Role of AF-6 nuclear signaling in regulating dendritic spine morphology

描述（由申请人提供）：大多数兴奋性神经元输入发生在树突棘;这些结构的重塑导致正常神经发育和加工所必需的神经元功能的同时变化。此外，这种现象的失调是许多神经和神经退行性疾病的基础。除了在突触处的信号传导之外，到细胞核的信号传导通路被认为对于树突棘形态和神经元可塑性的变化是重要的。然而，调节脊柱调制的信号转导网络还没有得到很好的理解。AF-6是一种含有PDZ结构域的蛋白质，在粘附连接处大量表达，并参与树突棘重塑。AF-6上游的通路，包括NMDA受体（NMDAR）、雌激素（E2）和N-钙粘蛋白依赖性信号传导，已经通过AF-6依赖性通路与调节树突棘形态相关。在我们的初步数据中，我们证明了这些途径也可以诱导AF-6易位到细胞核。因此，AF-6是一个理想的候选人介导树突棘形态发生的信号在不同的亚细胞区室。本建议的目的是确定差异AF-6易位在调节培养的皮层神经元树突棘形态发生的作用。首先，我们将通过免疫细胞化学和蛋白质印迹技术来表征AF-6在NMDAR、E2和N-cadherin介导的信号传导中的时间依赖性定位。我们还将通过过表达截短或突变的AF-6构建体来评估各种AF-6结构域在其易位中的作用。接下来，我们将使用免疫细胞化学来确定由NMDAR、E2和N-钙粘蛋白介导的信号传导或改变的AF-6结构的过表达诱导的棘表达的变化。最后，我们将干预AF-6易位，然后评估脊柱形态学的改变。为此，我们将使用专门修饰的AF-6构建体来防止AF-6定位于突触或细胞核，并且我们将使用细胞核定位信号来迫使AF-6进入细胞核。这些研究的结果将促进对调节树突棘形态发生的信号通路的更好理解，并提供对健康和疾病中控制突触可塑性的机制的深入了解。 公共卫生相关性：该项目的目标是了解脑细胞如何响应来自其他脑细胞的信号而发生变化。这些变化对正常的大脑功能至关重要，但在各种大脑疾病中也会受到干扰。这些研究的结果将帮助我们更好地了解某些大脑疾病的原因，并可能发现新的治疗形式。

DESCRIPTION (provided by applicant): The majority of excitatory neuronal input occurs at dendritic spines; remodeling of these structures leads to concurrent changes in neuronal function essential for normal neural development and processing. Furthermore, dysregulation of this phenomenon underlies numerous neurological and neurodegenerative disorders. In addition to signaling at synapses, signaling pathways to the nucleus are thought to be important for changes in dendritic spine morphology and neuronal plasticity. However, the signal transduction networks that regulate spine modulation are not well understood. AF-6 is a PDZ-domain-containing protein abundantly expressed at adhesion junctions and is involved in dendritic spine remodeling. Pathways upstream of AF-6, including NMDA receptor (NMDAR), estrogen (E2) and N-cadherin-dependent signaling, have been associated with modulating dendritic spine morphology via an AF-6 dependent pathway. In our preliminary data, we demonsrate that these pathways can also induce AF-6 translocation to the nucleus. Thus AF-6 is an ideal candidate to mediate dendritic spine morphogenesis by signaling in distinct subcellular compartments. The aim of this proposal is to determine the role of differential AF-6 translocation in regulating dendritic spine morphogenesis in cultured cortical neurons. First, we will characterize the time-dependent localization of AF-6 in NMDAR, E2 and N-cadherin-mediated signaling by using immunocytochemistry and Western-blotting techniques. We will also assess the role of the various AF-6 domains in its translocation by overexpressing truncated or mutated AF-6 constructs. Next, we will use immunocytochemistry to determine changes in spine expression induced by NMDAR, E2 and N-cadherin-mediated signaling or the overexpression of altered AF-6 constructs. Lastly, we will intervene with AF-6 translocation and then assess alterations in spine morphology. To this end, we will prevent AF-6 from localizing to synapses or the nucleus using specifically modified AF-6 constructs and we will force AF-6 into the nucleus using a nuclear localization signal. Results from these studies will foster a better understanding of the signaling pathways regulating dendritic spine morphogenesis and provide insight into the mechanisms governing synaptic plasticity in health and disease. PUBLIC HEALTH RELEVANCE: The goal of this project is to understand how brain cells change in response to signals from other brain cells. These changes are essential for normal brain function but also become disrupted in various brain disorders. Findings from these studies will help us to better understand the cause of certain brain disorders and potentially identify new forms of treatment.