Novel Optogenetic Tools for Controlling Neuronal Signaling in Living Brain

研究目标：开发和优化一套光遗传学工具，允许通过光时空操纵细胞内cAMP和cGMP信号。 背景/依据：cAMP和cGMP是细胞内信号传导的重要第二信使，介导信息的神经转导及其调节。药物试剂和基因操作已被用于研究其功能，然而，这些方法在空间和时间特异性方面受到限制。在这里，我们建议建立一套光遗传学工具，通过采用cAMP/cGMP信号的光活化酶来快速增加/减少第二信使信号。 研究方法：cAMP和cGMP由腺苷酸/鸟苷酸环化酶（AC/GC）从ATP/GTP合成，ATP/GTP随后被细胞内的磷酸二酯酶（PDE）降解。我们已经开始开发或优化一组可光活化的催化酶，以使用参与其合成（AC/GC）和降解（PDE）的基因工程化的可光活化的酶来增加或降低cAMP/cGMP水平。对于可光活化的AC/GC，我们已经利用了从细菌中分离的AC的内源性蓝光可活化形式。我们将验证/优化绿色或远红光激活的AC/GC以增加cAMP/cGMP。为了补充可光活化的环化酶，我们将设计可光活化的PDE 4和PDE 5以特异性降解cAMP和cGMP。为了双向增加/减少cAMP和cGMP，我们将验证/优化蓝光或绿色光可激活的AC/GC和远红光敏感的PDE 4/PDE 5的组合。此外，为了在脑深部组织内的单突触水平（~1 µm3）靶向光激活，我们将利用双光子激光激发，并使用不同波长的双光子激发光将该技术扩展为双光激活。 目标： 1)通过建立一组光活化酶开发/优化cAMP/cGMP水平的光依赖性调节 2)建立控制cAMP/cGMP激活的双光子光遗传学方法 光遗传学工具将允许通过光对cAMP和cGMP水平进行非侵入性时空操纵，并以前所未有的方式为研究活体脑内快速相互作用的cAMP/cGMP功能提供新的强大工具。

RESEARCH GOAL: To develop and optimize a set of optogenetic tools that allow spatiotemporal manipulation of intracellular cAMP and cGMP signaling by light. BACKGROUND/RATIONALE: cAMP and cGMP are essential second messengers for intracellular signaling that mediate the neuro-transduction of information and its modulation. Pharmacological reagents and genetic manipulations have been used to study their functions, however, these approaches are limited in spatial and temporal specificity. Here we propose to establish a set of optogenetic tools to rapidly increase/decrease second messenger signaling by employing photoactivatable enzymes for cAMP/cGMP signaling. METHODOLOGY: cAMP and cGMP are synthesized by adenylyl/guanylyl cyclase (AC/GC) from ATP/GTP which is subsequently degraded by phosphodiesterases (PDEs) within cells. We have begun the development or optimization of a panel of photoactivatable catalytic enzymes to increase or decrease cAMP/cGMP level using genetically engineered photoactivatable enzymes that are involved in their synthesis (AC/GC) and their degradation (PDEs). For photoactivatable AC/GC, we have utilized an endogenous blue-light activatable form of AC that has been isolated from bacteria. We will validate/optimize green or far-red light activatable AC/GC to increase cAMP/cGMP. To complement the photoactivatable cyclases, we will design photoactivatable PDE4 and PDE5 to specifically degrade cAMP and cGMP. To bidirectionally increase/decrease cAMP and cGMP, we will validate/optimize the combination of blue or green light activatable AC/GC and far-red light sensitive PDE4/PDE5. Furthermore, for targeting the photoactivation at the single synapse level (~1 µm3) within deep brain tissues, we will utilize two-photon laser light excitation and expand this technique for dual photoactivation using different wavelengths of two-photon excitation light. AIMS: 1) Development/optimization of light-dependent regulation of cAMP/cGMP levels by establishing a set of photoactivatable enzymes 2) Establish two-photon optogenetic approaches to control cAMP/cGMP activation The optogenetic tools will allow for non-invasive spatiotemporal manipulation of cAMP and cGMP levels by light and provide novel powerful tools to study the rapid interactive cAMP/cGMP functions within living brain in an unprecedented way.