Multidimensional analysis of small GTPases as key molecules in the neural network formation by using activity-imaging

在本研究中，我们使用基于gfp的FRET探针在PC12细胞、DRG神经元和海马神经元中对Ras和Rho gtpase进行了活性成像。在NGF诱导的PC12细胞神经突生长过程中，活性成像和RNA干扰研究表明，NGF刺激后局部PIP3的积累将Vav2和Vav3招募到质膜上，从而激活了Rac1和Cdc42；这种依赖于vav2 /3的Rac1和Cdc42的激活促进了导致神经突生长的短过程的形成。我们还提供证据表明，在NGF刺激后，Vav2和Vav3参与了突起处pi3激酶的局部激活。因此，本研究首次证明了Vav蛋白是pip3依赖的正反馈回路的组成部分，该回路随形态变化局部循环。我们观察了在层粘胶蛋白诱导的DRG神经元和N1E-115细胞生长锥推进过程中RhoA/Rac1/Cdc42的活性。在生长锥的p域，Rac1和Cdc42活性较高。通过对生长锥外周RhoA下调的模型，我们发现p区RhoA活性高于c区和轴突轴，并且生长锥延伸部分RhoA活性维持在较高水平。在lpa处理的N1E-115细胞中，具有生长锥的发育良好的神经突显示RhoA激活，但保持其延伸的形态，直到它们被吸引到收缩的体细胞。因此，轴上RhoA的激活导致神经突缩回，而p结构域的高RhoA活性对于保持神经生长锥的扩散形态是必要的。我们利用高密度海马神经元培养可视化突触后位点Ras和Rho gtpase的活性。这项研究表明，这些gtpase的每个成员在突触后位点都有一种特殊的激活模式。

In this study, we performed the activity-imaging of Ras and Rho GTPases using GFP-based FRET probes in PC12 Cells, DRG neurons, and hippocampal neurons.1.During NGF-induced neurite outgrowth in PC12 cells, the activity-imaging and RNA interference study showed that local PIP3 accumulation after NGF stimulation recruited Vav2 and Vav3 to the plasma membrane and thereby activated Rac1 and Cdc42 ; this Vav2/3-dependent activation of Rac1 and Cdc42 promoted the formation of short processes leading to neurite outgrowth. We also provide evidence that Vav2 and Vav3 are involved in the local activation of PI3-kinase at the protrusions after NGF stimulation. Therefore, this study demonstrates that Vav proteins are constituents for the first time that Vav proteins are constituents of the PIP3-dependent positive feedback loop that cycles locally with morphological changes.2.We visualized RhoA/Rac1/Cdc42 activities during laminin-induced growth cone advance of DRG neurons and N1E-115 cells. The Rac1 and Cdc42 activities were high in the P-domain of growth cones. Against a model involving RhoA down-regulation at the periphery of protruding growth cones, we found that the RhoA activity was higher in the P-domain than in the C-domain and axon shaft, and that a high level of RhoA activity was maintained in the extending part of growth cones. In LPA-treated N1E-115 cells, well-developed neurites with growth cones showed RhoA activation, but sustained their extended morphology until they were drawn toward the contracting somata. Thus, RhoA activation in the shaft results in neurite retraction, whereas high RhoA activity in the P-domain is necessary to retain the spread morphology of nerve growth cone.3.We visualized the activities of Ras and Rho GTPases within the postsynaptic sites using high-density cultures of hippocampal neurons. This study showed that each member of these GTPases had a peculiar pattern of activation in the postsynaptic sites.