Wednesday, January 15, 2025
1/15/2025
PROJECT SUMMARY Hypothalamic AgRP hunger neurons and POMC satiety neurons use neuropeptides AgRP, NPY, and αMSH to regulate food intake, energy expenditure, and body weight. One important downstream target is the satiety- promoting MC4R receptor-expressing neurons in the paraventricular nucleus of the hypothalamus (PVHMC4R), which receive converging peptidergic signals from AgRP and POMC neurons. However, exactly how PVHMC4R neurons interpret and integrate these peptide signals remains unclear, especially regarding the involvement of the downstream second messenger cAMP. The central unknowns can be summarized into three questions: (i) How do hunger and satiety peptides alter cAMP in PVHMC4R neurons? (ii) How does cAMP regulate the spiking of PVHMC4R neurons? (iii) Where in a PVHMC4R neuron is cAMP signaling most influential in regulating PVHMC4R activity? The main barrier to answering these questions is the lack of tools to measure and manipulate cAMP in PVHMC4R neurons. I have recently overcome this barrier by helping develop and apply a molecular and optical toolset to measure, make, and degrade cAMP during behaviors. The studies and career development activity in this K99/R00 proposal are designed to provide me with the necessary training to initiate an independent research program that applies the new cAMP tools to understand peptide signaling in PVHMC4R neurons. I hypothesize that coordinated, opposing changes in AgRP and POMC neuron activity regulate spiking in PVHMC4R neurons via competing actions on cAMP signaling. To test this hypothesis, I propose the following aims: Aim 1: To image cAMP dynamics in PVHMC4R neurons with the optical sensor cADDis during experimentally induced and feeding-induced peptide release, and to compare cAMP signaling between fasted and fed states. Aim 2: To measure how directly increasing cAMP (using a new optogenetic tool, biPAC) and degrading cAMP (using an engineered phosphodiesterase, PDE4D3-Cat) changes plasticity and spiking of PVHMC4R neurons. Aim 3: To engineer and use subcellularly targeted cADDis, biPAC, PDE4D3-Cat in order to understand local cAMP signaling in PVHMC4R neurons. The proposed research will be mentored by Drs. Mark Andermann and Bradford Lowell, who will also provide guidance on becoming an independent researcher. I will learn new techniques in intraventricular drug delivery, whole-cell patch-clamp, and measurements of energy expenditure from my mentors as well as Drs. Alex Banks, Maria Lehtinen, and Joseph Majzoub. I will receive training in protein engineering from Drs. Shiqiang Gao, Georg Nagel, Bernardo Sabatini, and Gary Yellen. I will acquire new skills from the above experts and from courses and scientific meetings, combined with my existing expertise in cAMP research. This will enable me to understand when, where, and how cAMP is used to titrate PVHMC4R neurons activity. These results will fill an important knowledge gap in developing and understanding treatments for obesity.
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