Role of the alternative splicing factor Nova2 in the development and function of layer 5 pyramidal neurons

皮层锥体神经元是皮层中数量最多的神经元，第5层（L5）锥体神经元（PNs）是主要的输出层。这些神经元是唯一树突跨越所有六个皮质层的新皮质细胞类型，使它们成为皮质柱的主要整合者之一。哺乳动物大脑的层流结构是由在时间和空间上协调的神经元迁移波产生的。尽管最近的进展揭示了协调这些过程的分子机制，但RNA转录后调控在L5 PN发育和功能中的作用仍然不明确。Nova蛋白是第一个被发现的哺乳动物神经元特异性剪接因子。尽管大量数据显示nova2介导的神经元蛋白选择性剪接，但要确定每个剪接事件在体内的作用及其生理相关性一直具有挑战性。例外的发现是：a)通过Reelin通路中的关键分子Disabled-1的选择性剪接调节，Nova2是迁移神经元皮层分层所必需的；b) Nova2通过netrin受体DCC的选择性剪接介导轴突寻路。我们未发表的对Nova2KO小鼠L5 PNs的观察结果显示出明显的异常表型：1)皮质位置错误，2)体细胞形态/极性缺陷，3)树突发育受损，4)树突棘形成/密度异常，5)轴突找路受损，6)动作电位阈值升高。这些数据表明，受Nova2调节的蛋白质控制着神经元发育和功能的不同方面，并且Nova2KO小鼠代表了一种前所未有的工具，可以对体内正常皮质发育所需的选择性剪接事件进行分子解剖。因此，我们的假设是，nova2介导的关键结构蛋白和突触蛋白的选择性剪接是L5 PNs迁移、发育、内在电生理特性和突触功能所必需的。本研究的第一个目的是研究Nova2在L5 PNs形态学（包括极性、轴突和树突发育）中的作用，第二个目的是确定Nova2在L5 PNs固有电生理特性和突触功能中的作用。为了确定nova2介导的关键蛋白剪接如何协调L5 PNs的发育和功能，我们将使用多方面的方法，包括最先进的光学、电生理、遗传和分子工具。特别是，我们将利用我们的Nova2调控靶点数据库来选择在对照小鼠的L5 PNs中表达的特定剪接变体，我们将在两种完全或部分Nova2缺失的遗传小鼠模型中进行表型拯救实验。我们期望这些发现将揭示皮层发育的机制，并最终理解新皮层主要输出神经元的发育和功能。

Cortical pyramidal neurons are the most abundant neurons in the cortex, with layer 5 (L5) pyramidal neurons (PNs) being the main output layer. These neurons are the only neocortical cell type with dendrites spanning all six cortical layers, making them one of the main integrators in the cortical column. The laminar structure of the mammalian brain is generated by waves of neuronal migration that are coordinated in time and space. Although recent progress has shed light on the molecular mechanisms that orchestrate these processes, the role that post-transcriptional regulation of RNA plays in L5 PN development and function remains ill-defined. Nova proteins were the first mammalian neuron-specific splicing factors identified. Despite abundant data showing Nova2-mediated alternative splicing of neuronal proteins, it has been challenging to establish the role of each splicing event in-vivo and therefore its physiological relevance. The exceptions being the finding that a) Nova2 is required for cortical layering of migrating neurons through the alternative splicing regulation of Disabled-1, a key molecule in the Reelin pathway, and  b) Nova2 mediates axonal pathfinding via alternative splicing of the netrin receptor DCC. Our unpublished observations of L5 PNs in Nova2KO mice show distinctive abnormal phenotypes: 1) Cortical misplacement, 2) defects in somatic morphology/polarity, 3) impaired dendritic development, 4) aberrant dendritic spine formation/density, 5) impaired axonal pathfinding, and 6) increased action potential threshold. These data suggest that proteins regulated by Nova2 control different aspects of neuronal development and function, and that the Nova2KO mouse represents an unprecedented tool to do a molecular dissection of the alternative-splicing events necessary for normal cortical development in-vivo. Hence, our hypothesis is that Nova2-mediated alternative splicing of key structural and synaptic proteins is required for L5 PNs migration, development, intrinsic electrophysiological properties and synaptic function. The first aim of the proposal is to study the role of Nova2 in L5 PNs morphology including polarity, axonal and dendritic development and the second aim is to establish the role of Nova2 in L5 PNs intrinsic electrophysiological properties and synaptic function. To determine how Nova2-mediated splicing of key proteins orchestrates the development and function of L5 PNs we will use a multifaceted approach including state of-the-art optical, electrophysiological, genetic and molecular tools. In particular, we will tap into our database of Nova2-regulated targets to select specific splice variants to be expressed in L5 PNs of control mice and we will perform phenotype-rescue experiments in two genetic mouse models with total or partial Nova2 deletion. We expect that these findings will shed light into the mechanisms of cortical development and ultimately in understanding the development and function of the main output neuron of the neocortex.