创新药物TBN在ALS模型通过调控AMPK改善线粒体功能和激活自噬保护运动神经元的作用及机理

肌萎缩侧索硬化症(ALS)的运动神经元中SOD1和TDP-43聚集，引起线粒体功能异常和自噬障碍是运动神经元损伤的关键因素。AMPK是能量代谢和自噬的共同调控分子。激活AMPK能否延缓ALS病程？前期我们发现强自由基清除剂TBN在SOD1转基因小鼠能改善运动缺陷，减少运动神经元丢失。此外，TBN能改善线粒体功能和激活自噬。体外激酶实验TBN对AMPK活性无直接作用。最近研究证明AMPKα亚基氧化，抑制其被磷酸化激活。我们推测TBN通过氧化还原调控AMPK，激活其下游信号通路保护运动神经元。本项目拟在体内外ALS模型，采用分子药理学、染色质免疫共沉淀、氧化还原和磷酸化修饰蛋白组学等技术，研究TBN激活AMPK的分子机理，在ALS模型改善线粒体功能和激活自噬清除突变SOD1/TDP-43蛋白的作用及机理。本项目有望阐明TBN治疗ALS的机理，为研发具自主知识产权的ALS新药提供理论依据。

Accumulation of SOD1 and TDP-43 proteins in motor neurons cause mitochondrial dysfunction and autophagy deficit, and are key factors of motor neuron injury in amyotrophic lateral sclerosis (ALS). AMPK is a common molecular pathway that regulates energy metabolism and autophagy. Can AMPK activation delay the disease onset and progression of ALS? Preliminarily, we demonstrated that the strong free radical scavenger TBN (a nitrone derivative of tetramethylpyrazine) improved motor deficits and reduced motor neuron loss in SOD1 transgenic mice. In addition, TBN improved mitochondrial function and activated autophagy. Results from in vitro kinase experiments revealed that TBN had no direct action on AMPK activity. Recent studies demonstrated that AMPKα subunit could be oxidized, so as to inhibit its activation through phosphorylation. In sight of this knowledge, we propose that TBN protects motor neurons by redox modulation of AMPK to activate the downstream signaling pathway. Based on our preliminary data, in this study, we will employ appropriate in vitro and in vivo models of ALS. With the help of state-of the art technologies including molecular pharmacology, chromatin immunoprecipitation, redox and phosphorylated proteomics, we will elucidate the molecular mechanism underlying AMPK activation by TBN. We will also investigate the effects and mechanisms of TBN on improving mitochondrial function, and activating autophagy in ALS to promote clearance of mutant SOD1 and TDP-43 proteins. This project will establish the experimental basis and provide mechanistic rationale for developing TBN as a novel therapeutic agent for ALS.