Project Summary The grant “TR2 nuclear receptor in vitamin A signaling” (since 08/01/99) was initiated from studying retinoic acid (RA) signaling using an embryonic stem cell-specific orphan receptor TR2’s (NR2C1) as a model to understand RA’s action in developmental regulation. The long-term goal is to comprehensively understand RA signaling pathways in health and diseases. Results first established RA’s activity in chromatin-remodeling via RA receptors (RARs), Retinoid X receptors (RXRs) and TR2. In 2008, we reported a then-mysterious effect of RA that did not involve RAR/RXR - it occurred rapidly in the cytoplasm to modulate ERK signaling, and thus was proposed as “non-canonical”. Previous proposal (05/1/2017-04/30/2022) aimed to i) identify the mediator of this non-canonical activity of RA in modulating ERK signaling and synthetic RA-like ligands specific to this pathway, and ii) determine the physiological relevance. Results (published in 14 papers) have established Cellular Retinoic Acid Binding Protein 1 (CRABP1) as the mediator, characterized RA-CRABP1-RAF-MEK- ERK signaling pathway and CRABP1 ligands specific to this pathway, and revealed physiological relevance in maintaining neuron stem cell pool, adiposity and inflammation, all involving CRABP1-ERK regulation. Recently, a second signaling pathway also directly modulated by CRABP1 was uncovered, calcium/calmodulin- dependent protein kinase II (CaMKII), but in a different physiological context - excitable cells requiring tight regulation of CaMKII such as motor neurons (MNs) and cardiomyocytes. The principal hypothesis is, RA- CRABP1 signalosomes form in specific cellular environments to provide timely (rapid) modulatory mechanisms ensuring homeostatic propagation of intracellular signals that are critical to the survival/function of the cells. Pertinent to CRABP1-CaMKII, our recent data show that CRABP1 is highly expressed in MNs, and CRABP1 knockout (CKO) mice spontaneously develop age-dependent motor deficit resembling motor symptoms of MN degenerative disease Amyotrophic Lateral Sclerosis (ALS), preceded by morphological, structural, and functional deterioration of MNs and neuromuscular junctions (NMJs). We thus engineered a sequenced MN-muscle co-differentiation system on custom-fabricated Hydrogel to generate an in vitro, functional 3D NMJ model for molecular studies. Two aims are to i) dissect molecular mechanisms of RA-CRABP1-CaMKII signalsome action in NMJ and identify CRABP1 ligands specific to this pathway, and ii) determine the physiological action of RA-CRABP1-CaMKII signaling and its disease relevance/therapeutic applications. Aim 1 will employ engineered 3D NMJ model to dissect physiologically relevant signaling pathways and molecular mechanisms. Aim 2 will distinguish “non-canonical” from “canonical” activity of RA by comparing CRABP1 ligands and RA in maintaining healthy NMJs, and determine the therapeutic potential of targeting CRABP1 to improve motor function.