Mechanisms of Synaptic Depression: Focus on Rap Signaling Pathways

描述(申请人提供)：长期突触抑制(LTD)和去增强，是重复突触活动期后持续突触抑制的两种形式，是脊椎动物突触可塑性的广泛研究实例。LTD和去增强的细胞和分子机制可能会阐明神经发育、适应、学习和记忆的生理和病理现象。现在有令人信服的证据表明，重复的突触活动导致NMDA敏感的谷氨酸受体(NMDA-Rs)的激活和突触后AMPA敏感的谷氨酸受体(AMPA-Rs)在LTD和去增强期间从兴奋性突触中移除。然而，将NMDA-R活性与AMPA-R交易联系起来的生化途径在很大程度上是未知的。我们之前已经报道过，小GTP酶Rap1通过激活p38MAPK来控制LTD。在一项初步研究中，我们观察到小GTPase Rap2通过激活JNK来控制去增强。基于这些发现，我提出了一个新的模型，即Rap1和Rap2通过两条独立的信号通路来传递突触抑制信号。我们将在这个模型中测试三个假设，分别有三个目标，使用器官类型培养的海马片制备。这种准备使我们能够使用生理学、药理学和重组蛋白传递方法来操纵突触活性和信号分子的活性。我们将通过检测电生理标记的重组AMPA-R介导的电流，测量GluR1和GluR2基因敲除小鼠的突触反应，以及量化磷酸化或活跃的内源性信号分子和谷氨酸受体来测试这些操作的效果。结合这些方法，我们将确定：(1)Rap1-p38MAPK信号有限而Rap2-JNK信号去增强；(2)不同下游信号分子传递Rap1-p38MAPK和Rap2-JNK通路；(目的3)不同上游信号分子控制Rap1-p38MAPK和Rap2-JNK通路。由于控制Rap信号通路的信号分子或酶的遗传缺陷会导致严重的精神发育迟缓，这项研究的发现也应该为可能有效治疗这些潜伏的精神疾病的新的遗传和药物策略提供更多的分子靶点。

DESCRIPTION (provided by applicant): Long-term synaptic depression (LTD) and depotentiation, the two forms of sustained synaptic depression after periods of repetitive synaptic activity, are extensively studied examples of vertebrate synaptic plasticity. The cellular and molecular mechanisms responsible for LTD and depotentiation will likely elucidate physiological and pathological phenomena of neural development, adaptation, learning and memory. There is now compelling evidence that repetitive synaptic activity leads to activation of NMDA-sensitive glutamate receptors (NMDA-Rs) and removal of postsynaptic AMPA-sensitive glutamate receptors (AMPA-Rs) from excitatory synapses during LTD and depotentiation. However, the biochemical pathways that link NMDA-R activity to AMPA-R trafficking are largely unknown. We have previously reported that small GTPase Rap1 controls LTD via activation of p38MAPK. In a preliminary study, we observed that small GTPase Rap2 controls depotentiation via activation of JNK. Based on these findings, I proposed a new model that Rap1 and Rap2 signal synaptic depression via two independent signaling pathways. We will test three hypotheses in this model with three aims, respectively, using an organotypic culture hippocampal slice preparation. This preparation allows us to manipulate synaptic activity and signaling molecules' activity using physiology, pharmacology and recombinant protein delivery methods. We will assay the effects of these manipulations by examining electrophysiologically tagged recombinant AMPA-R-mediated currents, measuring synaptic responses in GluR1 and GluR2 knockout mice, as well as quantifying phosphorylated or active endogenous signaling molecules and glutamate receptors. Combining these approaches, we will determine whether: (Aim 1) Rap1-p38MAPK signals LTD whereas Rap2-JNK signals depotentiation; (Aim 2) different downstream signaling molecules relay Rap1-p38MAPK and Rap2-JNK pathways; and (Aim 3) different upstream signaling molecules control Rap1-p38MAPK and Rap2-JNK pathways. Because genetic defects in signaling molecules or enzymes controlling Rap signaling pathways lead to severe mental retardation, the findings from this study should also suggest additional molecular targets for novel genetic and pharmacological strategies that may efficaciously treat these insidious mental diseases.