Project Summary This project is focused on engineering a novel innervated microphysiological system (MPS) of the young and aged brain-gut axis (GBA) in health and disease. The GBA is comprised of neural circuits where the vagus nerve connects enteric nervous system (ENS) neurons that reside in the gut to central nervous system (CNS) neurons, thus allowing for bi-directional communication. There is a need for simplified models of the neural pathways from gut-to-brain where the underlying mechanisms of action in health and disease are not well understood. The vagus nerve, predominantly sensory neurons located in the Nodose Ganglia, transduces dietary cues and CNS function by gut bacteria and has been implicated in neurotransmittable neurodegenerative diseases such as Parkinson’s Disease (PD). Thus, an MPS that captures key components of the human brain-gut axis (GBA), including primary central hind brain neurons, vagal neurons, and enteric neurons, would be a valuable tool for understanding and manipulating pathway function in young and aged tissue and for advancing discovery and therapies via neuromodulation. The approach here describes validation of a laser-fabricated, cut & assembled MPS towards a humanized GBA. Our multidisciplinary team has worked together to (1) establish a prototype GBA-MPS, (2) validate 3D neural compartments within assembled MPS, (3) demonstrate primary human neurosphere sourcing, and (4) create a transgenic model of gut innervating nodose neurons. Two aims will be pursued. Aim 1 will engineer an MPS with real-time sensing of GBA function via live electrophysiology and imaging, followed by time-course and end point characterization of neural function in young and aged populations compared to static and monocultures to ensure appropriate fate and phenotype. Aim 2 will validate the utility of the platform to create a disease model PD phenotype by pre formed fibrils (PFFs) in the enteric neurons of the young and aged MPS, and harness photostimulation of opsin expressing transgeneic nodose neurons, the circuit lynchpin, to evalutate neuromodulation as a method to slow PD spread from the gut to the brain compartment. Successful completion of the first ever MPS that simulates human GBA will accelerate the mechanistic study of brain-gut communication in health and disease and advance therapeutic target discovery by enabling analysis of neural biology and neurodegenerative disorders an accessible and instrumented MPS platform.