Enteric neuronal loss is responsible for intestinal dysmotility in several conditions (e.g. aging, diabetes mellitus, and slow transit constipation). Neuronal nitric oxide synthase (nNOS)-expressing neurons are critical to proper gastrointestinal motility. The mechanism underlying nNOS neuronal susceptibility to injury is largely unknown. Saturated fatty acids (SFA) are incorporated in cell membrane inducing the formation of lipid rafts, that regulate signaling from membrane-bound proteins such as Toll like receptors. Our preliminary studies show that (i) WD feeding for 12 weeks leads to loss of nitrergic enteric neurons and reduction of colonic motility in conventional, but not germ free or TLR4-/- mice; (ii) Less nitrergic neurons correlates with delayed colonic motility; (iii) In vitro palmitate and LPS enhance nitrergic neuronal loss in a lipid rafts dependent fashion; (iv) Palmitate and LPS can lead to activation of NLRP3 inflammasome and caspase-11, and subsequently pyroptotic nitrergic neuronal loss; NFκB over activation contributes to nNOS neuronal loss. We hypothesize that palmitate enhance LPS action through the TLR4 dimerization in lipid rafts, facilitating TLR4 signaling and NFκB activation in myenteric neurons in an ROS dependent fashion. This leads to activation of NLRP3 inflammasomes through canonical and non-canonical pathways and subsequent nitrergic enteric neuronal damage and colonic dysmotility. To test this hypothesis, we propose the following inter-related but independently achievable aims: Specific Aim 1: To determine the role of lipid rafts and ROS in SFA and TLR4/NFκB signaling in enteric neurons. We will determine whether TLR4 recruitment into lipid rafts is necessary and sufficient for SFA/LPS-induced TLR4 activation and signaling and if this is dependent on ROS production. Using inhibitors and gene silencing we will dissect out the role of ROS in SFA-mediated TLR4 activation of NFκB in enteric neurons Specific Aim 2: To understand the role of NLRP3 inflammasomes in nitrergic neuronal loss. We will establish the critical role of NLRP3 inflammasomes in mediating SFA/LPS- induced TLR4/NFκB activation and enteric neuroinflammation using both In vitro and in-vivo models. We will examine the effect of SFA and LPS on oligomerization of NLRP3 inflammasome components leading to activation of caspase-1/caspase-11 and pyroptotic neuronal cell death. In conditional nitrergic IKK2-/-, NLRP3-/-, Caspase 1-/- and Caspase 11-/- mice, we will determine their effects on enteric neurons and motility fed a regular diet (RD) or WD. For the gain-of-function studies, we will determine the effect of inducible nitrergic NLRP3 or IKK2 overexpression on enteric neurons and motility using nNOS-Cre-ERT/Nlrp3A350VneoR mice or nNOS-Cre- ERT/Ikk2CA mice. These studies will elucidate a novel mechanism in the pathogenesis of enteric neuronal dysfunction as well as provide “proof of principle” for targeted therapies to prevent or treat gastrointestinal ...