亨廷顿病模型小鼠大脑内超氧阴离子自由基的时空分布及其相关信号通路的双光子荧光成像研究

Huntington's disease (HD) is a fatal neurodegenerative disease. Because of its unclear pathogenesis, there is no active drugs for changing the natural course of HD. The pathological findings indicate that redox imbalance plays a critical role in HD. Superoxide anion radical (O2˙¯) as important intracellular reactive oxygen species, regulate the redox balance and multiple signaling pathways by dynamic changing of O2˙¯ level in time and subcellular regions. Hence it will be conducive to solving puzzle of HD pathogenesis by exploring the temporal and spatial dynamic regulation of intracellular O2˙¯. However, due to special properties of O2˙¯including wide distribution, short life and high reactivity, it is difficult to achieve dynamic, reversible, and in situ detection of O2˙¯ fluctuations in subcellular by present methods. In order to solve the above problems, this project will develop novel two-photon fluorescent probes, which are precisely located in the cell membrane, cytoplasm and mitochondria respectively, as well as possessing highly selective, highly sensitive and dynamically reversible properties towards O2˙¯. With assistance of two-photon microscopy, the spatial and temporal distributions of O2˙¯ in mouse brain at subcellular level will be visualized during the development of the disease. Furthermore, O2˙¯mediated signaling pathway and HD molecular mechanism will be revealed for the early diagnosis and precise treatment of HD.

亨廷顿舞蹈症（HD）是一种致命的神经退行性疾病，因发病机制尚不明确，导致目前没有药物可以改变HD的病情恶化过程。研究表明，氧化还原失衡是HD最重要的致病因素。超氧阴离子自由基（O2˙¯）通过其浓度在亚细胞区域随时间的动态变化，调控氧化还原平衡和多种信号通路。因此，探究亚细胞器内O2˙¯的时空分布及信号通路，有助于解开HD的分子机制之谜。但是由于体内O2˙¯具有分布广、寿命短、反应活性高等特点，目前的检测手段难以实现动态、可逆检测亚细胞水平上O2˙¯的浓度变化。为解决上述问题，本项目拟发展三种分别精准定位于细胞膜、细胞质、线粒体，且能够高选择性、动态、可逆响应O2˙¯的新型双光子荧光探针，建立双光子荧光成像分析O2˙¯在不同细胞器内浓度水平变化的新方法，揭示HD疾病小鼠大脑内O2˙¯的三维时空分布及相关的信号通路，为HD疾病的早期诊断和精准治疗提供新策略。

生物体内超氧阴离子（O2˙¯）的时空变化与多种疾病的发生发展密切相关。在亚细胞水平及活体水平可视化O2˙¯对生物学和医学研究有重要意义。但由于缺少时空示踪细胞器内O2˙¯水平的工具，限制了对相关疾病分子机制的阐明。因此，我们设计合成了三种精准靶向线粒体或高尔基体检测O2˙¯的荧光探针，建立了活体、细胞及亚细胞器多尺度范围的实时示踪O2˙¯的分析方法。实现从百纳米到百微米、从二维到四维时空多级别多维度的成像分析。利用该分析方法，我们发现了机体应对可能致病的外界刺激时，高尔基体和线粒体内O2˙¯及其他活性小分子的浓度均随时间的进程而异常升高。O2˙¯浓度达到阈值后，可激活高尔基体和线粒体内的多条凋亡通路。该分析方法具有普适性，能够探查多种疾病与O2˙¯相关的分子机制，也为今后探寻疾病治疗靶点提供了可靠的方法。