压电离子通道蛋白PIEZO1在高眼压视网膜神经节细胞损伤中 参与自噬与凋亡调控的分子机制研究

Ocular hypertension can induce irreversible degeneration of retinal ganglion cells (RGCs) is a major factor of glaucoma, a worldwide leading cause of blindness. However, the molecular mechanism remains unclear. Piezo1, a new type of mechanosensitve channels (MS channel), is mainly responsible for pressure and shear stress perception. Piezo1 participate in several diseases caused by stress, such as cartilage degeneration under pressure and hypertension, but there is no report on glaucoma. In our study, Piezo1 was upregulated during RGCs cultured in high pressure. The change of Piezo1 and mTOR is exactly the same during the time after optic nerve crush. Piezo1 knockdown in cells resulted in down-regulation of mTOR activity and up-regulation of autophagy, activation of Piezo1 in PC12 cells resulted in more apoptosis. Given the facts above, we suppose that Piezo1 can be activated in RGC by IOP, inhibits autophagy through mTOR pathway, interaction with apoptosis process and resulted in glaucomatous RGC degeneration. In this project, we will knockdown Piezo1 by RNAi in iPS-induced RGCs, primary RGCs, mouse ONC model and mouse high IOP model. Firstly, analysis the function of Piezo1 in regulation of autophagy and apoptosis in RGCs degeneration. Secondly, detect whether Piezo1 regulate autophagy by mTOR pathway, and how Piezo1 regulate mTOR pathway. Thirdly, block or improve mTOR activity to study how autophagy and apoptosis interaction during RGCs degeneration. In conclusion, our study will develop new ideas for study of RGCs degeneration and protection, and provide new Drug target for glaucoma therapy.

高眼压可引起RGC不可逆致损伤、导致视神经慢性退化直至失明，是形成青光眼的主要致病因素。Piezo1是近年发现的专职应力感受的新型机械敏感性离子通道，参与多种应力相关疾病，如软骨应力劳损和高血压等，但在高眼压青光眼中未见报道。前期数据显示Piezo1在RGC压力培养后表达升高，导致轴突损伤和胞体死亡增加；视神经损伤模型中Piezo1波动剧烈且与mTOR变化趋势一致；细胞敲降Piezo1可降低mTOR活性提高自噬水平，减少死亡。我们假设病理性高眼压在RGC中激活Piezo1，通过活化mTOR抑制自噬，与凋亡互相作用，导致RGC青光眼性退化。本项目拟在诱导分化RGC、小鼠高眼压模型等体内外模型中敲降Piezo1，分析其在RGC损伤中调控自噬和凋亡的表型；探讨其通过何种途径调控自噬；研究其介导的自噬调控与凋亡间的互作关系。为青光眼RGC退化和保护机制研究提供科学依据，为临床治疗提供潜在药物靶点。

病理性高眼压可诱导视网膜神经节细胞（Retinal Ganglion Cells, RGCs）不可逆损伤，引起特征性的视神经萎缩和视野缺损，是青光眼疾病的主要病理特征。难以有效的控制RGC进行性死亡、促进其再生和修复，是目前青光眼临床和基础研究的瓶颈。高眼压引起的RGC损伤是多因素共同调控的复杂过程，阐明其分子调控机制以及视神经的保护、修复和再生机制是目前亟待解决的科学问题。Piezo1是2010年首次发现的专职应力感受的新型机械敏感性离子通道，在应力应答，细胞发育、分化和凋亡中起到关键的调节作用。Piezo1参与多种应力相关疾病，如软骨应力劳损和高血压等，但在高眼压青光眼中未见报道。本项目拟利用hiPSCs定向分化来源RGCs、高压细胞培养装置和高眼压动物模型等体内外模型，结合siRNA转染、AAV-Piezo1-shRNA病毒感染等手段敲降Piezo1，小分子抑制剂抑制相关下游靶点，探讨Piezo1在高眼压下调控RGCs损伤保护的生物学作用。揭示Piezo1在高眼压RGCs损伤后自噬和凋亡动态平衡中作用机制，为全新药物靶点的开发提供科学依据。研究发现，Piezo1在小鼠视网膜发育过程中表达量呈整体下降的趋势，其表达位置从全层广泛表达逐步向视网膜内层集中，最后主要表达于GCL层。hiPSCs定向分化为神经元的过程中Piezo1表达先升高后下降，NPC阶段表达量最高。慢性高眼压导致Piezo1通道激活，钙离子内流增加，通过活化CaMKⅡ磷酸化和抑制自噬作用最终诱导RGC进行性死亡。AAV-shPiezo1玻腔注射敲降Piezo1表达、GsMTx4玻腔注射抑制Piezo1通道活性、KN93腹腔注射抑制CaMKⅡ磷酸化或RAPA腹腔注射激活细胞自噬功能等方法均可有效抑制RGC死亡，发挥慢性高眼压视网膜神经损伤的保护作用。进而，在神经元体外高压培养体系中验证，高压培养激活细胞中Piezo1表达，对神经元的突触和胞体带来损伤，siRNA敲降Piezo1表达、GsMTx4、KN93或RAPA等方法均可提高神经元高压培养下的突出完整性和胞体存活率。上述研究结果为青光眼等视神经退行性疾病的发病机制研究拓宽了理论边界，为新的治疗药物靶点开发提供了坚实的科学依据和数据支持。