Enteric neuronal loss is responsible for intestinal dysmotility in significant clinical conditions including aging, diabetes mellitus, and constipation. The overarching goal of this renewal application is to further delineate the neuronal degeneration pathway triggered by a Western fat diet (WD). Preliminary data demonstrate that primary mouse enteric neurons and isolated human enteric neurons exposed to PA have increased cellular iron levels and increased ferritin expression. PA-induced ferroptosis is prevented by the ferroptosis inhibitor, ferrostatin 1. Free iron catalyzes the increase in neuronal ROS and membrane lipid peroxidation. WD-fed mice have neuronal loss and delayed colonic motility have increased ferritin expression in myenteric ganglia. Thus, we hypothesize that WD-induced colonic neurodegeneration and resultant colonic dysmotility occur from increased intracellular labile iron that leads to enteric neuron ferroptosis through enhanced mitochondrial stress and impaired mitochondrial function. We will perform in vitro studies in primary mouse enteric neurons, an enteric neuronal cell line (IM-FEN) and isolated human ganglion networks, and in vivo in the WD-fed mouse model and ob/ob mice. To test this hypothesis, we propose the following Specific Aims: Specific Aim 1: To establish the mechanistic role of excess iron in SFA/WD-induced enteric neuronal loss. Using primary mouse enteric neuronal cultures and enteric neuronal cell lines, we will demonstrate that SFA increase β-oxidation and expression of iron transporters (Tfr1, DMT1 and ZIP14) resulting in elevated iron uptake and intracellular labile iron levels leading to ferroptosis. Mice fed a WD and ob/ob mice will be assessed for enteric neuronal ferritin expression and ferroptosis. The necessity of iron in enteric neuronal loss will be assessed by determining if enteric neuronal specific knockdown of Tfr1 in WD-fed mice prevent the WD-induced neuronal loss. Specific Aim 2: To determine that increased mitochondrial iron triggers ROS production leading to mitochondrial dysfunction and SFA/WD-induced enteric neuronal degeneration. Our preliminary data demonstrate that SFA increase mitochondrial iron and ROS and trigger mitochondrial dysfunction. We will determine the effects of SFA on mitochondrial ROS, function, structure, mass and dynamics in enteric neurons. We will determine whether MitoQ (an inhibitor of mitochondrial ROS) and DFP (a mitochondrial iron chelator) prevent SFA-induced enteric neuronal cell death. A novel AAV targeting enteric neurons will deliver the antioxidant Nfe2l2 (Nrf2) to mice to see if preserving mitochondrial function prevents neuronal ferroptosis. Specific Aim 3: To demonstrate the translational relevance of iron overload in isolated human myenteric ganglia and in colonic myenteric ganglia of obese individuals. The clinical relevance of the mouse findings will be validated in human studies. Using freshly isolated networks of human myenteric ganglia from l...