Neuritin (NT) 在神经元发育和突触可塑性中发挥重要作用，但其作用机理尚未明确。申请人与梅教授前期合作研究揭示NT可通过激活胰岛素受体和ERK/mTOR上调Kv4.2表达和钾通道密度，但它调控Kv4.2基因转录机制以及对神经元兴奋性影响还需深入研究。申请人近期的研究显示CaN-NFATc4 通路参与神经元功能。梅教授实验室发现小鼠过表达NT后，皮层神经元Kv4.2表达量明显增加，动作电位发放频率降低；但在NFATc4敲除小鼠上，NT的上述作用消失。因此，利用申请人提供的Nfatc4-/-小鼠和多种NFAT专用实验材料以及合作者电生理等技术，我们拟共同探讨：1）NT是否通过Ca-NFAT通路调节Kv4.2表达和神经元兴奋性， 2）NT如何调节CaN活性，3）NT如何调控NFATc4介导的Kv4.2基因转录。我们将发挥两个实验室的优势，阐明NT调控Kv4.2表达和神经元兴奋性的机制。

The goal of this application is to couple my expertise in signaling and gene transcription with Dr. Yan-Ai Mei’s established research interests in neurobiology to understand the molecular basis of neuronal excitability. Specifically, we will focus on the interplays of calcineurin phosphatase (CaN), mitogen-activated protein kinases (MAPKs), and transcription factor NFAT as downstream effectors of neuritin (NT), a previously underappreciated neurotrophic factor.. NT plays an important role in neuronal development, synaptic plasticity and survival. Dr. Mei and I recently published that NT increased expression of voltage-gated potassium channel Kv4.2 and subsequent IA densities. Mechanistically, NT activates the ERK-branch of MAPKs via an emerging role of the heterologous insulin receptor complex. . Similar to NT, CaN is highly expressed in brain and regulates neuronal growth and survival.We showed that the CaNNFATc4 pathway is important for neuronal development. The role of CaNNFATc4 pathway in neuronal excitability is not known; although previous studies showed that NFAT modulates the excitability in heart by regulating the cardiac expression of several potassium channel subunits, including Kv4.2. . The goal of this proposal is to elucidate the molecular basis of NT in neuronal excitability. Given that NT increases Kv4.2 expression, which is a target of NFAT, we propose that NT channels through the CaNNFATc4 pathway to mediate Kv4.2 expression and subsequent IA densities. . Specific questions that we will address in this application are: 1) Do Nfatc4-/- primary neurons exhibit defects in NT-mediated neuronal excitability?2) How does NT modulates intracellular calcium for CaN activation?3) How does NT regulates NFATc4-mediated Kv4.2 gene transcription?. To address these questions, we sent the NFATc4 mice to Dr. Mei’s laboratoryto prepare primary neurons and to examine electrophysiological propertiesin vivo.Our recent data indicate that ablation of NFATc4 abrogated NT-mediatedneuronal excitability. We will continue to examine the molecular basis of NT in NFAT activation using valuable reagents andCaN/NFAT mice acquired from my previous studies. In sum, we will exploit the expertise of both laboratories to elucidate the molecular basis of NT-mediated neuronal excitability. .

Neuritin是一种重要的神经营养因子，参与调节神经发育、突触可塑性和神经元存活。我们之前的研究表明，neuritin上调瞬时外向钾电流（IA）通道Kv4.2α-亚基表达和增加IA密度，部分通过激活胰岛素受体（IR）信号通路，但neuritin诱导Kv4.2表达的分子机制以及生理意义仍然不清楚。在本合作课题的研究中，我们发现Ca2 +依赖的钙调神经磷酸（CaN）活化的T细胞核因子c4（NFATc4）是neuritin诱导Kv4.2转录表达和增加小脑颗粒神经元（CGNs）的IA密度所必需的。Neuritin提高细胞内Ca2 +水平、增加Kv4.2表达和IA密度的效应对CaN抑制剂敏感，并且在Nfatc4 - / -小鼠中该作用消除，但对Nfatc2 - / -小鼠颗粒细胞的上述作用没有影响。在表达有IR的CGNs和HeLa细胞，neuritin刺激也明显增加NFATc4在核中的积累。 此外，NFATc4招募到Kv4.2基因启动子位点也通过荧光素酶报告基因和染色质免疫沉淀检测发现。而且，从腺相关病毒介导的neuritin过度表达的小鼠皮层神经元上获得的数据表明，neuritin减少神经元兴奋性和增加树突小棘形成的作用在Nfatc4- / -小鼠中同样被消除。这些结果提示，CaN / NFATc4信号通路在neuritin调节神经元兴奋性活动中具有不可或缺的作用。我们的研究对阐明下游分子信号传导对于神经突在神经元功能障碍中的潜在治疗应用具有重要意义。