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Mechanism of Enteric Neuropathy

肠神经病变的机制

基本信息

批准号：
9765742
负责人：
Shanthi K Srinivasan
金额：
$ 45.14万
依托单位：
EMORY UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-08-01 至 2023-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9765742
关键词：
Affect Aging Binding Proteins CASP1 gene Caspase Cell Death Cell Line Cell membrane Cholesterol Congenital Megacolon Constipation Data Diabetes Mellitus Diet Dimerization Disease Endotoxins Enteral Enteric Nervous System Gastrointestinal Motility Gene Silencing Genes Goals High Fat Diet Human In Vitro Infection Inflammasome Inflammatory Injury Intake Intestinal Motility Intestines Knowledge Lead Lipopolysaccharides Mediating Membrane Membrane Microdomains Modeling Mus Nerve Degeneration Neuronal Dysfunction Neurons Neuropathy Nitrergic Neurons Nitric Oxide Synthase Type I Palmitates Pathogenesis Pathway interactions Predisposition Production Reactive Oxygen Species Role Saturated Fatty Acids Signal Transduction Sphingolipids Sterility TLR4 gene Testing Toll-like receptors Volatile Fatty Acids Work cell motility experimental study feeding gain of function in vivo in vivo Model inhibitor/antagonist microbial motility disorder neuroinflammation neuron loss new therapeutic target novel overexpression prevent recruit targeted treatment western diet

项目摘要

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.

肠神经元损失是在几种情况下（例如衰老、糖尿病、和慢传输型便秘）。表达神经元型一氧化氮合酶（nNOS）的神经元对正常的胃肠蠕动。nNOS神经元对损伤易感性的机制在很大程度上尚不清楚。饱和脂肪酸（SFA）被掺入细胞膜，诱导脂筏的形成，来自膜结合蛋白如Toll样受体的信号传导。我们的初步研究表明，（i）WD 喂养12周导致氮能肠神经元的损失和结肠运动性的降低，而非无菌或TLR 4-/-小鼠;（ii）较少的氮能神经元与延迟的结肠运动相关;（iii）体外棕榈酸盐和LPS以脂筏依赖的方式增强氮能神经元的损失;（iv）棕榈酸盐和LPS 可导致NLRP 3炎性体和半胱天冬酶-11的活化，随后导致焦亡性氮能 NF-κB的过度激活导致nNOS神经元的丢失。我们假设棕榈酸盐通过脂筏中的TLR 4二聚化增强LPS作用，促进TLR 4信号传导和NFκB 以ROS依赖的方式激活肌间神经元。这导致NLRP 3的激活炎性小体通过经典和非经典途径和随后的氮能肠神经元损伤和结肠动力障碍。为了验证这一假设，我们提出了以下相互关联但独立可实现的目标：具体目标1：确定脂筏和ROS在SFA和肠神经元中的TLR 4/NFκB信号传导。我们将确定是否TLR 4招募到脂筏，必需和足够的SFA/LPS诱导的TLR 4激活和信号，如果这是依赖于ROS 生产使用抑制剂和基因沉默，我们将剖析ROS在SFA介导的TLR 4 NFκB在肠神经元中的活化具体目的2：了解NLRP 3炎性小体在肠神经元中的作用。氮能神经元丢失。我们将确定NLRP 3炎性小体在介导SFA/LPS-1中的关键作用。使用体外和体内模型诱导TLR 4/NFκB活化和肠神经炎症。我们将检查SFA和LPS对NLRP 3炎性体组分寡聚化的影响， caspase-1/caspase-11的活化和焦亡性神经元细胞死亡。在条件性氮能IKK 2-/-，NLRP 3-/-， Caspase 1-/-和Caspase 11-/-小鼠，我们将确定它们对肠神经元和运动的影响，饮食（RD）或WD。对于功能获得性研究，我们将确定诱导型氮能NLRP 3或NLRP 4的作用。使用nNOS-Cre-ERT/Nlrp 3A 350 VneoR小鼠或nNOS-Cre-ERT/Nlrp 3A 350 VneoR小鼠的肠神经元上IKK 2过表达和运动性 ERT/Ikk 2CA小鼠。这些研究将阐明一种新的机制，在发病机制肠神经元以及为预防或治疗胃肠道功能障碍的靶向治疗提供“原理证明”。运动障碍