Garita-Hernandez Marcela, Guibbal Laure, Toualbi Lyes, Routet Fiona, Chaffiol Antoine, Winckler Celine, Harinquet Marylin, Robert Camille, Fouquet Stephane, Bellow Sebastien, Sahel José-Alain, Goureau Olivier, Duebel Jens, Dalkara Deniz
Institut de la Vision, Sorbonne Université, INSERM, CNRS, Paris, France.
BioAxial, Paris, France.
Front Neurosci. 2018 Nov 2;12:789. doi: 10.3389/fnins.2018.00789. eCollection 2018.
Optogenetic technologies paved the way to dissect complex neural circuits and monitor neural activity using light in animals. In retinal disease, optogenetics has been used as a therapeutic modality to reanimate the retina after the loss of photoreceptor outer segments. However, it is not clear today which ones of the great diversity of microbial opsins are best suited for therapeutic applications in human retinas as cell lines, primary cell cultures and animal models do not predict expression patterns of microbial opsins in human retinal cells. Therefore, we sought to generate retinal organoids derived from human induced pluripotent stem cells (hiPSCs) as a screening tool to explore the membrane trafficking efficacy of some recently described microbial opsins. We tested both depolarizing and hyperpolarizing microbial opsins including CatCh, ChrimsonR, ReaChR, eNpHR 3.0, and Jaws. The membrane localization of eNpHR 3.0, ReaChR, and Jaws was the highest, likely due to their additional endoplasmic reticulum (ER) release and membrane trafficking signals. In the case of opsins that were not engineered to improve trafficking efficiency in mammalian cells such as CatCh and ChrimsonR, membrane localization was less efficient. Protein accumulation in organelles such as ER and Golgi was observed at high doses with CatCh and ER retention lead to an unfolded protein response. Also, cytoplasmic localization was observed at high doses of ChrimsonR. Our results collectively suggest that retinal organoids derived from hiPSCs can be used to predict the subcellular fate of optogenetic proteins in a human retinal context. Such organoids are also versatile tools to validate other gene therapy products and drug molecules.
光遗传学技术为在动物体内剖析复杂神经回路和利用光监测神经活动铺平了道路。在视网膜疾病中，光遗传学已被用作一种治疗方式，用于在光感受器外段丧失后使视网膜恢复活力。然而，如今尚不清楚众多微生物视蛋白中哪些最适合用于人类视网膜的治疗应用，因为细胞系、原代细胞培养物和动物模型无法预测微生物视蛋白在人类视网膜细胞中的表达模式。因此，我们试图从人类诱导多能干细胞（hiPSC）中生成视网膜类器官，作为一种筛选工具，以探索一些最近描述的微生物视蛋白的膜转运效率。我们测试了去极化和超极化的微生物视蛋白，包括CatCh、ChrimsonR、ReaChR、eNpHR 3.0和Jaws。eNpHR 3.0、ReaChR和Jaws的膜定位最高，这可能是由于它们额外的内质网（ER）释放和膜转运信号。对于未经过改造以提高在哺乳动物细胞中转运效率的视蛋白，如CatCh和ChrimsonR，膜定位效率较低。在高剂量使用CatCh时，观察到内质网和高尔基体等细胞器中的蛋白质积累，内质网滞留导致未折叠蛋白反应。此外，在高剂量使用ChrimsonR时观察到细胞质定位。我们的结果共同表明，源自hiPSC的视网膜类器官可用于预测人类视网膜环境中光遗传学蛋白的亚细胞命运。此类类器官也是验证其他基因治疗产品和药物分子的通用工具。
