Neuroregeneration in the Enteric Nervous System

 描述(申请人提供)：这些研究的总体目标是了解肠神经再生的机制，以便开发针对肠神经系统(ENS)内源性再繁殖的治疗方法来治疗肠神经病变。肠神经病变可导致多种消化系统疾病，包括特发性胃轻瘫、慢性假性肠梗阻(CIPO)、先天性巨结肠、查加斯病、失弛缓症以及可能的慢传输型便秘，其特征是肠神经元受损或丢失。现有的肠道神经病动物模型，即遗传性无神经节细胞病，其特点是包括神经元和神经胶质细胞在内的神经脊细胞完全丧失。虽然这种完全丧失是严重的先天性人类肠道神经病，但与人类大多数肠道神经病不同，大多数肠道神经病的特点是神经元丢失不那么严重，而且重要的是，基本上排除了神经脊前体细胞的内源性神经再生。克服了这一局限性，我们开发了一种新的低剂量，而不是传统的高剂量苯扎氯铵(BAC)在小鼠小肠模型，以诱导50%的神经元损失，并强大的神经再生。这是迄今为止唯一一个显示出强大的内源性神经再生的动物模型。利用该模型和新的转基因小鼠品系和其他体外方法，我们将检验肌间神经丛的再生是通过SRY相关的同源盒转录因子2(SOX2)依赖的机制将肠神经胶质细胞转分化为神经元所介导的总体假设。我们的总体假设将通过针对两个特定目标的实验来检验。具体目标1将确定BAC治疗后新神经元的来源和功能，因为目前，再生神经元的细胞起源和功能尚不清楚。这一目标将通过检验三个假设来实现：1.1)表达胶质纤维酸性蛋白(GFAP)的肠道细胞衍生的新神经元；1.2)胶质细胞直接转分化为神经元；1.3来自肠道胶质细胞的神经元具有功能和多样性。肠神经胶质细胞作为肠神经元内可操纵的内源性来源将是一个重大的进步，因为在ENS中，神经胶质细胞的数量比神经元多4：1，并且不断地由结构性胶质生成补充。具体目标2将确定促进肠神经再生的信号通路。这一目标将通过检验三个假设来实现：2.1)SOX2在胶质细胞中的表达是产生新神经元的必要条件和充分条件；2.2)骨形态发生蛋白2(BMP2)诱导肠神经胶质细胞表达SOX2；以及2.3)SOX2通过去除Re1沉默转录因子(REST)介导的神经基因抑制而将肠神经胶质细胞重新编程为神经元。拟议的研究结果，包括用于遗传谱系追踪、克隆分析和分子靶向的新型转基因小鼠品系的形态和分子特征，将提供 从机制上理解肠神经再生，并为肠神经病变的治疗提供新的治疗方法的基础。

 DESCRIPTION (provided by applicant): The overall goal of these studies is to understand the mechanisms of enteric neuroregeneration in order to develop therapies targeted toward endogenous repopulation of the enteric nervous system (ENS) for the treatment of enteric neuropathy. Enteric neuropathy, which contribute to numerous digestive diseases including idiopathic gastroparesis, chronic intestinal pseudoobstruction (CIPO) Hirshsprung's disease, Chagas' disease, achalasia, and possibly slow transit constipation, are characterized by damage or loss of enteric neurons. Existing animal models of enteric neuropathy, i.e., genetic aganglionosis are characterized by complete loss of neural crest cells including neurons and glia. This complete loss, while recapitulating severe congenital human enteric neuropathy, is in contrast to most enteric neuropathy in humans, which are characterized by less severe neuronal loss, and importantly essentially precludes endogenous neural regeneration by neural crest precursors. Overcoming this limitation, we developed a novel low-dose, instead of the traditional high-dose, benzalkonium chloride (BAC) model in the murine small intestine to induce a loss of 50% of neurons, and robust neuroregeneration. It is the only animal model to date that demonstrates robust endogenous neuroregeneration. Using this model with novel transgenic mouse strains and additional in vitro approaches, we will test the overall hypothesis that regeneration of the myenteric plexus is mediated by transdifferentiation of enteric glia to neurons via a SRY-related homeobox transcription factor 2 (SOX2)- dependent mechanism. Our overall hypothesis will be tested by experiments directed at two specific aims. Specific Aim 1 will determine the source and functionality of new neurons following BAC treatment because currently, the cellular origin and function of regenerating neurons are not understood. This aim will be met by testing three hypotheses: 1.1) new neurons derive from enteric cells that express glial fibrillary acidic protein (GFAP); 1.2) glia directly transdifferentiate into neurons; and 1.3 neurons derived from enteric glia are functional and diverse. Enteric glia as a manipulable endogenous source of enteric neurons would be a significant advance because glia outnumber neurons 4:1 in the ENS and are continually replenished by constitutive gliogenesis. Specific Aim 2 will determine the signaling pathways that contribute to enteric neuroregeneration. This aim will be met by testing three hypotheses: 2.1) SOX2 expression in glia is necessary and sufficient to generate new neurons; 2.2) bone morphogenic protein 2 (BMP2) induces SOX2 expression in enteric glial cells; and 2.3) SOX2 reprograms enteric glia to neurons by removing RE1-silencing transcription factor (REST)-mediated repression of neural genes. Results of the proposed studies, involving morphological and molecular characterization of novel transgenic mouse strains for genetic lineage tracing, clonal analysis, and molecular targeting, will provide a mechanistic understanding of enteric neuroregeneration and provide the basis for novel therapeutic approaches for the treatment of enteric neuropathy.