Role of Micro-RNAs in Neuronal Circuit Formation and Function

描述（由申请人提供）：近90%的人类基因组由不翻译成蛋白质的非编码区组成。 以前被称为“垃圾”DNA，最近变得明显的是，这些DNA中的一些被用来制造小的约22个核苷酸的单链RNA，称为微RNA（miRNA）。 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，在发育早期通过敲除合成miRNAs所需的关键RNAseIII酶Dicer来去除miRNAs，在原肠胚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持续表达，并且很明显它们继续在细胞功能中发挥重要的调节作用。 最大种类的miRNAs在神经系统中表达，包括皮质，这使得了解miRNAs在正常皮质功能以及精神和行为障碍中发挥的作用至关重要。 迄今为止，在培养的海马神经元中仅研究了少数miRNA，并且发现这些miRNA是适当的神经突生长和突触形态所需的。 重要的是，对可能靶点的计算分析表明，影响神经元存活和功能的许多信号通路受miRNA调控。 我们建议使用小鼠遗传学的力量，在锥体皮质神经元分化后，使用Dicer和miRNA途径DGCR8的另一个关键基因的条件性敲除，特异性地去除所有miRNA，然后询问miRNA在树突形态，突触棘和功能中起什么作用。 最后，我们将鉴定纯化的锥体神经元中表达的miRNAs的互补物，这可能是所观察到的表型的基础。 这些数据将为了解小RNA生物学在正常大脑功能和疾病中的作用提供第一个进展。 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层中的其他层特异性锥体神经元，以及我们已确定为影响突触传递或影响精神分裂症相关基因的候选者的特定miRNA。 公共卫生相关性。 小RNA途径是一个知之甚少，但重要的翻译抑制途径在哺乳动物神经元中大量表达。 这条通路可能对正常的神经元功能很重要，并且几乎肯定会影响各种行为和精神障碍，如自闭症和精神分裂症。 这些研究将是第一个提供详细的细胞和分子描述的小RNA途径在正常的神经元发育和功能在体内。

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.