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

描述（由申请人提供）：在海马神经元中，体细胞-树突CaV1.2 l型电压门控Ca2+通道（LTCC）在兴奋-转录（E-T）耦合中起作用。打开突触后神经元ltcc的去极化通过Ca2+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细胞核因子（NFAT）。由于LTCC转录调控是学习和记忆基础上兴奋性突触可塑性的持久形式所必需的，因此了解LTCC信号如何导致有效的、时空特异性的突触-核通信至关重要。突触到细胞核信号传导的一个基本问题是：树突突触后纳米结构域E-T耦合Ca2+信号的早期信号如何被转导成可靠地长距离传递到细胞核的信号？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。该AKAP通过两亲性α -螺旋基序锚定cAMP依赖性蛋白激酶（PKA），并通过非典型PxIxIT对接基序锚定Ca2+-钙调蛋白（CaM）激活的蛋白磷酸酶2b （calcineurin; CaN）。将PKA锚定在AKAP79/150上支持神经元LTCC电流振幅的增强，这是通过akap锚定的CaN进行的Ca2+依赖性反馈所强烈反对的。LTCC激活akap定位的CaN也是K+去极化触发的NFAT易位到细胞核和转录激活所必需的。然而，LTCC- akap - can - nfat通路的关键突触-核信号传导问题仍然存在：(1)AKAP79/150信号复合体是否在突触后谷氨酸受体激活激发的树突中特异性调节LTCC Ca2+内流；(2)树突中的Ca2+信号是否局部激活最终在细胞核中起作用的CaN-NFAT信号？(3)该信号通路调控的神经元靶基因有哪些；(4)这个过程是否参与突触可塑性？我们将在三个目标中探讨这些关键问题，这些目标依赖于Ca2+成像（Aim 1）， CaN和NFAT成像（Aim 2）和基因转录分析（Aim 3）的组合。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切片中，将研究AKAP79/150对LTCC Ca2+内流、CaN- nfat信号动力学和活性依赖基因转录的调控。该项目的总体目标是测试突触到核通信的中心假设，即突触后Ca2+信号在树突中局部重新编码为基于蛋白质的信号（例如NFAT），并传递给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.