MicroRNA mechanisms of Rett Syndrome

 描述（由申请人提供）：雷特综合症（RTT）是一种破坏性的神经发育障碍，也是已知的导致女孩自闭症的主要遗传原因。 X连锁基因MECP2（甲基-CpG结合蛋白2）的突变占绝大多数RTT病例。 MECP2 的神经生物学对于理解 RTT 机制和确定该疾病的治疗方法至关重要。 MeCP2 是一种基因表达的表观遗传调节剂，最近被证明与 microRNA 机制显着相互作用；这些相互作用是 MeCP2 机制的核心。多种证据表明 MeCP2 在大脑发育的连续阶段中发挥作用，包括产前神经发生、产后连接和功能发育以及经验依赖性突触可塑性。我们假设 MeCP2 的多效性作用是通过一组影响神经发生的早期调控 miRNA 在产前发育中介导的；在出生后发育过程中，通过一组不同的 miRNA 调节胰岛素样生长因子 1 (IGF1) 信号传导；在发育后期通过第三组 miRNA 影响突触功能和可塑性。该提案的目标是采用尖端的miRNA方法，结合干细胞、行为、双光子成像和靶向电生理学方法，揭示MeCP2相关miRNA在不同发育阶段的功能。在目标 1 中，我们将使用同基因人类 RTT 模型细胞系（目标 1a）、3-D 脑类器官（目标 1b）和小鼠模型（目标 1c）来研究 MeCP2 和下游 miRNA 介导的通路在产前神经发生中的作用。迄今为止，我们的研究结果表明 miR-199 和 -214 与 MeCP2 缺陷导致的产前神经发生的异常调节有关。我们将分析这些miRNA的功能机制和下游分子通路。在目标 2 中，我们将确定出生后 MeCP2 调节的 miRNA 对 IGF1 信号传导的影响及其在 RTT 治疗中的潜在作用。我们将在 Mecp2 缺陷小鼠中检查 LIN28a 和 BDNF 下游 miRNA let-7 家族的调节情况，以及它们调节 IGF1 表达的能力（目标 2a）。我们将研究使用 ß2 肾上腺素受体激动剂克伦特罗使分子改变水平正常化是否可以对 Mecp2 缺陷小鼠的生存和一系列表型产生积极影响（目标 2b），以及克伦特罗和 IGF1 之间的协同相互作用作为 RTT 的基于机制的有效联合疗法（目标 2c）。在目标 3 中，我们将研究 MeCP2 和晚期表达的 miRNA（例如 miR-132）在调节经验依赖性皮质可塑性中的作用。我们将确定恢复 Mecp2 突变小鼠视觉皮层中 miR-132 的表达是否可以恢复眼睛优势可塑性的正常年龄依赖性成熟（目标 3a）。我们还将检查 IGF1（以及随后的克伦特罗以及克伦特罗和 IGF1 的组合）是否上调 miR-132 表达，并可以通过其下游机制影响可塑性（目标 3b）。

 DESCRIPTION (provided by applicant): Rett Syndrome (RTT) is a devastating neurodevelopmental disorder and the leading known genetic cause of autism in girls. Mutations in the X-linked gene MECP2 (methyl-CpG binding protein 2) account for the vast majority of RTT cases. The neurobiology of MECP2 is fundamental to understanding the mechanisms of RTT and identifying therapeutics for the disorder. MeCP2 is an epigenetic modulator of gene expression that has recently been shown to interact significantly with microRNA machinery; these interactions are at the core of MeCP2 mechanisms. Multiple lines of evidence point to a role for MeCP2 in successive stages of brain development, including prenatal neurogenesis, postnatal development of connections and function, and experience-dependent synaptic plasticity. We hypothesize that the pleiotropic effects of MeCP2 are mediated in prenatal development via a set of early regulated miRNAs that influence neurogenesis; during postnatal development through a different set of miRNAs that regulate Insulin-like growth factor 1 (IGF1) signaling; and in late development into adulthood via a third set of miRNAs that influence synaptic function and plasticity. The goal of this proposal is to employ cutting-edge miRNA methodologies, in combination with stem cell, behavioral, two-photon imaging, and targeted electrophysiological approaches, to reveal the function of MeCP2-related miRNAs at different developmental stages. In aim 1, we will examine the role of MeCP2 and downstream miRNA-mediated pathways in prenatal neurogenesis, using isogenic human RTT model cell lines (aim 1a), 3-D cerebral organoids (aim 1b), and mouse models (aim 1c). Our findings to date implicate miR-199 and -214 in the aberrant regulation of prenatal neurogenesis as a result of MeCP2 deficiency; we will analyze the functional mechanisms and molecular pathways downstream of these miRNAs. In aim 2, we will determine the influence of postnatal MeCP2-regulated miRNAs on IGF1 signaling, and their potential role in RTT therapeutics. We will examine the regulation of LIN28a and the let-7 family of miRNAs downstream of BDNF, and their ability to regulate IGF1 expression, in Mecp2 deficient mice (aim 2a). We will investigate whether normalizing the levels of molecular alterations using the ß2 adrenergic receptor agonist clenbuterol can positively impact survival and a range of phenotypes in Mecp2 deficient mice (aim 2b), along with synergistic interactions between clenbuterol and IGF1 as a potent mechanism-based combination therapeutic for RTT (aim 2c). In aim 3, we will examine the role of MeCP2 and late-expressed miRNAs such as miR-132 in regulating experience-dependent cortical plasticity. We will determine whether restoring expression of miR-132 in the visual cortex of Mecp2 mutant mice can restore normal age-dependent maturation of ocular dominance plasticity (aim 3a). We will also examine whether IGF1 (and subsequently, clenbuterol and the combination of clenbuterol and IGF1) upregulates miR-132 expression, and can act through its downstream mechanisms to influence plasticity (aim 3b).