CRTC1 phosphorylation and transcriptional activity during hippocampal plasticity

海马可塑性过程中 CRTC1 磷酸化和转录活性

• 批准号: 8910152
• 负责人: Shivan Bonanno
• 金额: $ 3.47万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-01 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8910152
• 关键词: 14-3-3 Proteins; Acute; Affinity; Amino Acids; Binding; Binding Sites; Biochemical; Biology; Brain; CREB1 gene; Cell Line; Cell Nucleus; Cells; Chromatin; Complex; Coupling; DNA Binding; Disease; Doctor of Philosophy; Elements; Employee Strikes; Epitopes; Evolution; Fellowship; Gene Expression; Gene Targeting; Generations; Genetic Transcription; Glutamate Receptor; Glutamates; Goals; Hippocampus (Brain); Knock-in Mouse; Long-Term Depression; Long-Term Potentiation; Maps; Mass Spectrum Analysis; Memory; Mental disorders; Modeling; Modification; Molecular; Mus; Nervous system structure; Neuroblastoma; Neuronal Plasticity; Neurons; Nuclear; Nuclear Import; Pattern; Phosphopeptides; Phosphorylation; Post-Translational Protein Processing; Process; Protein Dephosphorylation; Proteins; Proteomics; Receptor Activation; Regulation; Research; Research Project Grants; Rest; Role; Sampling; Series; Signal Pathway; Signal Transduction; Slice; Specific qualifier value; Specificity; Stimulus; Synapses; Synaptic plasticity; Testing; Training; Transcriptional Regulation; Transgenic Mice; Travel; Viral; Viral Vector; Western Blotting; base; chromatin immunoprecipitation; experience; insight; long term memory; neuronal cell body; neuropsychiatry; nucleocytoplasmic transport; programs; promoter; public health relevance; research study; response; tool; transcription factor

 DESCRIPTION (provided by applicant): One of the most remarkable features of the nervous system is its capacity to undergo experience- dependent rewiring. Synaptic plasticity, the change in the number and efficacy of synaptic connections with experience, provides the basis for memory formation and storage in the brain. Long-lasting plasticity, such as that underlying long-term memory, requires new gene expression. Investigating how synaptically generated signals are transported from stimulated synapses to the nucleus to regulate transcription, the Martin lab previously uncovered a role for the synapse to nuclear transport of the transcriptional regulator CRTC1 (Ch'ng et al. 2012), a co-activator and regulator of CREB-dependent transcription. CRTC1 activity is regulated by desphosphorylation, with phosphorylated CRTC1 being cytoplasmically localized and dephosphorylated CRTC1 localizing to the nucleus. Thus, CRTC1 is anchored at synapses in unstimulated neurons, but undergoes rapid translocation to the nucleus in response to glutamatergic stimulation. In the nucleus, CRTC1 regulates the expression of CREB target genes. A striking finding that emerged from our studies was that CRTC1 underwent dramatic and complex changes in phosphorylation following neuronal stimulation. Initial mass spectrometric analyses revealed 50 amino acid residues in CRTC1 that are phosphorylated in the neuroblastoma N2a cell line. My fellowship research project aims to map the residues that undergo regulated dephosphorylation during long-term plasticity in mouse hippocampal neurons. As models of plasticity, I will study long-term potentiation (LTP) and long-term depression (LTD) of acute mouse hippocampal slices. My experiments test the hypothesis that distinct patterns of activity trigger distinct changes in the pattern of CRTC1 phosphorylation and that the differentially phosphorylated forms of CRTC1 regulate distinct programs of gene expression. In this way, the post-translational modification of CRTC1 can encode patterns of stimulation to produce appropriate changes in gene expression. To test this hypothesis, I propose two aims. In the first, I determine how LTP and LTD inducing stimuli alter the phosphorylation of CRTC1. In the second, I determine how these stimuli regulate the interaction of CRTC1 with specific transcription factors and how these complexes regulate downstream gene expression.

 描述(由申请人提供)：神经系统最显著的特征之一是其经历经验依赖的重新连接的能力。突触可塑性，即突触连接的数量和有效性随经验的变化，为大脑中记忆的形成和存储提供了基础。持久的可塑性，比如潜在的长期记忆，需要新的基因表达。马丁实验室在研究突触产生的信号是如何从受刺激的突触运输到细胞核以调节转录的过程中，先前发现了突触到转录调节因子CRTC1的核运输的作用(Chng等人。2012)，CREB依赖转录的共激活和调节因子。Crtc1的活性受去磷酸化的调节，磷酸化的CRTc1定位于细胞质，去磷酸化的CRTc1定位于细胞核。因此，CRTC1在未受刺激的神经元中锚定在突触上，但在谷氨酸能刺激下经历快速移位到细胞核。在细胞核中，CRTC1调控CREB靶基因的表达。从我们的研究中出现的一个惊人的发现是，CRTC1在神经元刺激后经历了戏剧性和复杂的磷酸化变化。最初的质谱分析显示，CRTC1中有50个氨基酸残基在神经母细胞瘤N2a细胞系中被磷酸化。我的研究员研究项目旨在定位在小鼠海马神经元长期可塑性过程中经历受调节的去磷酸化的残基。作为可塑性的模型，我将研究急性小鼠海马片的长时程增强(LTP)和长时抑制(LTD)。我的实验测试了这样一个假设，即不同的活动模式会引发CRTC1磷酸化模式的明显变化，并且CRTC1的不同磷酸化形式调节不同的基因表达程序。通过这种方式，CRTC1的翻译后修饰可以编码刺激模式，从而在基因表达方面产生适当的变化。为了检验这一假设，我提出了两个目标。首先，我确定了LTP和LTD诱导刺激如何改变CRTC1的磷酸化。在第二个实验中，我确定了这些刺激如何调控CRTC1与特定转录因子的相互作用，以及这些复合体如何调控下游基因的表达。