MOLECULAR PATHOGENESIS STUDIES OF RETT SYNDROME

项目摘要 Rett综合征(RTT)是一种出生后发病的严重神经发育障碍。因功能丧失所致 X连锁基因甲基CpG结合蛋白2(MECP2)突变，患有经典型RTT的女孩 通常为生命的头~12-18个月，达到预期的里程碑，但随后失去获得性 技能，而不是形成刻板印象，共济失调，失用，癫痫发作和许多其他 症状。主要减少MeCP2蛋白产量的较温和的突变会导致较轻微的突变 症状需要更长时间才能显现。RTT的各种小鼠模型(雄性MeCP2空白鼠，雌性 杂合子，携带特定致病突变的小鼠)忠实地复制了这一自然历史 (正常健康、退化和恶化)。令人惊讶的是，成年后该基因的缺失会复制完整的 表型，而MeCP2在成年空白小鼠中的表达挽救了它，表明MeCP2具有一定的AS- 尚未指明的对神经元维持至关重要的功能。尽管基因疗法似乎是最好的 希望，MeCP2在女性中的嵌合性(~50%的神经元将具有野生型MeCP2)呈现出一个困难 这一方法面临挑战，因为过量的MeCP2也会导致疾病(MECP2重复综合征[MDS]， 这完全是毁灭性的)。因此，在我们寻找可行的治疗方案时，我们遵循两个原则 原则：早期正常发育期为干预和拖延提供了机会之窗 发病，以及MeCP2丢失和症状出现(或症状逆转，在上下文中)之间的滞后 拯救MDS)意味着有一连串的分子事件，如果我们能绘制它们的话，应该会允许我们 确定可作为治疗靶点和治疗反应生物标志物的关键基因或途径。 在先前发现穹隆深部脑刺激改善RTT小鼠的学习和记忆后，我们 假设症状前期的运动训练也可能刺激神经回路 并延迟症状的出现。事实上，事实证明是这样的。我们现在试图追踪分子的级联 以及在MeCP2耗尽之后但在症状出现之前发生的细胞变化，以定义 当我们通过强化训练或提高刺激大脑时，RTT的发病机制和反应的生物标志物 MeCP2水平在亚型突变的背景下。在我们的第一个目标中，我们将确定转录 发生在特定任务神经元中的变化，这些神经元对高强度训练做出反应，功能得到改善， 电生理学和形态学。在目标2中，我们将追踪分子、表观遗传和细胞的级联。 急性MeCP2耗尽后几周内的变化，最终导致神经系统 功能障碍。在目标3中，我们将在两个不同的RTT小鼠模型中上调MeCP2水平，每个模型都携带 降低MeCP2水平的突变，以确定改善的程度。

Project Summary Rett syndrome (RTT) is a severe neurodevelopmental disorder with postnatal onset. Caused by loss-of-function mutations in the X-linked gene methyl CpG binding protein 2 (MECP2), girls with classic RTT develop normally for the first ~12-18 months of life, reaching the expected milestones, but then lose their acquired skills over a period of weeks and instead develop stereotypies, ataxia, apraxia, seizures, and a host of other symptoms. Milder mutations that primarily reduce the amount of MeCP2 protein produced cause milder symptoms that take longer to manifest. Various mouse models of RTT (male Mecp2 nulls, female heterozygotes, mice bearing specific disease-causing mutations) faithfully reproduce this natural history (normal health, regression, and deterioration). Strikingly, deletion of the gene in adulthood reproduces the full phenotype, whereas expression of Mecp2 in adult null mice rescues it, demonstrating that MeCP2 has some as- yet-unspecified function that is critical for neuronal maintenance. Although gene therapy would seem the best hope, the mosaic nature of MeCP2 in females (~50% of neurons will have wild-type MeCP2) presents a difficult challenge for this approach, as too much MeCP2 also causes disease (MECP2 duplication syndrome [MDS], which is every bit as devastating). Therefore, in our search for viable therapeutic options, we are guided by two principles: the period of early normal development provides a window of opportunity to intervene and delay onset, and the lag between loss of MeCp2 and appearance of symptoms (or reversal of symptoms, in the context of rescuing MDS) means there is a cascade of molecular events that, if we could but map them, should allow us to identify key genes or pathways that could serve as therapeutic targets and biomarkers of treatment response. Having previously found that forniceal deep brain stimulation improves learning and memory in RTT mice, we hypothesized that motor training during the presymptomatic period might also stimulate the neural circuits and delay symptom onset. Indeed, this proved to be the case. We now seek to trace the cascade of molecular and cellular changes that occur after MeCP2 depletion but before the onset of symptoms to define initiators of RTT pathogenesis and biomarkers of response as we stimulate the brain through intensive training or raise MeCP2 levels in the context of hypomorphic mutations. In our first aim, we will identify the transcriptional changes that take place in task-specific neurons that respond to intensive training with improved function, electrophysiology, and morphology. In aim 2, we will trace the cascade of molecular, epigenetic, and cellular changes that follow for several weeks after acute MeCP2 depletion, and that eventually lead to neurological dysfunction. In aim 3, we will upregulate MeCP2 levels in two distinct RTT mouse models, each carrying mutations that reduce MeCP2 levels, to determine the extent of improvement.