Normalizing E:I imbalance in Rett Syndrome by Modulation of Late Response Genes

项目总结和职业发展摘要 Rett综合征(RTT)是一种破坏性的神经发育障碍，目前尚无治疗方法 可行的治疗策略。大多数RTT病例是由于转录中的功能突变丢失所致 被称为甲基CpG结合蛋白2(MECP2)的因子和MeCP2(MeCP2-KO)的敲除导致RTT- 就像啮齿动物的表型和病理一样。RTT模型小鼠的大脑以获得性 小头畸形，不是由神经萎缩引起的，而是突触减少和随后的 神经节的简化。因此，临床前治疗策略获得了最多的 注意是那些旨在使突触密度正常化的东西，通常是通过替换晚期反应来实现的 (LR)或对其受体的直接调制。在这种能力下，RTT-鼠标模型是指导 治疗设计；然而，考虑到将数据从小鼠转换为人类的相关挑战，我们 最近采取了一种不同的策略。利用8名RTT患者的皮质和小脑组织，我们 进行RNA测序(SEQ)分析以筛选植根于人类的新的干预点 患者数据，然后在小鼠身上反向模拟相关的命中。令人兴奋的是，我们发现了毒碱类的乙酰胆碱 受体4(M4)基因在RTT患者中显著降低。这一发现是显著的，因为M4-调节器 已被证明可以挽救精神分裂症等与RTT和M4重叠的疾病的症状域- KO小鼠的认知和社会缺陷与MeCP2-KO小鼠相似。这增加了一种令人兴奋的可能性 M4表达和信号的减弱可能是RTT患者潜在的认知和社会表型。 在目标1中，我们建议进一步开发初步数据，表明认知和社会表型在 MeCP2-KO小鼠对M4阳性变构调节剂(PAM)有反应。在目标2中，我们建议 通过对来自中国的尸检样本进行RNA-SEQ分析，最终将M4信号与患者的RTT联系起来 已被临床诊断为RTT，但没有MECP2(MECP2-)突变的患者 突变阴性)。最后，在目标3中，我们建议将我们的M4-PAM与谷氨酸能调节剂联合给药 和GABA能信号，以确定是否可以挽救一个互补的症状域。 我的职业目标是成为一个实验室的负责人，专注于儿童疾病的研究 神经系统，我已经确定了四个训练不足的领域，我建议补救以帮助 在这一角色上取得长期成功。这四个领域是：1)电生理学培训2)接下来的培训 世代测序分析3)临床接触RTT和4)导师经验。要删除这些文件，请执行以下操作 技术和概念障碍，我已经制定了一项培训计划，将正式的说教培训与 亲力亲为的指导，目标是将这些弱点转化为优势。预计该项目的完成 拟议的项目和培训计划将使我处于获得终身教职的理想位置 因此，我相信这份申请非常适合K01职业发展奖。

PROJECT SUMMARY AND CAREER DEVELOPMENT ABSTRACT Rett syndrome (RTT) is a devastating neurodevelopmental disorder for which there are currently no viable treatment strategies. The majority of RTT cases result from loss of function mutations in a transcription factor known as methyl CpG binding protein 2 (MECP2), and knockout of Mecp2 (Mecp2-KO) results in RTT- like phenotypes and pathologies in rodents. The brains of RTT-model mice are characterized by acquired microcephaly, which results not from neural atrophy, but rather a reduction in synapses and the subsequent simplification of the neuronal arbor. As such, the preclinical treatment strategies that have garnered the most attention are those designed to normalize synaptic density, often through the replacement of late response (LR) or direct modulation of their receptors. In this capacity RTT-mouse models are valuable tools to guide therapeutic design; however, given the challenges associated with translating data from mice to humans, we have recently adopted a different strategy. Using cortical and cerebellar tissue from 8 RTT patients, we performed RNA-sequencing (seq) analysis to screen for novel points of intervention that are rooted in human patient data, and then back-modeled relevant hits in mice. Excitingly, we identified the muscarinic acetylcholine receptor 4 (M4) as a gene significantly decreased in RTT patients. This finding is salient, as M4-modulators have been shown to rescue symptom domains in diseases like schizophrenia that overlap with RTT and M4- KO mice have cognitive and social deficits similar to Mecp2-KO mice. This raises the exciting possibility that attenuated M4 expression and signaling could be underlying cognitive and social phenotypes in RTT patients. In Aim 1, we propose to further develop preliminary data indicating that cognitive and social phenotypes in Mecp2-KO mice are responsive to M4 positive allosteric modulators (PAMs). In Aim 2, we propose to conclusively link M4-signaling to RTT in patients by perform an RNA-seq analysis on autopsy samples from patients that have been clinically diagnosed with RTT, but who do not have mutations in MECP2 (MECP2- mutation negative). Finally, in Aim 3 we propose co-administer our M4-PAM with modulators of glutamatergic and GABAergic signaling, to establish whether a complimentary symptom domains can be rescued. My career goal is to become the head of a lab with a research focus on pediatric diseases of the nervous system, and I have identified four areas of insufficient training that I propose to remedy to assist in achieving long term success in that role. The four areas are: 1) Training in electrophysiology 2) Training next generation sequencing analysis 3) Clinical exposure to RTT and 4) Mentor experience. To remove these technical and conceptual barriers, I have developed a training plan that integrates formal didactic training with hands-on mentorship, with the goal of turning these weaknesses into strengths. It is anticipated that completion of the proposed project and training plan will place me in an ideal position to receive a tenure track faculty position, and as such, I believe that this application is well suited for a K01 Career Development Award.