Novel Optogenetic Tools for Controlling Neuronal Signaling in Living Brain

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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.