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属性是其在整个成年期持续产生新生神经元的能力。这些新生神经元首先暴露于GABA能信号，因为它们从齿状回的亚颗粒层出现。这种活性驱动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，CREB信号如何转化为特定细胞的成熟、存活和功能整合到海马回路中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，它导致了一个不成熟的树突状乔木，其表型模仿CREB信号传导的中断。然而，作为一种microRNA，miR-132具有下调许多不同转录物的能力。该提案解决的重要问题是鉴定和表征有助于建立树突状乔木的特定miR-132靶标。在不了解特定细胞靶点的身份的情况下，我们无法完全理解miR-132所赋予的调控作用，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，以表征候选目标肝素结合上皮生长因子（Hb-EGF）的miR- 132的调节如何有助于新生神经元的树突状乔木。初步数据表明，Hb-EGF的水平显着下调，特别是在响应miR-132的活性。目的1利用能够靶向切除floxed小鼠模型的成年新生神经元中的miR-132位点来检查Hb-EGF如何在这些细胞中受miR-132调节。将评估Hb-EGF水平的变化如何影响树突状分支，然后我们将确定Hb-EGF的耗竭是否可以挽救成年新生神经元中miR-132活性被消融时发生的未成熟树突状表型。 其他miR-132直接靶点可能对成年新生神经元的成熟也很重要。为了鉴定可能与Hb-EGF起作用的直接miR-132靶点，我开发了一种分子miR- 132靶点陷阱，我将在Aim 2中将其开发为筛选。该方法利用显性负性RNAi诱导的沉默复合物（RISC）-微小RNA途径中的中心组分，其桥接微小RNA及其靶标的识别-以稳定和捕获相关的mRNA靶转录物，否则所述mRNA靶转录物将通过去腺苷化和核酸外切酶活性而衰减。 公共卫生相关性：为了解决成人神经发生与人类衰老，疾病和损伤的关系，对这一过程的分子机制有一个清晰而详细的了解是至关重要的。多年来，各种细胞因子参与了成年新生神经元的建立和成熟，然而，活性依赖性microRNA-132在这一过程中的重要性的揭示提供了一个独特的机会，以更深入地充实重要和特定的分子途径。该提案汇集了来自体外分子研究的详细初步数据-这些数据揭示了针对miR-132的特定核苷酸的机制见解-以及对所确定的调控如何影响整个生物体背景下这些新生神经元发育的重要体内分析。

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.