Neuroregeneration in the Enteric Nervous System

 描述（由申请方提供）：这些研究的总体目标是了解肠神经再生的机制，以开发针对肠神经系统（ENS）内源性再增殖的治疗方法，用于治疗肠神经病。肠神经病变的特征在于肠神经元的损伤或丧失，其导致许多消化系统疾病，包括特发性胃轻瘫、慢性肠假性梗阻（锡波）、先天性巨结肠、恰加斯病、贲门失弛缓症和可能的慢传输型便秘。现有的肠神经病动物模型，即，遗传性无神经节细胞症的特征在于包括神经元和神经胶质的神经嵴细胞的完全丧失。这种完全丧失，虽然重演了严重的先天性人类肠神经病，但与人类中的大多数肠神经病相反，人类中的大多数肠神经病的特征在于不太严重的神经元丧失，并且重要的是基本上排除了神经嵴前体的内源性神经再生。为了克服这一局限性，我们开发了一种新的低剂量苯扎氯铵（BAC）模型，而不是传统的高剂量苯扎氯铵（BAC）模型，在小鼠小肠中诱导50%的神经元损失和强大的神经再生。它是迄今为止唯一一个证明了强大的内源性神经再生的动物模型。使用这种新的转基因小鼠品系和其他体外方法的模型，我们将测试的整体假设，即肠神经丛的再生介导的转分化的肠神经胶质细胞通过SRY相关的同源框转录因子2（SOX 2）依赖性机制的神经元。我们的总体假设将通过针对两个具体目标的实验进行检验。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。这一目标将通过测试三个假设来实现：1.1）新的神经元来源于表达胶质细胞酸性蛋白（GFAP）的肠细胞; 1.2）胶质细胞直接转分化为神经元;以及1.3来源于肠胶质细胞的神经元是功能性和多样性的。肠神经胶质细胞作为肠神经元的可操纵内源性来源将是一个重大的进步，因为神经胶质细胞在ENS中的数量超过神经元4：1，并通过组成性胶质细胞生成不断补充。具体目标2将确定有助于肠神经再生的信号通路。这一目标将通过测试三个假设来实现：2.1）神经胶质中的SOX 2表达是产生新神经元所必需的且足够的; 2.2）骨形态发生蛋白2（BMP 2）诱导肠神经胶质细胞中的SOX 2表达;以及2.3）SOX 2通过去除RE 1沉默转录因子（REST）介导的神经基因抑制将肠神经胶质重编程为神经元。所提出的研究结果，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学和分子表征，将提供一个新的研究方向。 本发明提供了对肠神经再生的机制理解，并为治疗肠神经病的新治疗方法提供了基础。

 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.