Developing Novel Chemo-optogenetic Tools for in Vivo Applications

项目总结/摘要 光遗传学和化学-光遗传学是利用光调节细胞活动的有力工具。这些工具 通过提供询问神经回路功能的必要手段来加速神经科学研究。 用于视网膜疾病的光遗传学疗法的临床试验已经在进行中。其中的一些局限性 目前的工具包括通常很小的光诱导电流，它们操纵特定细胞的能力有限 深层组织中的活性，需要强大的转基因表达以防止非法生理效应，以及安全性 对长期外源转基因表达的担忧。我开发的新型化学光遗传学工具 解决其中许多问题。我以前开发了一种新的化学光遗传学工具， 电导TRPA 1通道，其适用于调节神经元和非神经元细胞活性， vivo.我还开发和执行了一个小分子屏幕的基础上斑马鱼光诱导 运动反应，并发现了针对内源性脊椎动物蛋白质的分子光开关。我是 表征从该筛选中鉴定的两个命中（Aim 1，K99相）。一种是阶梯式化疗 基于TRPA 1通道的光遗传学系统。这个新系统将允许光控制通道 ON/OFF，进一步增强TRPA 1效用。第二个是基于TRPV 1的化学光遗传学系统 频道我的化学光遗传学工具开发计划的下一阶段是增强TRPA 1通道 对钠的选择性，同时保持其高通道电导（Aim 2，K99/R 00相）。这将提供 生理上更相关的光诱导动作电位的产生。我也会把斑马鱼 特异性鉴定内源性蛋白质靶向分子的光诱导运动反应筛选测定 具有近红外范围内的光谱的光电开关（Aim 3，R 00相）。近红外光的使用允许 用于更深地渗透到组织中以及用于与现有光遗传学工具和生物传感器成像的兼容性。 总的来说，我提出的研究将产生新的化学-光遗传学工具， 通道电导，更深组织中的光控开/关活动，并且不需要或需要低水平的 外源基因表达我的研究还将为发现新的化疗药物创造一个平台， 模拟自然细胞活动的光遗传致动器。我开发的下一代工具将增强我们的 解剖生物过程的能力，如大脑的复杂神经网络，并加速 光遗传学的潜在临床应用。我的导师、顾问和合作者组成的多元化团队， 选择我是为了确保我的成功，并在与此项目相关的相关领域进一步培训 例如离子通道生物学、化学生物学、电生理学、光遗传学和神经科学。我的训练 计划将装备我的技术技能和知识，开发新的化学光遗传致动器，在 体内神经科学的应用和超越，并为成功过渡到一个 独立研究员。

Project Summary/Abstract Optogenetics and chemo-optogenetics are powerful tools for modulating cell activities with light. These tools accelerate neuroscience research by providing the necessary means for interrogating neural circuit function. Clinical trials of optogenetic therapy for retinal diseases are already underway. Some of the limitations of these current tools include a generally small light-induced current, their limited ability to manipulate specific cell activity in deep tissue, the need for robust transgene expression to illicit physiological effects, and safety concerns over long-term exogenous transgene expression. The novel chemo-optogenetic tools I develop will address many of these issues. I previously developed a novel chemo-optogenetic tool based on the high conductance TRPA1 channel which is suitable for modulating both neuronal and non-neuronal cell activity in vivo. I have also developed and performed a small molecule screen based on the zebrafish light-induced motion response and discovered molecular photoswitches that target endogenous vertebrate proteins. I am characterizing two hits identified from this screen (Aim 1, K99 phase). One is a step-function chemo- optogenetic system based on the TRPA1 channel. This new system will allow for light-controlled channel ON/OFF, further enhancing TRPA1 utility. The second is a chemo-optogenetic system based on the TRPV1 channel. The next phase of my chemo-optogenetic tool-development program is to enhance TRPA1 channel selectivity for sodium while preserving its high channel conductance (Aim 2, K99/R00 phase). This will provide a more physiologically relevant light-induced generation of action potentials. I will also extend the zebrafish light-induced motion response screening assay to specifically identify endogenous protein-targeting molecular photoswitches with spectra in the near infrared range (Aim 3, R00 phase). The use of near infrared light allows for deeper penetration into tissues and for compatibility with existing optogenetic tools and biosensor imaging. Overall, my proposed research will generate novel chemo-optogenetic tools with improvements to unitary channel conductance, light-controlled ON/OFF activity in deeper tissue, and require no or low levels of exogenous gene expression. My research will also create a platform for the discovery of novel chemo- optogenetic actuators that mimic natural cell activity. The next generation tools I develop will enhance our ability to dissect biological processes such as the complex neuronal network of the brain and accelerate the potential clinical use of optogenetics. My diverse team of mentors, advisors and collaborators have been chosen to both ensure my success and to further my training in the relevant areas associated with this project such as ion channel biology, chemical biology, electrophysiology, optogenetics and neuroscience. My training plan will equip me with technical skills and knowledge for developing novel chemo-optogenetic actuators for in vivo neuroscience applications and beyond, and provide a foundation for a successful transition into an independent researcher.