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，但是microRNA也在成熟神经元中表达，其中它们被提出来控制突触可塑性。这是一个很有吸引力的概念，因为蛋白质翻译的局部调节对于这一过程至关重要，而microRNA可以调节树突生长和棘形成，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，部分原因是多聚核糖体在这个区室中的存在一直存在争议。我们认为，在这个建议，轴突生长的microRNA调节并不一定需要翻译发生在轴突和棕榈酰化酶，在神经元的其他地方的microRNA调节，可以直接运输的关键信号分子轴突膜。这项提议的重点是miR-134，一种最初以其“活性依赖性”和调节树突棘大小的能力为特征的microRNA。使用一组比率microRNA传感器，我们意外地发现，成熟皮层神经元中的miR-134活性仅限于抑制性生长抑素（SST）产生的中间神经元，这与miR-134功能的广泛观点相矛盾。负责限制miR-134表达SST-中间神经元的机制是未知的，我们提出这是通过miR-134前体的细胞特异性加工来实现的。我们将确定神经元产生成熟的功能性miR-134的能力是否是由于转录或转录后机制，鉴定与前体相互作用的RNA结合蛋白，并使用miR-Glo（一种新型荧光测定法）测试这些因素是否以细胞特异性方式影响miR-134前体的加工。使用一种名为RISC-trap的新方法，旨在捕获mRNA降解前的microRNA-mRNA相互作用，我们发现miR-134靶向棕榈酰化酶DHHC 9，该酶控制Ras向细胞膜的运输。我们将测试抑制性SST中间神经元中DHHC 9的miR-134调节以及随后Ras的棕榈酰化是否控制Ras向轴突生长锥的运输，并因此控制SST中间神经元中的轴突形态。我们假设miR- 134的活性调节对轴突生长有负面影响，并且与这些细胞特有的轴突分支模式有关。microRNA如miR-134调节棕榈酰化酶的能力，从而调节信号分子如Ras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.