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

大多数兴奋性神经元的输入发生在树突棘；这些结构的重塑导致正常神经发育和处理所必需的神经元功能的同步变化。此外，这一现象的失调是许多神经和神经退行性疾病的基础。除了突触上的信号外，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也很重要。然而，调节脊椎调节的信号转导网络还不是很清楚。AF-6是一种含有PDZ结构域的蛋白，在黏附连接处大量表达，参与树突棘重建。包括NMDAR(NMDAR)、雌激素(E2)和N-钙粘素依赖信号在内的AF-6上游信号通路通过AF-6依赖通路与调节树突棘形态有关。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移位到细胞核。因此，AF-6是通过在不同的亚细胞室中信号转导树突棘形态发生的理想候选者。这项建议的目的是确定差异的AF-6易位在调节培养的皮质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技术研究AF-6在NMDAR、E2和N-钙粘素介导的信号转导中的时间依赖性定位。我们还将通过过度表达截短或突变的AF-6结构来评估各种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-钙粘素介导的信号或改变的AF-6结构过表达诱导的脊柱表达的变化。最后，我们将干预AF-6易位，然后评估脊柱形态的变化。为此，我们将使用特殊修改的AF-6结构防止AF-6定位到突触或细胞核，并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控树突棘形态发生的信号通路，并为健康和疾病中调控突触可塑性的机制提供洞察力。

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.