The development of cortico-cerebellar circuits in a genetic form of intellectual disability

项目总结 智能障碍(ID)是一种普遍存在的神经发育障碍，没有有效的药物治疗 治疗。肌萎缩侧索硬化症常常与运动问题并存，而这些临床疾病之间共同风险的性质 人们对其表现形式还没有完全了解。在ID高危基因中，有显著的运动受累 X连锁基因DDX3X。DDX3X的突变几乎只影响女性。DDX3X编码一种RNA 解旋酶调节信使核糖核酸的翻译，它在神经发育中的作用刚刚开始显现。这个 DDX3X突变引起的电路水平变化尚不清楚。迫切需要填补这些空白。 因为了解DDX3X在大脑回路形成中的作用可能会为破译 肌萎缩侧索硬化症及其合并运动问题。为了解决这一未得到满足的需求，一种模拟DDX3X损失的鼠标 功能突变(DDX3X+/-)在本实验室获得并鉴定。我们的长期目标是绘制 并将其作为治疗靶点进行评估。总体目标是研究 DDX3X在形成有助于认知和运动控制的皮质-小脑回路中的作用。中环 假说认为，DDX3X调节大脑皮质-小脑回路的发育，辅助认知和 运动功能。其基本原理是，一旦我们了解了基于ID的电路级驱动机制 精准疗法可以被开发出来。核心假设将通过追求两个具体目标来检验： 1)确定DDX3X突变小鼠小脑中改变的神经群；以及，2)测试 DDX3X突变小鼠行为缺陷的皮质-小脑交流。在目标1下， 将使用单细胞RNA测序捕获DDX3X+/-小鼠的小脑种群。的体系结构 DDX3X+/-小鼠的小脑皮质将用小脑种群的免疫染色进行分析 在胚胎和出生后的生命中验证的细胞特异性标记。形态发生和 DDX3X+/-Purkinje细胞的突触发生将使用单胚胎原代培养进行检测。在目标2下， DDX3X+/-小鼠的皮质-桥脑-小脑通路将通过同时追踪皮质脑桥来分析。 以及桥脑-小脑的联系。将产生Purkinje特异性DDX3X+/-条件性小鼠并对其进行测试 使用公认的行为范式进行认知和运动行为。这个建议很有新意 因为它将通过映射小脑细胞和 受DDX3X突变影响的电路及其与行为的关系。它还具有创新性，因为它 连接发育生物学、细胞生物学、行为神经科学和单细胞转录学。这个 应用是重要的，因为它将促进我们对ID和共病运动障碍的理解，并看起来 尤其是被忽视的女性。它还具有重要意义，因为它将为 小脑发育，大脑功能的基本过程。这些结果预计将产生 积极的影响，因为它们将为更好的预防和治疗艾滋病的方法铺平道路。

PROJECT SUMMARY Intellectual disability (ID) is a prevalent neurodevelopmental disorder with no effective pharmacological treatment. ID is often co-morbid with motor problems, and the nature of shared risk between these clinical manifestations is not fully understood. Amongst the ID high-risk genes with significant motor involvement is the X-linked gene DDX3X. Mutations in DDX3X affect almost exclusively females. DDX3X encodes an RNA helicase regulating mRNA translation, and its role in neurodevelopment is just beginning to emerge. The circuit-level changes induced by DDX3X mutations are unknown. There is a critical need to fill these gaps because understanding the role of DDX3X in the formation of brain circuits might offer a new key to decipher ID and its co-morbidity with motor problems. To address this unmet need, a mouse modeling DDX3X loss-of- function mutations (Ddx3x+/-) was generated and characterized in our laboratory. The long-term goal is to map the circuit-level drivers of ID and assess them as therapeutic targets. The overall objective is to examine the role of DDX3X in shaping cortico-cerebellar circuits contributing to cognition and motor control. The central hypothesis is that DDX3X regulates the development of cortico-cerebellar circuits subserving cognitive and motor function. The rationale is that, once we understand the circuit-level drivers of ID, mechanism-based precision therapeutics can be developed. The central hypothesis will be tested by pursuing two Specific Aims: 1) Identify the neural populations altered in the cerebellum of Ddx3x mutant mice; and, 2) Test the role of cortico-cerebellar communication in behavioral deficits of Ddx3x mutant mice. Under Aim 1, the alterations in cerebellar populations of Ddx3x+/- mice will be captured using single-cell RNA sequencing. The architecture of the cerebellar cortex in Ddx3x+/- mice will be analyzed using immunostaining of cerebellar populations with authenticated cell-specific markers during embryonic and postnatal life. The morphogenesis and synaptogenesis of Ddx3x+/- Purkinje cells will be examined using single-embryo primary cultures. Under Aim 2, the cortico-ponto-cerebellar pathway will be analyzed in Ddx3x+/- mice by simultaneously tracing cortico-pons and ponto-cerebellar connections. Purkinje-specific Ddx3x+/- conditional mice will be generated and tested for cognition and motor behavior using well-established behavioral paradigms. This proposal is innovative because it will define the neurobiology of a largely unknown ID gene by mapping the cerebellar cells and circuits affected by DDX3X mutations, and their relationship with behavior. It is also innovative because it bridges developmental biology, cellular biology, behavioral neuroscience, and single-cell transcriptomics. The application is significant because it will advance our understanding of ID and co-morbid motor deficits, and look specifically at females, which have been neglected. It is also significant because it will shed new light on cerebellar development, a fundamental process for brain function. These results are expected to have a positive impact because they will pave the way for better methods for prevention and treatment of ID.