利用光遗传学技术在ON型双极细胞上重建视网膜退行变性疾病的视觉功能

The outcomes of retinal degenerative diseases are usually the apoptosis of photoreceptor cells, such as retinal pigmentation and age-related macular degeneration，which both are hot topics in fundus ophthalmology research. The retinal neuroprotection after the apoptosis of photoreceptor cells and even the reconstruction of visual signal transmission become extremely important. We had previously studied the retinal neuroprotection effect mediated by cannabinoids in degenerative diseases of the retina, which lays a certain foundation for the research on the function of bipolar cells. We next propose to restore light capability by optogenetics tools. This project intends to carry out the following series of studies by means of electrophysiological recording:1）The newly developed photosensitive ion channel protein will be introduced into bipolar cells located downstream of photoreceptor cells to convert light-insensitive ON bipolar cells to a light-sensitive state. The combined pattern of light-sensitive proteins converts OFF bipolar cells to light-sensitive state as well.2) A targeting motif will be added in the vector to target either the central area or surround area of these ON bipolar cells. The center-surround receptive field will recreated that further permits improvement of visual spatial processing for restored vision.3) The light response of bipolar cells and downstream ganglion cells will recorded by single cell electrophysiological patch clamp.Our study will provide new therapeutic strategy for vision restoration for retinal degenerative diseases.

视网膜色素变性和黄斑变性等退行性疾病主要特征是光感受器细胞凋亡，而下级神经网络结构正常，因此光感受器细胞凋亡后的视网膜保护乃至视觉信号传导重建异常重要。申请人前期成果研究了视网膜退行性疾病中大麻素受体介导的神经保护作用，对双极细胞的研究有一定的基础。本项目拟进一步联合光遗传学技术，重建视网膜视觉信号传导，开展以下系列研究：1）对光感受器细胞下级的双极细胞引入最新研发光敏离子通道蛋白，使ON型双极细胞获得“感光”能力；组合的光敏蛋白模式使OFF型双极细胞也恢复电活动。2）通过引入定位基序（targeting motif），以模拟中心/周边视觉神经元感受野的方式分布光敏蛋白，提高双极细胞的视觉信号加工能力。3）用单细胞电生理膜片钳的方法，记录双极细胞和下游神经节细胞的对光反应，鉴定视网膜视觉信号传导的重建。申请人拟通过本项目的研究，为视网膜退行性疾病的治疗提供新的理论基础。

光感受器细胞凋亡后的视网膜色素变性和老年黄斑变性等常见致盲眼底病，本项目拟联合光遗传学技术，重建视网膜视觉信号传导。应用基因工程技术改造光敏蛋白PsChR，筛选出Ca2+通透性更高的突变体PsCatCh2.0，并在非洲爪蟾蜍卵母细胞中，验证PsCatCh2.0的光敏感性及动力学特性。PsCatCh2.0的最佳激活波长在470nm左右，可以在对视网膜安全的光强阈值范围内产生电流反应，具有高光敏感性；PsCatCh2.0可以稳定的响应高频率的光刺激，超过视觉信号需要的24Hz，满足光遗传学恢复视觉功能的动力学需求。进一步在rd1小鼠上，通过玻璃体腔注射病毒rAAV2/2-CMV-PsCatCh2.0-EGFP导入PsCatCh2.0，取视网膜切片进行全细胞模式膜片钳，记录表达PsCatCh2.0的视网膜神经节细胞的对光反应及响应频率。取视网膜和脑片进行免疫荧光染色，评估c-Fos和Arc在视网膜、上丘和视觉皮层V1区的表达；记录老鼠V1区闪光视觉诱发电位；最后进行明暗箱和视动反应这两个行为学实验，评估治疗后rd1小鼠的视觉行为学的恢复。结果显示PsCatCh2.0能有效的改善模型鼠视力，具有很好的临床意义前景。

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