Model Animal Research Center, State Key Laboratory of Pharmaceutical Biotechnology, Department of Neurology of the Affiliated Nanjing Drum Tower Hospital of Nanjing University Medical School, Nanjing University, Nanjing, 210061, China.
Model Animal Research Center, State Key Laboratory of Pharmaceutical Biotechnology, Department of Neurology of the Affiliated Nanjing Drum Tower Hospital of Nanjing University Medical School, Nanjing University, Nanjing, 210061, China.
J Genet Genomics. 2019 Feb;46(2):87-96. doi: 10.1016/j.jgg.2019.02.003. Epub 2019 Feb 23.
As a critical guanine nucleotide exchange factor (GEF) regulating neurite outgrowth, Trio coordinates multiple processes of cytoskeletal dynamics through activating Rac1, Cdc42 and RhoA small GTPases by two GEF domains, but the in vivo roles of these GEF domains and corresponding downstream effectors have not been determined yet. We established multiple lines of knockout mice and assessed the respective roles of Trio GEF domains and Rac1 in axon outgrowth. Knockout of total Trio in cerebellar granule neurons (CGNs) led to an impaired F-actin rearrangement of growth cone and hence a retarded neurite outgrowth. Such a retardation was reproduced by inhibition of GEF1 domain or knockdown of Cdc42 and restored apparently by introduction of active Cdc42. As Rac1 deficiency did not affect the neurite outgrowth of CGNs, we suggested that Trio GEF1-mediated Cdc42 activation was required for neurite outgrowth. We established a GEF2-knockout line with deletion of all Trio isoforms except a cerebella-specific Trio8, a short isoform of Trio without GEF2 domain, and used this line as a GEF2-deficient animal model. The GEF2-deficient CGNs had a normal neurite outgrowth but abolished Netrin-1-promoted growth, without affecting Netrin-1 induced Rac1 activation. We thus suggested that Trio GEF1-mediated Cdc42 activation rather than Rac1 activation drives the F-actin dynamics necessary for neurite outgrowth, while GEF2 functions in Netrin-1-promoted neurite elongation. Our results delineated the distinct roles of Trio GEF domains in neurite outgrowth, which is instructive to understand the pathogenesis of clinical Trio-related neurodevelopmental disorders.
作为调控神经突生长的关键鸟嘌呤核苷酸交换因子（GEF），Trio 通过两个 GEF 结构域激活 Rac1、Cdc42 和 RhoA 小 GTPases，协调细胞骨架动力学的多个过程，但这些 GEF 结构域及其相应的下游效应物的体内作用尚未确定。我们建立了多种敲除小鼠品系，并评估了 Trio GEF 结构域和 Rac1 在轴突生长中的各自作用。小脑颗粒神经元（CGNs）中 Trio 的完全敲除导致生长锥中 F-肌动蛋白重排受损，从而导致神经突生长迟缓。GEF1 结构域的抑制或 Cdc42 的敲低可再现这种迟缓，而活性 Cdc42 的引入则明显恢复了这种迟缓。由于 Rac1 缺陷并不影响 CGNs 的神经突生长，因此我们认为 Trio GEF1 介导的 Cdc42 激活对于神经突生长是必需的。我们建立了一个 GEF2 敲除系，该系缺失了除小脑特异性 Trio8 以外的所有 Trio 同工型，Trio8 是一种缺乏 GEF2 结构域的 Trio 短同工型，并将该系用作 GEF2 缺陷动物模型。GEF2 缺陷的 CGNs 具有正常的神经突生长，但取消了 Netrin-1 促进的生长，而不影响 Netrin-1 诱导的 Rac1 激活。因此，我们认为 Trio GEF1 介导的 Cdc42 激活而不是 Rac1 激活驱动了神经突生长所必需的 F-肌动蛋白动力学，而 GEF2 则在 Netrin-1 促进的神经突伸长中发挥作用。我们的结果描绘了 Trio GEF 结构域在神经突生长中的不同作用，这有助于理解临床 Trio 相关神经发育障碍的发病机制。
