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

描述(申请人提供)：在海马神经元中，体树突状CaV1.2 L型电压门控性钙通道在兴奋-转录(E-T)偶联中起作用。突触后神经元中开放LTCC的去极化通过钙调节的激酶和磷酸酶激活转录因子cAMP反应元件结合蛋白(CREB)和活化T细胞的核因子(NFAT)。由于LTCC转录调控是作为学习和记忆基础的持久形式的兴奋性突触可塑性所必需的，因此理解LTCC信号如何导致高效的、时空特异性的突触与核通信是至关重要的。突触到核信号的一个基本问题是：E-T中的早期信号是如何耦合树突突触后纳米结构域中的钙离子信号转换成可靠地远距离传递到细胞核的信号的？突触后支架蛋白A-激酶锚定蛋白(AKAP)79/150通过修饰的亮氨酸拉链(LZ)基序与CaV1.2结合。这个AKAP通过一个两亲性的α-螺旋基序锚定cAMP依赖的蛋白激酶(PKA)，并通过一个非典型的PxIxIT对接基序锚定钙-钙调蛋白(CaM)激活的蛋白磷酸酶2B(CaN；CaN)。将PKA锚定到AKAP79/150上支持神经元LTCC电流幅度的增强，而这种增强通过AKAP锚定的CaN被钙依赖的反馈有效地对抗。K+去极化触发的NFAT转位到细胞核和激活转录也需要AKAP定位的CaN的LTCC激活。然而，对于LTCC-AKAP-CAN-NFAT通路来说，关键的突触到核的信号转导问题仍然存在：(1)AKAP79/150信号复合体是否调节突触后谷氨酸受体激活所激活的树突中的LTCC钙内流；(2)树突中的这些钙信号是否局部激活了最终作用于细胞核的CAN-NFAT信号；(3)受该信号通路调控的神经元靶基因是什么；以及(4)这一过程是否参与了突触可塑性？我们将在三个目标上探索这些关键问题，这三个目标依赖于结合钙离子成像(目标1)、CaN和NFAT成像(目标2)和基因转录分析(目标3)。在表达改变PKA锚定、CaN锚定或LZ结构域结合的AKAP突变体的神经元或脑片中，将研究AKAP79/150对LTCC钙内流、CaN-NFAT信号动力学和活性依赖基因转录的调节。该项目的总体目标是检验突触到核通讯中的一个中心假说，即突触后钙信号在树突中被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。

DESCRIPTION (provided by applicant): 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 alpha-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.