Optogenetic Light Sensors in Human Retinal Organoids.
Optogenetic Light Sensors in Human Retinal Organoids.
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DOI：
10.3389/fnins.2018.00789
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发表时间：
2018
影响因子：
4.3
通讯作者：
Dalkara D
中科院分区：
文献类型：
--
作者：
Garita-Hernandez M;Guibbal L;Toualbi L;Routet F;Chaffiol A;Winckler C;Harinquet M;Robert C;Fouquet S;Bellow S;Sahel JA;Goureau O;Duebel J;Dalkara D
光遗传学技术为解剖复杂的神经回路和利用光监测动物的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，以在光感受器外节丧失后使视网膜恢复活力。然而，目前尚不清楚微生物视蛋白的多样性中哪些最适合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不能预测人类视网膜细胞中微生物视蛋白的表达模式。因此，我们试图产生源自人类诱导多能干细胞（hiPSC）的视网膜类器官，作为筛选工具来探索一些最近描述的微生物视蛋白的膜运输功效。我们测试了去极化和超极化微生物视蛋白，包括 CatCh、ChrimsonR、ReaChR、eNpHR 3.0 和 Jaws。 eNpHR 3.0、ReaChR 和 Jaws 的膜定位最高，可能是由于它们额外的内质网 (ER) 释放和膜运输信号。对于未经过改造以提高哺乳动物细胞运输效率的视蛋白（例如 CatCh 和 ChrimsonR），膜定位效率较低。在高剂量下观察到蛋白质在 ER 和高尔基体等细胞器中积累，CatCh 和 ER 保留导致未折叠的蛋白质反应。此外，在高剂量的 ChrimsonR 下观察到细胞质定位。我们的结果总体表明，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多功能工具。
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.
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影响因子：
4.3
作者：
Bedbrook CN;Yang KK;Rice AJ;Gradinaru V;Arnold FH
通讯作者：
Arnold FH
影响因子：
25
作者：
Lin, John Y.;Knutsen, Per Magne;Muller, Arnaud;Kleinfeld, David;Tsien, Roger Y.
通讯作者：
Tsien, Roger Y.
影响因子：
25
作者：
Kleinlogel, Sonja;Feldbauer, Katrin;Bamberg, Ernst
通讯作者：
Bamberg, Ernst
影响因子：
8
作者：
Khabou, Hanen;Garita-Hernandez, Marcela;Dalkara, Deniz
通讯作者：
Dalkara, Deniz
影响因子：
8.8
作者：
Belle, Morgane;Godefroy, David;Chedotal, Alain
通讯作者：
Chedotal, Alain