RNA Processing-Mediated Mechanisms of CNS Dysfunction in Myotonic Dystrophy

强直性肌营养不良(dystrophia myotonica，DM)是一种常染色体显性遗传病，诊断为 全球1：8000人的流行率，影响到身体的多种组织，包括骨骼肌， 心脏，以及与这一提议相关的中枢神经系统(CNS)。DM1是由CTG扩张引起的 DMPK基因3‘端非编码区重复序列。在老鼠身上有大量的证据 DM1模型和人类DM1死后组织支持RNA介导的疾病机制 毒性核内CUG RNA焦点隔离物粘虫(MBNL)正常发挥作用的RNA结合蛋白 对转录后基因调控的各个方面起着至关重要的作用。我们的一个主要差距是 我们的理解是，我们不知道哪些RNA加工缺陷是DM1中特定损伤的基础 大脑功能。最近的工作和我们的新发现一起表明，RNA编码的错接 突触蛋白与DM1的中枢神经系统功能障碍有关。我们的中心假设是中枢神经系统表型 直接归因于MBNL介导RNA加工的丧失和MBNL活性和/或恢复 剪接可以恢复大脑功能。我们的目标是彻底了解RNA介导的加工 DM1中中枢神经系统功能障碍的机制，并以此开发和严格评估新的治疗方法 战略。这项提案的总体目标是同时使用候选和基因组范围的方法， 应用于MBNL KO小鼠和一种新的基于AAV9的神经元小鼠模型，与RNAseq分析相比较 以评估特定剪接事件在驱动症状方面的作用，并 全面识别错接和RNA加工方面的变化。目标1将描述如何 GABRG2、GRIN1和SNAP25剪接事件的失调与分子、细胞和行为有关 DM1中观察到的表型。Aim 2将开发一种新的基于AAV9的小鼠模型来阐明这组RNA 在神经元中处理导致DM1表型的事件，通过转录图谱和重叠 人DM脑和DM小鼠模型脑。目标3将评估反义的程度 寡核苷酸或MBNL的表达可挽救大鼠的分子、细胞、生理和行为表型 DM1小鼠模型。这些研究将提供新的机制洞察力，以了解扰动如何具体到 RNA加工事件可导致强直性肌营养不良的中枢神经系统症状，并提供更广泛的 全面查看DM CNS中发生的所有转录组变化。拟议的工作意义重大， 因为没有分子变化与DM中枢神经系统的任何表型相关联。这提供了以下框架 未来的治疗努力旨在纠正中枢神经系统缺陷。

Myotonic dystrophy (dystrophia myotonica, DM) is an autosomal dominant genetic disease, with a diagnosed prevalence of 1:8000 people worldwide, that affects multiple tissues of the body, including skeletal muscle, heart, and related to this proposal, the central nervous system (CNS). DM1 is caused by expanded CTG repeats in the 3' UTR of dystrophia myotonica protein kinase (DMPK). There is substantial evidence in mouse DM1 models and human DM1 postmortem tissue to support an RNA-mediated disease mechanism where toxic intranuclear CUG RNA foci sequester Muscleblind (MBNL) RNA binding proteins that normally play crucial roles to regulate various aspects of post-transcriptional gene regulation. A major gap in our understanding is that we do not know which RNA processing defects underlie specific impairments in DM1 brain function. Recent work together with our new findings suggests that missplicing of RNAs encoding synaptic proteins is responsible for CNS dysfunction in DM1. Our central hypothesis is that CNS phenotypes are directly attributed to loss of MBNL mediated RNA processing and that restoration of MBNL activity and/or splicing can restore brain function. Our goal is to gain a thorough understanding of RNA processing-mediated mechanisms of CNS dysfunction in DM1 and use this to develop and rigorously evaluate novel therapeutic strategies. The overall objectives of this proposal are to use both candidate and genome wide approaches, applied to MBNL KO mice and a new AAV9 based neuronal mouse model, compared to RNAseq analysis of human postmortem brain, to evaluate the role of specific splicing events to drive symptoms, and to comprehensively identify changes in missplicing and RNA processing. Aim 1 will characterize how dysregulation of GABRG2, GRIN1, and SNAP25 splicing events is linked to molecular, cellular, and behavioral phenotypes observed in DM1. Aim 2 will develop a new AAV9 based mouse model to elucidate the set of RNA processing events in neurons that cause DM1 phenotypes, through transcriptional profiling and overlap of human DM brains with DM mouse model brains. Aim 3 will assess the extent to which antisense oligonucleotides or MBNL expression can rescue molecular, cellular, physiologic and behavioral phenotypes in DM1 mouse models. These studies will provide new mechanistic insights into how perturbations to specific RNA processing events can lead to CNS symptoms in myotonic dystrophy, and provide a broader comprehensive view of all transcriptome changes occurring in the DM CNS. The proposed work is significant, as no molecular changes have been linked to any phenotypes in the DM CNS. This provides the framework for future therapeutic efforts aimed at correcting CNS defects.