular NAD+ level as an aging indicator and screen the anti-aging candidate compounds. This study will provide a novel tool for basic biomedical research, as well as new insights into the index and drugs for organismal aging, thus it possesses both important scientific significance and economic significance.

伴随机体损伤的积累，生物体发生代偿性的代谢重编程和细胞衰老，这是器官衰老的决定性环节。NAD+作为胞内一个核心代谢物，它的水平常常随着器官衰老而显著降低。NAD+的减少加速了器官衰老，而补充NAD+则可延缓衰老的进程。因此，探索NAD+水平作为预测器官衰老的指标以及搜寻上调NAD+水平的先导化合物是本领域的热点问题。然而，利用酶学、色谱和质谱等传统生化分析方法测定NAD+存在严重的技术缺陷，更难以用于高通量筛选。本项目将开发高时空分辨检测NAD+代谢的遗传编码荧光探针，并在单细胞、亚细胞和活体水平建立衰老过程研究的前沿NAD+成像技术，评价NAD+水平作为器官衰老预测指标的潜力，进而将NAD+探针应用于抗衰老化合物的筛选。本项目不仅为生命医学基础研究提供创新性的工具，也为器官衰老的评价以及相关药物的开发提供新的技术支撑和研究手段，因此本研究具有重要的科学意义及社会经济意义。

围绕“器官衰老与器官退行性变化的机制”这一重大研究计划，项目负责人聚焦组织器官衰老和退行性变化过程中氧化还原代谢这一关键指标变化，发展新技术与新方法。在培育项目的资助下，我们开发了系列高性能的NAD+、NADH、NADPH遗传编码的荧光探针，实现了在活细胞、亚细胞和活体上对氧化还原代谢的高时空监测与成像，发展了“全景式”氧化还原代谢成像技术。基于NAD+荧光探针，进一步建立了小鼠、人不同衰老模型的监测方法，通过高通量筛选发现了3个有效调控NAD+的化合物，并分析了多个有助于延缓衰老进程的化合物作用机制。相关工作在国际著名刊物Nature Methods、Nature Protocols、Developmental Cell、Cell Metabolism等一流杂志发表SCI论文12篇，受邀在Annual Review of Analytical Chemistry、Antioxidants and Redox Signaling、Neuroscience Bulletin、《中国科学: 生命科学》等国内外学术期刊撰写综述，申请、授权专利16项。研究结果在国际上产生重要影响，被国际同行评价为“革命性”技术，相关技术已被国内外100多个一流机构实验室跟踪应用。本项目不仅为生命医学研究提供创新性的工具，也为器官衰老的评价以及相关药物的开发提供新的技术支撑和研究手段，具有重要的科学意义及社会经济意义。