A Drosophila Model of Neuronal MECP2 Function

甲基CpG结合蛋白2(MECP2)是一种广泛存在于中枢神经系统的多功能基因表达调控因子。在人类中，MECP2的功能缺失和功能获得突变都会导致智力低下和运动障碍，被归类为Rett综合征(RTT，功能丧失)或MECP2重复综合征(MDS，功能获得)。目前还没有针对这些疾病的有效治疗方法。虽然经典的已知MeCP2通过与甲基化的DNA结合来抑制转录，但许多其他的基因调节功能已经被确定。在细胞水平上，MeCP2的错误调控会导致突触和树突的缺陷。MeCP2在神经元中的功能研究主要依赖于MeCP2缺失、突变和过度表达的小鼠模型，但在小鼠模型中进行大规模的筛选和体内验证是耗时的。本研究的目的是以果蝇为模型系统，研究MECP2在神经元中的特定细胞和分子功能。果蝇基因组的甲基化程度很低，因此为专门研究hMECP2的非甲基DNA结合功能提供了独特的机会。我们已经证明，人MECP2(HMECP2)在果蝇运动神经元中的表达会导致树突结构和运动行为的明显缺陷，正如MeCP2在人类和小鼠中的功能获得一样。这些效应依赖于特定的MECP2蛋白结构域，可以通过基因相互作用实验来挽救；因此，它们是由hMECP2功能获得而不是一般毒性引起的。我们利用RNA测序技术在果蝇中鉴定了45个与hMECP2表达有差异调控的基因和120个转录异构体。Kibra是一种与人类学习和记忆相关的基因，在这张屏幕上被确定为首要目标。Kibra还激活河马激活级联(HKC)，这是一条参与树突生长调节的途径。初步研究表明，Kibra基因的敲除和hMECP2的共表达可以挽救树突缺陷。第一个目标将检验MECP2诱导的树突状细胞和行为缺陷依赖于Kibra上调的假设。我们将定量证实Kibra基因的敲除是否可以挽救hMECP2在果蝇中表达的树突和行为效应。此外，我们还将在小鼠原代神经元培养中进行实验，以确定这些机制在哺乳动物神经元中是否保守。第二个目的将检验hMECP2增加HKC激活的假设，以及这种激活介导了hMECP2表达所观察到的树突状和行为缺陷。研究这些在果蝇中发现的新的MECP2靶点有助于阐明MeCP2的破坏是如何导致神经系统和行为缺陷的，并最终可能导致未来RTT和MDS的治疗策略。

Methyl-CpG binding protein 2 (MECP2) is a widely abundant, multifunctional regulator of gene expression in the CNS. In humans, both loss- and gain-of-function mutations of MECP2 cause mental retardation and motor dysfunction classified as either Rett Syndrome (RTT, loss-of-function) or MECP2 Duplication Syndrome (MDS, gain-of-function). There are currently no effective treatments for these conditions. While Mecp2 is classically known to repress transcription by binding to methylated DNA, many additional gene regulatory functions have been identified. At the cellular level, mis-regulation of Mecp2 leads to synaptic and dendritic defects. Research on Mecp2 function in neurons has relied primarily on mouse models of Mecp2 deletion, mutation, and over-expression, but large-scale screening and in vivo validation in the mouse model is time consuming.The objective of this proposal is to use Drosophila melanogaster as a model system to study specific cellular and molecular functions of MECP2 gain-of-function in neurons. The Drosophila genome is sparsely methylated and therefore provides a unique opportunity to specifically examine non-methyl DNA binding functions of hMECP2. We have shown that expression of human MECP2 (hMECP2) in Drosophila motoneurons leads to distinct defects in dendritic structure and motor behavior, as reported with Mecp2 gain-of-function in humans and mice. These effects are dependent on specific MECP2 protein domains and can be rescued by genetic interaction experiments; hence, they are caused by gain of hMECP2 function and not general toxicity. We used RNA-sequencing technology to identify 45 genes and 120 transcript isoforms differentially regulated with hMECP2 expression in Drosophila. Kibra, a gene associated with learning and memory in humans, was identified as a top target from this screen. Kibra also activates the Hippo kinase cascade (HKC), a pathway involved in the regulation of dendritic growth. Pilot studies indicate that knockdown of kibra together with co-expression of hMECP2 rescue the dendritic defects. The first aim will test the hypothesis that MECP2-induced dendritic and behavioral defects depend on up-regulation of kibra. We will confirm quantitatively if knockdown of kibra can rescue the dendritic and behavioral effects of hMECP2 expression in Drosophila. We will additionally conduct experiments in mouse primary neuron cultures to determine whether these mechanisms are conserved in mammalian neurons. The second aim will test the hypothesis that hMECP2 increases activation of the HKC, and that this activation mediates the dendritic and behavioral defects observed with hMECP2 expression. Investigating these novel MECP2 targets identified in Drosophila can help elucidate how disruption of MeCP2 leads to nervous system and behavioral defects and can ultimately lead to future therapeutic strategies for RTT and MDS.