Mechanisms of Synaptic Depression: Focus on Rap Signaling Pathways

描述（由申请人提供）：长期突触抑制（LTD）和去增强（depotentiation）是重复突触活动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。负责LTD和去增强的细胞和分子机制可能会解释神经发育，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，在LTD和去增强期间，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）从兴奋性突触中移除。然而，将NMDA-R活性与AMPA-R运输联系起来的生化途径在很大程度上是未知的。我们之前报道过小的GTPase Rap1通过激活p38MAPK来控制LTD。在初步研究中，我们观察到小的GTPase Rap2通过激活JNK来控制去增强。基于这些发现，我提出了Rap1和Rap2通过两个独立的信号通路信号突触抑制的新模型。我们将在这个模型中测试三个假设，分别有三个目标，使用器官型培养海马切片制备。这种制备使我们能够利用生理学、药理学和重组蛋白递送方法来操纵突触活动和信号分子的活动。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敲除小鼠的突触反应，以及量化磷酸化或活性内源性信号分子和谷氨酸受体来分析这些操作的效果。结合这些方法，我们将确定：（Aim 1） Rap1-p38MAPK信号有限而Rap2-JNK信号去增强；（目的2）不同的下游信号分子介导Rap1-p38MAPK和Rap2-JNK通路；和（Aim 3）不同的上游信号分子控制Rap1-p38MAPK和Rap2-JNK途径。由于控制Rap信号通路的信号分子或酶的遗传缺陷导致严重的智力低下，本研究的发现也应该为新的遗传和药理学策略提供额外的分子靶点，可能有效地治疗这些潜在的精神疾病。

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.