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

神经元钙调神经磷酸酶-NFAT突触-核信号转导机制 项目摘要/摘要 海马神经元体树突状CaV1.2 L型电压门控性钙通道 (LTCC)在激发-转录(E-T)偶联中起作用。中打开LTCC的去极化 突触后神经元激活转录因子cAMP反应元件结合蛋白 (CREB)和核因子(NFAT)通过钙调节激酶和 磷酸酶。因为LTCC转录调控对于长效的形式是必需的 兴奋性突触可塑性是学习和记忆的基础，了解 LTCC信号导致了高效的、时空特异性的突触到核的通讯。 突触到核信号传递中的一个重要问题是：早期信号是如何 在E-T偶联中--突触后树突纳米域中的钙信号--转化为信号 能够可靠地远距离传递到原子核？ 突触后支架蛋白A-激酶锚定蛋白(AKAP)79/150结合于 CaV1.2通过修饰的亮氨酸拉链(LZ)基序。这个AKAP将阵营和- 依赖蛋白激酶(PKa)，通过两亲性α-螺旋基序和钙-钙调蛋白 (CaM)激活的蛋白磷酸酶-2B(CaN；CaN)，通过非典型的PxIxIT对接 Motif。PKA锚定到AKAP79/150支持神经元LTCC电流的增强 幅度被钙离子依赖的反馈通过AKAP锚定的CAN有效地对抗。 K+去极化触发的NFAT也需要AKAP局部化CAN的LTCC激活 转位到细胞核，激活转录。然而，关键突触到细胞核 LTCC-AKAP-CAN-NFAT通路的信号问题仍然存在：(1)AKAP79/150 信号复合体调节突触后兴奋的树突中LTCC钙离子内流 谷氨酸受体激活；(2)树突内的这些钙信号是否局部激活了CaN-NFAT 最终作用于细胞核的信号；(3)神经靶基因受什么调控 这一信号通路；以及(4)这一过程是否参与了突触的可塑性？我们会 从三个目标探索这些关键问题，这三个目标依赖于结合钙离子成像(AIM 1)、CaN和NFAT成像(目标2)和基因转录分析(目标3)。AKAP79/150 LTCC钙离子内流、CaN-NFAT信号动力学和活性依赖基因的调控 将在表达AKAP突变体的神经元或脑片中进行转录研究 PKA锚定、Can锚定或LZ结构域结合。该项目的总体目标是测试 突触到核通讯的一个中心假说是突触后钙信号是 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 以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.