Development of innovative genetically encoded tools to dissect melanocortin-4 receptor signaling in neuronal circuits

PROJECT SUMMARY/ABSTRACT The absence of genetically encoded tools capable of activating endogenous melanocortin-4 receptor (MC4R) in the brain with cell selectivity and temporal control has created a critical gap in the ability to study the neural circuits regulating feeding behavior and energy homeostasis. The long-term goal of this project is to map the melanocortin system microcircuitry by activating MC4R in specific neuronal populations to study effects on feeding behavior and physiology. Pharmacological methods lack cell-type specificity and spatial resolution, while existing chemogenetic and optogenetic techniques do not replicate natural MC4R signaling events. The overall objective of this application is to engineer genetically encoded tools capable of achieving high spatiotemporal control over MC4R activation. The rationale for this project is that surface-tethered protein switches can be employed to control the activity of the MC4R agonist alpha-melanocyte stimulating hormone (Į-MSH). Specific Aim 1 will engineer a chemogenetic controlled Į-MSH (ccMSH) for drug-dependent activation of MC4R using chemically activated protein domains (CAPs) to cage Į-MSH. The ccMSH will be optimized for high drug dependence and assessed in mammalian cell cultures to validate the system's functionality. It will then be applied to mouse models to observe behavioral and physiological responses to chemical-dependent MC4R activation. Specific Aim 2 will develop optogenetically controlled Į-MSH (ocMSH) for light-dependent activation of MC4R using the light-oxygen-voltage sensing (LOV) domain to cage Į-MSH. Directed evolution will be used to improve the membrane trafficking and photoactivation of the LOV domain. The effectiveness of ocMSH will be characterized in cell cultures and tested in mouse models to assess the impact of light-gated MC4R activation on behavior and physiology. Upon successful completion, this project will introduce innovative and novel chemogenetic and optogenetic tools that will enable detailed studies of the melanocortin system in vivo, providing high spatiotemporal resolution of endogenous MC4R activation. The anticipated outcomes will significantly impact our understanding of the regulation of energy homeostasis and feeding behavior, contributing significantly to the development of therapeutic strategies for treating obesity and related metabolic disorders.