Role of Micro-RNAs in Neuronal Circuit Formation and Function

描述(申请人提供)：近90%的人类基因组由非编码区组成，这些非编码区没有翻译成蛋白质。以前被称为“垃圾DNA”的DNA最近变得很明显，其中一些DNA被用来制作小型应用程序。称为微RNA(MiRNAs)的22核苷酸单链RNAs。在过去的几年里，研究人员发现，这些miRNAs不仅对各种过程很重要，而且是必不可少的。事实上，通过敲除合成miRNAs所需的关键RNAseIII酶来去除发育早期的miRNAs，在原肠形成的非常早期阶段是致命的。重要的是，这些miRNAs在哺乳动物的整个生命过程中不断表达，显然它们继续在细胞功能中发挥重要的调节作用。MiRNAs的种类最多的是神经系统，包括皮质，因此了解miRNAs在正常皮质功能以及精神和行为障碍中扮演的角色是至关重要的。到目前为止，在培养的海马神经元中只有少数miRNA被研究，这些miRNA被发现是正常的轴突生长和突触形态所必需的。重要的是，对可能靶点的计算分析表明，许多影响神经元存活和功能的信号通路都受到miRNAs的调控。我们建议利用小鼠遗传学的力量，在锥体皮质神经元分化后，使用条件敲除DICER和另一个miRNA途径的关键基因Dgcr8来特异性地移除所有miRNAs，然后询问miRNAs在树突形态、突触棘和功能中扮演什么角色。最后，我们将确定在纯化的锥体神经元中表达的miRNAs的互补，这可能是观察到的表型的基础。这些数据将为理解小RNA生物学在正常大脑功能和疾病中的作用提供第一条途径。这些初步研究将使我们能够开发和完善工具，将这些研究扩展到皮质中其他特定层的锥体神经元，以及我们已经确定的影响突触传递或影响精神分裂症相关基因的特定miRNAs。与公共卫生相关。小RNA途径是一种知之甚少但在哺乳动物神经元中大量表达的重要翻译抑制途径。这条通路可能对正常的神经元功能很重要，几乎可以肯定的是，它会在自闭症和精神分裂症等各种行为和精神障碍中受到影响。这些研究将是第一次对体内正常神经元发育和功能中的小RNA途径提供详细的细胞和分子描述。

DESCRIPTION (provided by applicant): Nearly 90% of the human genome is composed of non-coding regions that are not translated into proteins. Previously referred to as "junk" DNA it has recently become apparent that some of this DNA is used to make small app. 22-nucleotide single stranded RNAs known as micro-RNAs (miRNAs). Within the last several years researchers have discovered that these miRNAs are not only important for a variety of processes, but essential. Indeed, removing miRNAs early in development by knocking out the key RNAseIII enzyme required for their synthesis, Dicer, is lethal at the very early stages of gastrulation. Importantly, these miRNAs are continuously expressed throughout the life of mammals and it is clear that they continue to play important regulatory roles in cellular function. The greatest variety of miRNAs are expressed in the nervous system, including the cortex, making it paramount to understand what role the miRNAs play in normal cortical function and mental and behavioral disorders. To date only a few miRNAs has been investigated in hippocampal neurons in culture and these miRNA was found to be required for proper neurite outgrowth and synapse morphology. Importantly computational analyses of likely targets indicate numerous signaling pathways that impact neuronal survival and function are regulated by miRNAs. We propose to use the power of mouse genetics to specifically remove all miRNAs from pyramidal cortical neurons after they have differentiated using a conditional knock out of Dicer and another key gene for the miRNA pathway DGCR8 and then ask what role miRNAs play in dendrite morphology, synaptic spines, and function. Finally, we will identify the complement of miRNAs that are expressed in purified pyramidal neurons that may underlie the observed phenotypes. These data will provide the first inroad to understanding the role of small RNA biology in normal brain function and disease. These initial studies will allow us to develop and perfect the tools to extend these studies to other, layer specific, pyramidal neurons in the cortex and to specific miRNAs that we have identified as candidates for impacting synaptic transmission or affecting schizophrenia-linked genes. PUBLIC HEALTH RELEVANCE. The small RNA pathway is a poorly understood, but essential translational repression pathway heavily expressed in mammalian neurons. This pathway is likely to be important for normal neuronal function and is almost certainly affected in a variety of behavioral and mental disorders such as autism and schizophrenia. These studies will be among the first to provide a detailed cellular and molecular description of the small RNA pathways in normal neuronal development and function in vivo.