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

AF-6核信号在调节树突棘形态中的作用

• 批准号: 8050676
• 负责人: JON-ERIC VANLEEUWEN
• 金额: $ 3.34万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8050676
• 关键词: Adhesions; Affect; Binding; Biochemical; Brain; Brain Diseases; Cell Nucleus; Data; Dendritic Spines; Development; Disease; Dominant-Negative Mutation; Estrogens; Fostering; Goals; Health; Individual; MLLT4 gene; Mediating; Morphogenesis; Morphology; Mutate; N-Cadherin; N-Methyl-D-Aspartate Receptors; Neurodegenerative Disorders; Neuronal Plasticity; Neurons; Nuclear; Nuclear Localization Signal; Nuclear Translocation; Pathway interactions; Process; Proteins; RNA Interference; Receptor Activation; Regulation; Relative (related person); Role; Scaffolding Protein; Signal Pathway; Signal Transduction; Stimulus; Structure; Synapses; Synaptic plasticity; Techniques; Time; Vertebral column; Western Blotting; afadin; brain cell; excitatory neuron; genetic regulatory protein; hippocampal pyramidal neuron; imaging modality; immunocytochemistry; insight; interdisciplinary approach; knock-down; molecular imaging; nervous system disorder; neurodevelopment; novel; overexpression; postsynaptic; prevent; response

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.

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神经元功能的同步变化，这对正常的神经发育和处理至关重要。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。除了突触的信号外，通往细胞核的信号通路被认为对树突棘形态和神经元可塑性的变化很重要。然而，调控脊柱调节的信号转导网络尚不清楚。AF-6是一种含有pdz结构域的蛋白，在粘附连接处大量表达，并参与树突脊柱重塑。AF-6的上游通路，包括NMDA受体（NMDAR）、雌激素（E2）和n-钙粘蛋白依赖信号，通过AF-6依赖通路调节树突棘形态。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是通过不同亚细胞区室的信号传导介导树突棘形态发生的理想候选物质。本研究的目的是确定AF-6的不同易位在调节培养皮层神经元树突棘形态发生中的作用。首先，我们将利用免疫细胞化学和Western-blotting技术表征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断或突变的AF-6结构体来评估各种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NMDAR、E2和n- cadherin介导的信号或AF-6结构改变的过表达诱导的脊柱表达变化。最后，我们将干预AF-6易位，然后评估脊柱形态的改变。为此，我们将使用特异性修饰的AF-6结构来阻止AF-6定位到突触或细胞核，我们将使用核定位信号迫使AF-6进入细胞核。这些研究的结果将有助于更好地理解调节树突棘形态发生的信号通路，并为健康和疾病中控制突触可塑性的机制提供见解。