DESCRIPTION (provided by applicant): The proteins in synapses are the fundamental regulators of synaptic plasticity, which ultimately controls the neural circuits that underlie behavior. A major advance in our understanding of how synaptic connectivity is linked to animal behavior comes from transcranial two-photon imaging of dendritic spines in living animals. However, despite the advances made by two-photon microscopy, most experiments have been observational. Researchers lack the ability to directly manipulate the protein content at specific synapses and spines, hindering their efforts to decipher the roles of synaptic proteins in learning, memory, behavior, and disease. In this application, we describe a novel method to use two-photon irradiation to control protein synthesis in a spine- specific manner in awake animals. This method is called LIPS: light-induced protein synthesis. LIPS utilizes a "light-responsive" mRNA encoding a protein of interest. This mRNA is designed so that it is not translated under normal conditions. However, upon two-photon irradiation of a dendrite or spine, a light-activated protein is recruited to the 5'UTR of the mRNA, resulting in localized translation. Thus, LIPS will allow protein synthesis to be achieved with unprecedented spatial and temporal resolution in the brains of live animals. In order to develop a simple and robust optogenetic technology to allow researchers to study the role of essentially any protein on synaptic function, the specific aims of this application are (1) To generate and optimize RNA aptamers that recruit light-activated forms of two Arabidopsis phytochromes: Cry2 and PhyB. These experiments will result in the generation of the first light-regulated RNA-protein interactions; (2) To optimize LIPS in cortical neurons. To make light-regulated mRNAs, we will incorporate these aptamers into specific mRNAs. We will then optimize a two-photon irradiation protocol for controlling protein synthesis in dendrites and spines in live animals; (3) To use LIPS to dissect the role of FMRP and FMRP domains in spine remodeling. The experiments in this aim are designed to investigate the role of FMRP in regulating dendritic spine dynamics at the level of individual dendritic spines in the cortex of live mice. Here we will use LIPS to directly interrogate how varying the level of FMRP in spines correlates with spine turnover and we will determine if LIPS-mediated restoration of FMRP in Fmr1 KO dendrites results in restoration of spine stability. Together, these experiments will provide insight into how FMRP controls spine remodeling in living mice. In summary, LIPS provides unprecedented spatiotemporal control of protein expression within a neuron. LIPS will transform two photon studies by enabling researchers to control the protein composition of spines and dendrites and monitor the effects of specific proteins on processes like dendritic spine morphology and synaptic plasticity.

PUBLIC HEALTH RELEVANCE: The proteins in synapses fundamentally influence synaptic plasticity and dendritic spines, and their alterations are frequently the cause of diverse neurological and mental health diseases. Although powerful technologies such as transcranial two-photon microscopy enable researchers to directly monitor specific spines and synapses in awake animals, researchers lack the ability to directly manipulate the protein composition at these sites, preventing them from deciphering the roles of synaptic proteins in disease pathogenesis. The goal of this application is to develop a simple and robust optogenetic technology to allow researchers to control protein expression in specific dendrites, spines, and/or synapses in order to determine the effect of essentially any protein on synaptic and spine function.