Targets of microRNA-132 in adult neurogenesis

描述（由申请人提供）：哺乳动物海马体的一个显著特征是其在成年期持续产生新生神经元的能力。当这些新生神经元从齿状回的亚颗粒层出现时，它们首先暴露于gaba能信号。这种活动驱动creb依赖的信号通路，改变新生神经元内的基因表达，因此是其成熟和存活所必需的。然而，关于creb信号如何转化为特定细胞进入海马回路的成熟、存活和功能整合的确切机制仍然知之甚少。我们确实知道，体内成年新生神经元成熟的重要CREB靶点之一是非编码转录物microRNA-132 （miR-132）。当我们切除成年新生神经元中的miR-132位点时，它导致了一个未成熟的树突乔木，其表型上复制了CREB信号的破坏。然而，作为一种microRNA， miR-132具有下调多种转录本的能力。该提案解决的重要问题是鉴定和表征有助于树突乔木建立的特定miR-132靶点。在不了解特定细胞靶点的身份的情况下，我们不能完全理解miR-132所赋予的调节作用，只知道它的活性是这一过程所必需的。通过初步筛选和小鼠模型的可用性，我处于一个独特的位置来描述miR- 132对候选靶点肝素结合上皮生长因子（Hb-EGF）的调节如何促进新生神经元的树突树突。初步数据表明，Hb-EGF水平在miR-132活性下特异性显著下调。Aim 1利用能够靶向切除floxed小鼠模型成年新生神经元中的miR-132位点的优势，研究这些细胞中miR-132如何调节Hb-EGF。我们将评估Hb-EGF水平的变化如何影响树突树突化，然后我们将确定Hb-EGF的消耗是否可以挽救在成年新生儿神经元中miR-132活性消退时发生的未成熟树突表型。其他miR-132的直接靶点可能对成年新生神经元的成熟也很重要。为了确定可能与Hb-EGF一起起作用的miR-132直接靶标，我开发了一种分子miR-132靶标陷阱，我将在Aim 2中开发成一种筛选方法。该方法利用显性负rna诱导沉默复合体（RISC）——microRNA途径中的一个中心成分，连接microRNA及其靶标的识别——来稳定和捕获相关的mRNA靶标转录本，否则这些转录本将通过死基化和外切酶活性而衰减。

DESCRIPTION (provided by applicant): One distinguishing attribute of the mammalian hippocampus is its ability to continuously produce newborn neurons throughout adult life. These newborn neurons are first exposed to GABAergic signals as they emerge from the subgranule layer of the dentate gyrus. This activity drives a CREB-dependent signaling pathway that alters the gene expression within that newborn neuron, and as such, is required for its maturation and survival. Nevertheless, the exact mechanism is still poorly understood as to how CREB-signaling translates into the maturation, survival, and functional integration of that particular cell into the hippocampal circuitry. We do know that one of the important CREB targets for the maturation of adult newborn neurons in vivo is non-coding transcript microRNA-132 (miR-132). When we excised the miR-132 locus in adult newborn neurons it resulted in an immature dendritic arbor that phenocopied disruption of CREB signaling. Nevertheless as a microRNA, miR-132 has the ability to downregulate a number of diverse transcripts. The important issue that this proposal addresses is the identification and characterization of specific miR-132 targets that contribute to this establishment of the dendritic arbor. Without understanding the identity of specific cellular targets, we cannot fully appreciate the regulation conferred by miR-132, and only know that its activity is required for this process. Through preliminary screens and the availability of a mouse model, I am in a unique position to characterize how the regulation of candidate target Heparin-binding Epithelial Growth Factor (Hb-EGF) by miR- 132 contributes to the dendritic arbor of newborn neurons. Preliminary data demonstrate that levels of Hb-EGF are dramatically downregulated specifically in response to miR-132 activity. Aim 1 takes advantage of being able to target excision of the miR-132 locus in the adult newborn neurons of the floxed mouse model to examine how Hb-EGF is regulated by miR-132 in these cells. Changes in Hb-EGF levels will be evaluated for how they affect dendritic arborization, and then we will determine whether depletion of Hb-EGF can rescue the immature dendritic phenotype that occurs with miR-132 activity is ablated in adult newborn neurons. It is likely that other miR-132 direct targets are also important for maturation of the adult newborn neuron. To identify direct miR-132 targets that may function with Hb-EGF, I have developed a molecular miR- 132 target trap that I will develop into a screen in Aim 2. This approach takes advantage of a dominant- negative RNAi-Induced Silencing Complex (RISC)- a central component in the microRNA pathway that bridges the recognition of microRNAs and their targets-to stabilize and capture associated mRNA target transcripts that would otherwise decay via deadenylation and exonuclease activity. PUBLIC HEALTH RELEVANCE: In order to address how adult neurogenesis relates to human aging, disease, and injury, it is absolutely vital to have a clear and detailed understanding of the molecular mechanisms underlying this process. Over the years various cellular factors have been implicated in the establishment and maturation of adult newborn neurons, however, the revelation that activity- dependent microRNA-132 is important in this process provides a unique opportunity to flesh out significant and specific molecular pathways in greater depth. This proposal brings together detailed preliminary data from in vitro molecular studies-which reveal mechanistic insight down to the specific nucleotides that are targeted my miR-132-with important in vivo analysis of how the identified regulation affects the development of these newborn neurons within the context of a whole organism.