Mechanisms of Neuronal Calcineurin-NFAT Synapse-to-Nucleus Signaling

神经元钙调神经素-NFAT突触-核信号转导机制 项目总结/摘要 在海马神经元中，体-树突状CaV1.2 L型电压门控Ca 2+通道 （LTCC）在兴奋-转录（E-T）偶联中的功能。去极化打开LTCC， 突触后神经元激活转录因子cAMP反应元件结合蛋白 （CREB）和活化的T细胞的核因子（NFAT）通过Ca 2+调节的激酶， 磷酸酶因为LTCC转录调控是持久形式的转录所必需的， 兴奋性突触可塑性是学习和记忆的基础，了解如何是至关重要的 LTCC信号传导导致有效的、时空特异性的突触-核通信。 在突触到细胞核的信号传递中，一个至关重要的问题是： 在E-T偶联中-树突状突触后纳米结构域中的Ca 2+信号-转换为信号 能可靠地长距离传递到细胞核 突触后支架蛋白A-激酶锚定蛋白（AKAP）79/150结合 CaV1.2通过修饰的亮氨酸拉链（LZ）基序。这种AKAP锚定cAMP- 依赖性蛋白激酶（PKA），通过一个两亲性α-螺旋基序，和Ca 2 +-钙调蛋白 （CaM）-活化蛋白磷酸酶-2B（钙调神经磷酸酶; CaN），通过非典型PxIxIT对接 母题PKA对AKAP 79/150的修饰支持神经元LTCC电流的增强 通过AKAP锚定的CaN的Ca 2+依赖性反馈有力地对抗的幅度。 K+去极化触发的NFAT也需要LTCC激活AKAP定位的CaN 易位到细胞核并激活转录。然而，关键的突触到细胞核 对于LTCC-AKAP-CaN-NFAT通路仍然存在信号传导问题：（1）AKAP 79/150 信号复合物特异性调节突触后兴奋的树突中LTCC Ca 2+内流 谷氨酸受体激活;（2）树突中的这些Ca 2+信号是否局部激活CaN-NFAT 最终作用于细胞核的信号传导;（3）哪些神经元靶基因受 这一信号通路;（4）这一过程参与突触可塑性？我们将 在三个目标中探索这些关键问题，这些目标依赖于Ca 2+成像（Aim 1）、CaN和NFAT成像（Aim 2）和基因转录分析（Aim 3）。AKAP 79/150 LTCC Ca 2+内流、CaN-NFAT信号动力学和活性依赖性基因的调节 将在表达AKAP突变体的神经元或脑切片中研究转录， PKA锚定、CaN锚定或LZ结构域结合。本项目的总体目标是测试 突触-细胞核通讯中的一个中心假设，即突触后Ca 2+信号是 在树突中局部重新编码为基于蛋白质的信号（例如，NFAT），并传递到细胞核 来控制可塑性相关基因的表达。

Mechanisms of Neuronal Calcineurin-NFAT Synapse-to-Nucleus Signaling Project Summary/Abstract In hippocampal neurons, somato-dendritic CaV1.2 L-type voltage-gated Ca2+ channels (LTCC) function in excitation-transcription (E-T) coupling. Depolarizations that open LTCCs in postsynaptic neurons activate the transcription factors cAMP-response element binding protein (CREB) and nuclear factor of activated T-cells (NFAT) through Ca2+-regulated kinases and phosphatases. Because LTCC transcriptional regulation is required for long-lasting forms of excitatory synaptic plasticity that underlie learning and memory, it is crucial to understand how LTCC signaling leads to efficient, spatiotemporally specific synapse-to-nucleus communication. A question of fundamental importance in synapse-to-nucleus signaling is: how are early signals in E-T coupling―Ca2+ signals in dendritic postsynaptic nanodomains―transduced into signals that are reliably relayed over long distances to the nucleus? The postsynaptic scaffold protein A-kinase anchoring protein (AKAP) 79/150 binds to CaV1.2 through a modified leucine zipper (LZ) motif. This AKAP anchors both the cAMP- dependent protein kinase (PKA), via an amphipathic α-helical motif, and the Ca2+-calmodulin (CaM)-activated protein phosphatase-2B (calcineurin; CaN), via an atypical PxIxIT docking motif. Anchoring of PKA to AKAP79/150 supports enhancement of neuronal LTCC current amplitude that is potently opposed by Ca2+-dependent feedback through AKAP-anchored CaN. LTCC activation of AKAP-localized CaN is also required for K+ depolarization-triggered NFAT translocation to the nucleus and activation of transcription. However, key synapse-to-nucleus signaling questions remain for the LTCC-AKAP-CaN-NFAT pathway: (1) does the AKAP79/150 signaling complex regulate LTCC Ca2+ influx specifically in dendrites excited by postsynaptic glutamate receptor activation; (2) do these Ca2+ signals in dendrites locally activate CaN-NFAT signaling that ultimately acts in the nucleus; (3) what are the neuronal target genes regulated by this signaling pathway; and (4) is this process engaged during synaptic plasticity? We will explore these crucial questions in three aims that rely upon a combination of Ca2+ imaging (Aim 1), CaN and NFAT imaging (Aim 2), and gene transcription analyses (Aim 3). AKAP79/150 regulation of LTCC Ca2+ influx, CaN-NFAT signaling dynamics, and activity-dependent gene transcription will be investigated in neurons or brain slices expressing AKAP mutants that alter PKA anchoring, CaN anchoring, or LZ domain binding. The overall goal of this project is to test a central hypothesis in synapse-to-nucleus communication that postsynaptic Ca2+ signals are locally re-coded in dendrites as protein-based signals (e.g., NFAT), and relayed to the nucleus to control plasticity-associated gene expression.