DECIPHERING THE MECHANISTIC BASIS OF INFRARED VISION FOR OPTOGENETIC APPLICATIONS

 描述(申请人提供)：光发生致动器是可以被光调节的离子通道或泵，从而允许以高空间和时间精确度开启和关闭神经元活动。光遗传学为盲人患者恢复视力带来了巨大的希望，但目前的治疗策略需要应用高强度的蓝绿光，这构成了视网膜光损伤的重大风险。因此，对使用短波长光的依赖是安全和有效地实施视网膜疾病光遗传疗法的主要障碍。这一障碍可以通过使用红移激发光谱的光致动器来克服。红光能量较低，因此对视网膜的损害较小。因此，研究人员试图开发红移光致动器，并通过光学工程在红移致动器方面取得了相当大的进展。然而，为了将光生致动器的工作范围扩展到近红外(&gt；700 nm)，需要新的正交化方法。本提议的目标是引入一种新的红移光生电子致动器的仿生策略：红移发色团替代。这种方法是对视蛋白工程的补充，是基于迁徙鱼类使用的一种策略，以使在浑浊的水中能够更好地视力。当鲑鱼从开阔的海洋迁徙到内陆河流(那里的入射光发生显著红移)时，它们从以视网膜为视觉发色团，转向具有红移光谱特性的4-双脱氢视网膜。这种生色团的开关导致鱼的视觉在光谱敏感度上发生戏剧性的红移，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视网膜转化为3，4-脱氢视网膜的酶，并在活体内与哺乳动物神经元中的光遗传致动器共表达，从而使其作用光谱红移。在特定的目标1中，我们利用斑马鱼和牛蛙的转录组图谱来鉴定介导这种转化的酶，然后在体内表征其功能。在目标2中，我们将在体内将该酶与红移的光遗传致动器共表达，以赋予不起作用的小鼠光感受器对近红外光(&gt；700 nm)的敏感性。这种方法的一个关键特征是，生色团替代可以与哺乳动物神经系统任何部分的任何现有致动器的使用相结合。因此，这一提议有望对光遗传学领域产生广泛影响。

 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.