Mechanism of Enteric Neuropathy

肠神经元丢失在几种情况下导致肠道运动障碍(如衰老、糖尿病、 和慢传输性便秘)。神经元型一氧化氮合酶(NNOS)表达的神经元对于 胃肠动力。NNOS神经元损伤易感性的机制在很大程度上是未知的。 饱和脂肪酸(SFA)结合在细胞膜上，诱导脂筏的形成，调节 来自膜结合蛋白的信号，如Toll样受体。我们的初步研究表明：(I)WD 饲喂12周可导致大鼠肠道氮能神经元的丧失和结肠动力的降低， 但不是无菌或TLR4-/-小鼠；(Ii)氮能神经元较少与结肠运动延迟有关；(Iii)在体外 棕榈酸酯和脂多糖以脂筏依赖的方式增加氮能神经元的丢失；(Iv)棕榈酸酯和脂多糖 可导致NLRP3炎症体和caspase-11的激活，从而导致焦性氮能 神经元丢失；核因子κB的过度激活导致神经元丢失。我们假设棕榈树 脂筏中TLR4二聚化增强内毒素作用，促进TLR4信号转导和核因子κB 肌间神经元以ROS依赖的方式激活。这会导致NLRP3的激活 炎性小体通过典型和非典型途径以及随后的氮能肠神经元 损伤和结肠动力障碍。为了检验这一假设，我们提出了以下相互关联但 可独立实现的目标：具体目标1：确定脂筏和ROS在SFA和 肠神经细胞中TLR4/NFκB信号转导。我们将确定TLR4在脂筏中的募集是否 SFA/LPS诱导的TLR4激活和信号传递的必要条件和充分条件，以及这是否依赖于ROS 制作。通过使用抑制剂和基因沉默，我们将剖析ROS在SFA介导的TLR4中的作用 NLRP3炎性小体在肠神经元特异性κB激活中的作用 氮能神经元丢失。我们将确定NLRP3炎性小体在介导SFA/内毒素- 用体外和体内模型诱导TLR4/NFκB活化和肠神经炎。我们会 检测SFA和内毒素对NLRP3炎性小体成分齐聚的影响 Caspase-1/caspase-11的激活与焦虑性神经细胞死亡在条件性氮能IKK2-/-、NLRP3-/-中， Caspase1-/-和Caspase11-/-小鼠，我们将测定它们对肠道神经元和运动功能的影响 饮食(RD)或WD。对于功能增益研究，我们将确定诱导性氮能NLRP3或 NNOS-CRE-ERT/Nlrp3A350VneR小鼠或nNOS-CRE-ERT/Nlrp3A350VneR小鼠和nNOS-Cre-ERT/Nlrp3A350VneR小鼠肠上皮细胞IKK2过表达及运动 ERT/Ikk2CA小鼠。这些研究将阐明肠神经细胞发病的新机制。 以及为预防或治疗胃肠道疾病的靶向治疗提供“原则证明” 运动性障碍。

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 motility disorders.