CRTC1 phosphorylation and transcriptional activity during hippocampal plasticity

 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性重新布线的能力。突触可塑性，即突触连接的数量和效力随经验的变化，为大脑中记忆的形成和储存提供了基础。持久的可塑性，如长期记忆，需要新的基因表达。研究突触产生的信号如何从刺激的突触转运到细胞核以调节转录，Martin实验室先前揭示了转录调节因子CRTC1（CREB依赖性转录的共激活因子和调节因子）的突触到细胞核转运的作用（Ch'ng等人，2012）。CRTC1活性受去磷酸化调节，磷酸化CRTC1定位于细胞质，去磷酸化CRTC1定位于细胞核。因此，CRTC1被锚定在未受刺激的神经元中的突触处，但是响应于突触能刺激而经历快速易位到细胞核。在细胞核中，CRTC1调节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在神经元刺激后磷酸化发生了戏剧性和复杂的变化。最初的质谱分析揭示了CRTC1中的50个氨基酸残基在神经母细胞瘤N2a细胞系中被磷酸化。我的奖学金研究项目旨在绘制在小鼠海马神经元长期可塑性过程中经历调节去磷酸化的残基。作为可塑性研究的模型，我将研究急性期小鼠海马脑片的长时程增强（LTP）和长时程抑制（LTD）。我的实验测试的假设，不同的活动模式触发不同的变化，在模式的CRTC1磷酸化和差异磷酸化形式的CRTC1调节不同的程序的基因表达。通过这种方式，CRTC1的翻译后修饰可以编码刺激模式，从而在基因表达中产生适当的变化。为了验证这个假设，我提出了两个目标。在第一，我确定如何LTP和LTD诱导刺激改变CRTC1的磷酸化。在第二，我确定这些刺激如何调节CRTC1与特定的转录因子的相互作用，以及这些复合物如何调节下游基因的表达。

 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.