Role of kalirin-7 and Rac1 in synaptic plasticity

描述（由申请人提供）：发育和突触可塑性过程中树突棘数量和形状的活动依赖性变化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至关重要。我们的长期目标是了解调节树突棘活性依赖性可塑性的信号机制。Rho样小GTP酶是肌动蛋白细胞骨架和棘形态发生的中心调节因子。我们以前确定了Rac 1-鸟嘌呤-核苷酸交换因子（GEF）kalirin-7作为神经元中棘形态发生的关键调节因子。然而，目前尚不清楚kalirin-7和Rac 1是否受突触活动的调节，以及它们是否调节活动依赖性的脊柱结构可塑性。在初步数据部分，我们表明：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酪氨酸磷酸化受突触活性调节; 3）在培养的神经元中，结构可塑性可以通过NMDA受体依赖性机制诱导; 4）NMDA受体诱导的棘结构可塑性由含PDZ结构域的蛋白AF-6介导，其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Rac 1调节活性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）检查突触活性依赖性调节Kalirin-7酶促GEF活性和Rac 1与GTP结合; 2）检查Kalirin-7与NMDA受体的关联以及突触活性对Kalirin-7和Rac 1的突触易位的调节; 3）通过延时成像和光漂白后荧光恢复（FRAP）可视化EGFP标记的kalirin-7和Rac 1在神经元中的实时易位; 4）评估kalirin-7和Rac 1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物。

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.