Role of kalirin-7 and Rac1 in synaptic plasticity

描述(申请人提供)：树突棘的数量和形状在发育过程中的活动依赖性变化和突触可塑性对于神经元回路的形成、学习和记忆以及精神发育迟滞和精神疾病的病因学都是必不可少的。我们的长期目标是了解调节树突棘活性依赖可塑性的信号机制。Rho样小GTP酶是肌动蛋白细胞骨架和脊柱形态发生的中央调节因子。我们先前发现，rac1-鸟嘌呤-核苷酸交换因子Kalirin-7是神经元中脊髓形态发生的关键调节因子。然而，目前尚不清楚Kalirin-7和rac1是否受突触活动的调节，以及它们是否调节依赖活动的脊柱结构可塑性。在初步数据部分，我们发现：1)Kalirin-7向突触的募集受酪氨酸磷酸化和潜在的突触活性调节；2)Kalirin-7的酪氨酸磷酸化受突触活性调节；3)NMDA受体依赖的机制可以诱导培养神经元的结构可塑性；4)NMDA受体诱导的脊髓结构可塑性是由含有PDZ结构域的蛋白AF-6介导的，该蛋白在酵母双杂交筛选中与Kalirin-7相互作用。基于这些观察，我们假设Kalirin-7和rac1调节活性依赖的突触结构可塑性。为了验证这一假说，我们提出了以下具体目标：1)检测Kalirin-7酶催化的全球环境基金活性和Rac1与GTP结合的突触活性依赖的调节；2)检测Kalirin-7与NMDA受体的联系以及突触活性对Kalirin-7和rac1突触移位的调节；3)通过时延成像和光漂白后荧光恢复(FRAP)显示神经元中EGFP标记的Kalirin-7和rac1的实时移位；4)评估Kalirin-7和rac1在活性依赖的脊髓形态发生中的作用和要求。这些研究将使用大脑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.