DECIPHERING THE MECHANISTIC BASIS OF INFRARED VISION FOR OPTOGENETIC APPLICATIONS

 描述（由申请人提供）：光遗传致动器是可以通过光调节的离子通道或泵，从而允许以高空间和时间精度打开和关闭神经元活动。光遗传学在恢复失明患者的视力方面具有重要的前景，但目前的治疗策略需要应用高强度的蓝绿光，这会带来视网膜光损伤的重大风险。因此，对使用短波长光的依赖代表了安全有效地实施视网膜疾病光遗传学疗法的主要障碍。这个障碍可以通过使用具有红移激发光谱的光遗传学致动器来克服。红光能量较低，因此对视网膜的损害较小。因此，研究人员一直在寻求开发红移光遗传致动器，并且通过视蛋白工程在红移致动器中已经取得了相当大的进展。然而，为了将光遗传致动器的操作范围扩展到近红外（>700 nm），需要新的正交方法。本发明的目的是介绍一种用于红移光遗传致动器的新型仿生策略：红移发色团取代。这种方法是视蛋白工程的补充，并基于迁移鱼类在浑浊的水中实现更好视力的策略。当鲑鱼从开阔的海洋迁移到内陆河流（入射光明显红移）时，它们从使用视网膜作为其视觉发色团转变为具有红移光谱特性的4-二脱氢视网膜。这种发色团开关导致鱼的视蛋白光谱灵敏度发生戏剧性的红移，从而增强了动物看到长波长光的能力，从而使动物能够更深入地凝视浑浊的溪流。我们的目标是鉴定介导retinal转化为3，4-dodehydroretinal的酶，并在体内哺乳动物神经元中与光遗传致动器共表达，从而使其作用光谱红移。在特定目标1中，我们使用斑马鱼和牛蛙的转录组分析来鉴定介导这种转换的酶，然后表征其在体内的功能。在目标2中，我们将在体内共表达这种酶与红移光遗传学致动器，以赋予无功能的小鼠光感受器对近红外光（>700 nm）的敏感性。这种方法的一个关键特征是，发色团取代可以耦合到哺乳动物神经系统的任何部分中的任何现有致动器的使用。因此，这一提议有望对光遗传学领域产生广泛的影响。

 DESCRIPTION (provided by applicant): Optogenetic actuators are ion channels or pumps that can be regulated by light, thus permitting neuronal activity to be turned on and off with high spatial and temporal precision. Optogenetics holds significant promise for restoring vision to blind patients, but current treatment strategies require the application of high-intensity blue-green light, which poses a significant risk of retinal photodamage. Dependence on the use of short-wavelength light therefore represents a major barrier to safe and effective implementation of optogenetic therapy for retinal disease. This barrier can be surmounted by the use of optogenetic actuators with red-shifted excitation spectra. Red light is less energetic and therefore less damaging to the retina. Accordingly, researchers have sought to develop red-shifted optogenetic actuators, and considerable progress has been made in red-shifting actuators via opsin engineering. However, in order to extend the operational range of optogenetic actuators into the near-infrared (>700 nm), new orthogonal approaches are needed. The goal of the present proposal is to introduce a novel biomimetic strategy for red-shifting optogenetic actuators: red- shifted chromophore substitution. This approach is complementary to opsin engineering and is based on a strategy used by migrating fish to enable better vision in turbid water. When salmon migrate from the open ocean into inland streams (where incident light is significantly red-shifted), they switch from using retinal as their visual chromophore to , 4-didehydroretinal which has red-shifted spectral properties. This chromophore switch causes a dramatic red-shift of the fish's opsin spectral sensitivity, thereby enhancing the animal's abilityto see long-wavelength light and thus permitting the animal to peer more deeply into turbid streams. Our goal is to identify the enzyme mediating the conversion of retinal into 3, 4-dodehydroretinal, and to co-express it with optogenetic actuators in mammalian neurons in vivo, thereby red-shifting their action spectra. In Specific Aim 1, we use transcriptome profiling in zebrafish and bullfrog to identify the enzyme mediating this conversion, and then characterize its function in vivo. In Aim 2, we will co-express this enzyme with red-shifted optogenetic actuators in vivo to endow non-functioning mouse photoreceptors with sensitivity to near-infrared light (>700 nm). A key feature of this approach is that chromophore substitution can be coupled to the use of any existing actuator in any part of the mammalian nervous system. Thus, this proposal promises to have a widespread impact on the field of optogenetics.