Deconvoluting microRNA expression in brain

描述（由申请人提供）：在理解microrna对神经元发育的影响方面已经取得了相当大的进展，但microrna也在成熟神经元中表达，它们被认为控制突触可塑性。这是一个吸引人的概念，因为蛋白质翻译的局部调控对这一过程至关重要，而microrna可以调节树突生长和脊柱形成，这些特性是可塑性的基础。microrna促进轴突生长的观点受到的关注较少，部分原因是多核糖体在这个隔室中的存在一直存在争议。我们认为，轴突生长的microRNA调控并不一定需要轴突内的翻译，而棕榈酰化酶，由神经元中其他地方的microRNA调控，可以将关键信号分子运输到轴突膜。这项提议的重点是miR-134，这是一种microRNA，最初以其“活性依赖性”和调节树突棘大小的能力为特征。使用一组比例microRNA传感器，我们意外地发现，miR-134在成熟皮质神经元中的活性仅限于抑制性生长抑素（SST）产生的中间神经元，这与广泛持有的miR-134功能观点相矛盾。限制miR-134在sst -中间神经元表达的机制尚不清楚，我们认为这是通过miR-134前体的细胞特异性加工来完成的。我们将确定神经元产生成熟的、功能性的miR-134的能力是由于转录机制还是转录后机制，鉴定与前体相互作用的rna结合蛋白，并使用miR-Glo（一种新型荧光试验）测试这些因素是否以细胞特异性的方式影响miR-134前体的加工。使用一种名为RISC-trap的新方法，旨在捕获mRNA降解之前的microRNA-mRNA相互作用，我们发现miR-134靶向棕榈酰化酶DHHC9， DHHC9控制Ras转运到细胞膜。我们将测试miR-134对DHHC9在抑制性SST中间神经元中的调节，以及随后的Ras棕榈酰化，是否控制Ras向轴突生长锥的运输，从而控制SST中间神经元的轴突形态。我们假设miR- 134的活性调控对轴突生长有负面影响，并与这些细胞独特的轴突分支模式特征有关。microrna如miR-134调节棕榈酰化酶的能力，从而调节Ras等信号分子的膜运输，可能是突触可塑性的重要组成部分，特别是与轴突生长有关。

DESCRIPTION (provided by applicant): Considerable progress has been made understanding the effects of microRNAs on neuronal development, but microRNAs are also expressed in mature neurons where they have been proposed to control synaptic plasticity. This is an appealing concept because local regulation of protein translation is essential for this process and microRNAs can regulate dendritic growth and spine formation, properties that underlie plasticity. The idea that microRNAs contribute to axonal growth has received less attention, in part because the existence of polyribosomes in this compartment has been controversial. We argue in this proposal that microRNA regulation of axonal growth does not necessarily require translation to occur within the axon and that palmitoylation enzymes, regulated by microRNAs elsewhere in the neuron, can direct trafficking of key signaling molecules to axonal membranes. This proposal focuses on miR-134, a microRNA initially characterized by virtue of its "activity-dependence" and ability to regulate dendritic spine size. Using a set of ratiometric microRNA sensors, we found, unexpectedly, that miR-134 activity in mature cortical neurons was limited to inhibitory somatostatin (SST)-producing interneurons, contradicting a widely held view of miR-134 function. The mechanisms responsible for restricting miR-134 expression to SST-interneurons are unknown, and we propose that this is accomplished via cell-specific processing of the miR-134 precursor. We will establish whether the ability of neurons to generate mature, functional miR-134 is due to transcriptional or post-transcriptional mechanisms, identify RNA-binding proteins that interact with the precursor, and test whether these factors affect processing of the miR-134 precursor in a cell- specific manner using miR-Glo, a novel fluorescence assay. Using a new method termed RISC-trap designed to capture microRNA-mRNA interactions prior to mRNA degradation, we discovered that miR-134 targets the palmitoylation enzyme, DHHC9, which controls Ras trafficking to the cell membrane. We will test whether the miR-134 regulation of DHHC9 in inhibitory SST interneurons, and the consequent palmitoylation of Ras, controls Ras trafficking to axonal growth cones and, consequently, axon morphology in SST interneurons. We hypothesize that activity-regulation of miR- 134 negatively influences axon growth and is related to the unique axonal branching pattern characteristic of these cells. The ability of microRNAs such as miR-134 to regulate palmitoylation enzymes, and thereby membrane trafficking of signaling molecules like Ras, could be an important component of synaptic plasticity, particularly in relation to axonal growth.