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

皮质锥体神经元是皮质中最丰富的神经元，其中第5层（L5）锥体神经元（PN）是主要输出层。这些神经元是唯一一种树突跨越所有六个皮质层的新皮质细胞类型，使它们成为皮质柱中的主要整合者之一。哺乳动物大脑的层状结构是由在时间和空间上协调的神经元迁移波产生的。虽然最近的进展已经阐明了协调这些过程的分子机制，但RNA的转录后调控在L5 PN发育和功能中所起的作用仍然不明确。Nova蛋白是第一个被鉴定的哺乳动物神经元特异性剪接因子。尽管有大量的数据显示Nova 2介导的神经元蛋白的选择性剪接，但建立体内每个剪接事件的作用及其生理相关性一直具有挑战性。例外的发现是a）Nova 2是通过Disabled-1（Reelin途径中的关键分子）的选择性剪接调节的迁移神经元的皮质分层所需的，和B）Nova 2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Nova 2KO小鼠L5 PN的观察结果显示了独特的异常表型：1）皮质错位，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）轴突寻路受损，6）动作电位阈值增加。这些数据表明，由Nova 2调节的蛋白质控制神经元发育和功能的不同方面，并且Nova 2KO小鼠代表了一种前所未有的工具，可以对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Nova 2介导的关键结构和突触蛋白的选择性剪接是L5 PN迁移、发育、内在电生理特性和突触功能所必需的。该提案的第一个目的是研究Nova 2在L5 PN形态学中的作用，包括极性，轴突和树突发育，第二个目的是建立Nova 2在L5 PN内在电生理特性和突触功能中的作用。为了确定Nova 2介导的关键蛋白质剪接如何协调L5 PN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Nova 2调控靶点数据库来选择在对照小鼠的L5 PN中表达的特定剪接变体，并且我们将在两种具有全部或部分Nova 2缺失的遗传小鼠模型中进行表型拯救实验。我们希望这些发现将有助于了解皮层发育的机制，并最终了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 Our unpublished observations of L5 PNs in Nova2KO mice show distinctive abnormal phenotypes: 1) Cortical misplacement, 2) defects in somatic morphology/polarity, 3) impaired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.