Role of kalirin-7 and Rac1 in synaptic plasticity

描述（由申请人提供）：在发育过程中树突棘的数量和形状的活动依赖性变化和突触可塑性对于神经元回路的形成、学习和记忆以及智力迟钝和精神疾病的病因至关重要。我们的长期目标是了解调节树突棘活动依赖性可塑性的信号机制。rho样小gtpase是肌动蛋白细胞骨架和脊柱形态发生的中枢调节因子。我们之前发现rac1 -鸟嘌呤-核苷酸交换因子（GEF） kalirin-7是神经元脊柱形态发生的关键调节因子。然而，kalirin-7和Rac1是否受突触活动的调节，以及它们是否调节活动依赖性脊柱结构可塑性，目前尚不清楚。在初步数据部分，我们发现：1)kalilin -7向突触的募集受到酪氨酸磷酸化和潜在的突触活性的调节；2)钾氨酸-7酪氨酸磷酸化受突触活性调控；3) NMDA受体依赖机制可诱导培养神经元的结构可塑性；4) nmda受体诱导的脊柱结构可塑性是由含有PDZ结构域的蛋白AF-6介导的，在酵母2杂交筛选中，AF-6与kalirin-7相互作用。基于这些观察，我们假设kalirin-7和Rac1调节活动依赖性突触结构可塑性。为了验证这一假设，我们提出了以下具体目标：1)研究Rac1对kalirin-7酶GEF活性和gtp结合的突触活性依赖性调节；2)研究kaliin -7与NMDA受体的关联，以及通过突触活性调节kaliin -7和Rac1的突触易位；3)通过延时成像和光漂白后荧光恢复（FRAP）实时观察egfp标记的kalirin-7和Rac1在神经元中的易位；4)评估钾素-7和Rac1在活动依赖性脊柱形态发生中的作用和需求。这些研究将使用皮层和海马神经元的原代培养。

DESCRIPTION (provided by applicant): Activity-dependent changes in the number and shape of dendritic spines during development and synaptic plasticity are essential for the formation of neuronal circuits, in learning and memory, and in the etiology of mental retardation and mental illness. Our long-term objective is to understand the signaling mechanisms which regulate activity-dependent plasticity of dendritic spines. Rho-like small GTPases are central regulators of the actin cytoskeleton and spine morphogenesis. We previously identified the Rac1-guanine- nucleotide exchange factor (GEF) kalirin-7 as a key regulator of spine morphogenesis in neurons. However, it is not clear whether kalirin-7 and Rac1 are regulated by synaptic activity and whether they regulate activity-dependent spine structural plasticity. In the Preliminary data section, we show that: 1) kalirin-7 recruitment to synapses is regulated by tyrosine phosphorylation and potentially synaptic activity; 2) kalirin-7 tyrosine phosphorylation is regulated by synaptic activity; 3) structural plasticity can be induced in cultured neurons by an NMDA receptor-dependent mechanism; 4) NMDA-receptor-induced spine structural plasticity is mediated by the PDZ domain-containing protein AF-6, which interacted with kalirin-7 in a yeast 2-hybrid screen. Based on these observations, we hypothesize that kalirin-7 and Rac1 regulate activity-dependent synaptic structural plasticity. To test this hypothesis we propose the following specific aims: 1) to examine the synaptic activity-dependent regulation of kalirin-7 enzymatic GEF activity and GTP-binding by Rac1; 2) to examine the association of kalirin-7 with NMDA receptors and the regulation of synaptic translocation of kalirin-7 and Rac1 by synaptic activity; 3) to visualize the real-time translocation of EGFP-tagged kalirin-7 and Rac1 in neurons by time-lapse imaging and fluorescence recovery after photobleaching (FRAP); 4) to assess the role and requirement of kalirin-7 and Rac1 in activity-dependent spine morphogenesis. These studies will use primary cultures of cortical and hippocampal neurons.