小梁网GRP78-PERK-MAMs信号异常致眼压升高的分子机制

Malfunction of trabecular meshwork plays a vital role in pathological increase of intraocular pressure (IOP) in POAG, which is associated with abnormal regulation of mitochondria-associated endoplasmic reticulum membranes (MAMs), but the mechanisms are unknown. We previously confirmed that the expression of GRP78 in trabecular meshwork of POAG patients was down-regulated and co-localized with MAMs and PERK; and our preliminary results suggest that PERK signaling pathway mediated the elevation of IOP induced by trabecular meshwork injury. In the present project, human primary trabecular meshwork and human trabecular meshwork cell (HTMC) cell line were used to establish chronic oxidative damage in vitro model, and explore the following: to detect the expression and distribution of GRP78 in MAMs before and after injury; to explore the GRP78 signaling mechanisms of PERK-MAMs through using gene overexpression/knockdown, or receptor targeted agonists/antagonists and to analysis the endoplasmic reticulum stress, calcium ion distribution, mitochondrial function and apoptosis; furthermore, to ascertain the underlying mechanisms between GRP78-PERK-MAMs and chronic high IOP in the in vivo animal model. This study will elucidate the molecular mechanisms of GRP78-PERK-MAMs signaling pathway in regulating the increase of IOP caused by dysfunction of trabecular meshwork cells, in order to provide a new theory and potential therapeutic targets for the pathogenesis of POAG.

小梁网细胞功能损伤所致病理性眼压升高是POAG发病的重要原因，其发生与线粒体相关内质网膜（MAMs）钙调控功能异常密切相关，但分子机制未明。申请人前期研究证实POAG小梁网细胞GRP78表达下调，且与MAMs、PERK共定位；近期预实验提示PERK信号通路介导小梁网损伤致眼压升高。本课题拟采用人源小梁网细胞原代培养及HTMC细胞系，建立体外慢性氧化损伤模型，检测损伤前后MAMs区域GRP78的表达、分布；利用靶向受体激动剂/抑制剂，基因过表达/敲减等方法，分析内质网应激、钙离子分布、线粒体功能、细胞凋亡水平，探究GRP78对PERK-MAMs信号调控机制；进一步利用慢性高眼压动物模型，获得该通路介导小梁网损伤致眼压升高的确切体内证据。本研究将阐明“GRP78-PERK-MAMs”信号调控小梁网细胞功能损伤致眼压升高的分子机制，以期为POAG发病机制提供新的理论依据及潜在治疗靶点。

小梁网细胞功能损伤所致病理性眼压升高是POAG发病的重要原因，其发生与线粒体相关内质网膜（MAMs）钙调控功能异常密切相关，但分子机制未明。课题组前期研究证实POAG小梁网细胞GRP78表达下调，且与MAMs、PERK共定位，同时发现PERK信号通路介导小梁网损伤致眼压升高。本课题采用人源小梁网细胞原代培养及HTMC细胞系，建立体外慢性氧化损伤模型，检测损伤前后MAMs区域GRP78的表达、分布，利用靶向受体激动剂，分析内质网应激、钙离子分布、线粒体功能、细胞凋亡水平，研究发现内质网应激模型中，够HTMC和GTM3细胞的凋亡率增加，ATF4、eIF2a、CHOP mRNA的表达水平上调，且MAMs相关蛋白VDAC1和IP3R与GRP78表达水平呈正相关，提示内质网应激诱导剂同样能够诱导小梁网细胞发生氧化应激，而上调GRP78的表达可保护内质网应激引起的小梁网细胞损伤，GRP78的上述保护作用是通过调节eIF2a的表达，激活PERK-eIF2a-ATF4 /CHOP信号通路实现的。本研究为阐明“GRP78-PERK-MAMs”信号调控小梁网细胞功能损伤致眼压升高的分子机制提供崭新视角，以期为POAG发病机制提供新的理论依据及潜在治疗靶点。