MicroRNA mechanisms of Rett Syndrome

 描述(申请人提供)：Rett综合征(RTT)是一种毁灭性的神经发育障碍，是女孩自闭症的主要已知遗传原因。X连锁基因MECP2(甲基CpG结合蛋白2)突变占RTT病例的绝大多数。MECP2的神经生物学对于了解RTT的机制和确定治疗该疾病的方法是至关重要的。MeCP2是一种表观遗传基因表达调节剂，最近被证明与microRNA机制显著相互作用；这些相互作用是MeCP2机制的核心。多条证据表明，MeCP2在大脑发育的连续阶段中发挥作用，包括出生前神经发生、出生后连接和功能的发展，以及经验依赖的突触可塑性。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节的miRNAs在产前发育中介导的；在出生后发育期间通过一组不同的miRNAs调节胰岛素样生长因子1(IGF1)信号；在成年后期通过第三组miRNAs影响突触功能和可塑性。该提案的目标是利用尖端的miRNA方法，结合干细胞、行为学、双光子成像和靶向电生理方法，揭示MeCP2相关miRNAs在不同发育阶段的功能。在目标1中，我们将使用同基因的人RTT模型细胞系(目标1a)、三维脑器官(目标1b)和小鼠模型(目标1c)来研究MeCP2及其下游miRNA介导的通路在产前神经发生中的作用。到目前为止，我们的发现表明miR-199和-214参与了由于MeCP2缺乏而导致的产前神经发生的异常调控；我们将分析这些miRNAs下游的作用机制和分子通路。在目标2中，我们将确定出生后MeCP2调节的miRNAs对IGF1信号转导的影响，以及它们在RTT治疗中的潜在作用。我们将在MeCP2缺陷小鼠中检测Lin28a和BDNF下游的let-7家族miRNAs的调节，以及它们调节IGF1表达的能力(目标2a)。我们将研究，在MeCP2基因缺陷小鼠中，使用2肾上腺素能受体激动剂克伦特罗使分子改变水平正常化是否能积极影响小鼠的存活率和一系列表型(目标2b)，以及作为治疗RTT的一种有效的基于机制的组合(目标2c)，克伦特罗和IGF1之间的协同作用。在目标3中，我们将研究MeCP2和后期表达的miRNAs(如miR-132)在调节经验依赖的皮质可塑性中的作用。我们将确定在MeCP2突变小鼠的视皮层中恢复miR-132的表达是否可以恢复正常的眼优势可塑性的年龄依赖成熟(目标3a)。我们还将研究IGF1(以及随后的克伦特罗和克伦特罗和IGF1的组合)是否上调miR-132的表达，并是否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).