A new strategy for vision restoration based on melanopsin transduction mechanisms

项目总结 治疗视网膜疾病的一个紧迫挑战是在不可逆转的情况下恢复视网膜的视力 光感受器退化。理论上，即使在没有视杆和视锥的情况下，这种病变的视网膜应该仍然 能够使用感光色素通过固有的感光视网膜神经节细胞(IpRGC)感知光线 名为黑色素。然而，黑素介导的光反应的动力学是缓慢的，因此只有非常 较差的时间分辨率，因此在很大程度上限制了其提供成像信息的能力。对于 同样的原因，之前提出的一种视力恢复方法-利用病毒表达的 黑素使传统的视网膜神经节细胞(RGC)对光敏感-也受到缓慢的限制 黑素蛋白的反应动力学。我们最近的研究已经确凿地证明，黑素介导的 光传导受其下游成分的速度限制，可以通过病毒加速 表达的离子通道具有更快的动力学。我们建议通过以下方式开发一种新的视力恢复策略 通过调控光感受器下游转导成分加速黑素信号转导 退化的小鼠模型。我们有两个主要目标。在目标1中，我们将重点加强光响应 内源性黑素在M4和M5亚型的ipRGC中的表达，因为它们已被证明投射到 图像形成的大脑中心，使他们能够对图像形成的视觉做出贡献。我们发现， M4细胞对黑素光激活的反应是通过提高细胞内环核苷酸水平和 随后开放对cNMP敏感的HCN通道。我们新的初步数据表明，M5细胞也使用 HCN途径。我们进一步证明，依赖于HCN的信号通路的反应可以是 通过引入环核苷酸门控通道家族的成员CNGA2来加速，从而导致更快的 响应动力学和更大的幅度，证明了我们的战略概念。在目标2中，我们将恢复光线 病毒表达的黑素结合快速信号在传统视网膜神经节细胞中的反应 分子。我们的多电极阵列结果显示，同时表达黑素和CNGA2的细胞 与单独表达黑素相比，cRGCs提供更高的光敏感度和更快的上升阶段。vbl.使用 水基视觉引导迷宫试验中，我们进一步发现，外源性黑素和CNGA2不仅 在光感受器退化的动物中，恢复光敏感度的同时也提供了模式视觉。加在一起，这些 令人兴奋的初步观察增加了使用CNG通道和/或修改其他 黑素蛋白的光传导成分作为光感受器后恢复视力的治疗手段 退化。创新。到目前为止，所有提出的光遗传方法都集中在寻找更好的光感知 具有更高表达、更好的光敏感度和更快的响应动力学的蛋白质。我们是第一个利用 用于视力恢复的黑素下游成分和新型信号分子。这 代表了一种概念创新和一种开箱即用的视力恢复策略。

PROJECT SUMMARY A pressing challenge in the treatment of retinal diseases is to restore vision in the retina with irreversible photoreceptor degeneration. In theory, even in the absence of rods and cones, such diseased retina should still be able to sense light through intrinsically photosensitive retinal ganglion cells (ipRGCs) using a photopigment named melanopsin. However, the kinetics of melanopsin-mediated light response is slow, thus giving only very poor temporal resolution and therefore largely limiting its ability to provide image-forming information. For the same reason, a vision restoration approach previously proposed – which made use of virally expressed melanopsin to endow light sensitivity to conventional retinal ganglion cells (RGCs) – is also limited by the slow response kinetics of melanopsin. Our recent study has firmly established that melanopsin-mediated phototransduction is rate-limited by its downstream components and that it can be accelerated by virally expressed ion channels with faster kinetics. We propose to develop a novel vision restoration strategy by accelerating melanopsin signaling via manipulating downstream transduction components in photoreceptor degenerated mouse models. We have two major aims. In Aim 1, we shall focus on enhancing the light response of endogenous melanopsin in M4- and M5-subtypes of ipRGCs because they have been shown to project to image-forming brain centers, allowing them to contribute to the image-forming vision. We have discovered that M4-cells respond to melanopsin photoactivation by elevating intracellular levels of cyclic nucleotides (cNMP) and subsequent opening of cNMP-sensitive HCN channels. Our new preliminary data suggest that M5 cells also use the HCN pathway. We have further shown that the response of the HCN-dependent signaling pathway can be sped up by introducing a member of the cyclic nucleotide-gated channel family, CNGA2, resulting in faster response kinetics and larger amplitudes, proving the concept of our strategy. In Aim 2, we shall restore light response in conventional retinal ganglion cells using virally expressed melanopsin together with faster signaling molecules. Our multielectrode array results show that simultaneously expressing melanopsin and CNGA2 in cRGCs provides higher light sensitivities and faster rising phases than expressing melanopsin alone. Using water-based vision-guided maze tests, we have further found that exogenous melanopsin and CNGA2 not only restore light sensitivity but also confer pattern vision in photoreceptor-degenerated animals. Together, these exciting preliminary observations raise the prospect of using CNG channels and/or modifications to other phototransduction components of melanopsin as a therapeutic modality to restore vision following photoreceptor degeneration. Innovation. All optogenetic approaches proposed thus far focus on finding a better light-sensing protein with higher expression, better light sensitivity, and faster response kinetics. We are the first to exploit downstream components and novel signaling molecules of melanopsin for vision restoration purposes. This represents a conceptual innovation and an out-of-the-box strategy for vision restoration.