Trio调控小脑颗粒神经元轴突发育的机制研究

Trio, as a founding member of an intriguing family of Dbl guanine nucleotide exchange factors (GEFs), contains two GEF domains. It is a key factor regulating the activity of the three most studied Rho GTPases, Rac1, Cdc42 and RhoA. By constructing Trio nervous system specific knockout mice (Trioflxo/flox;Nestin-Cre), our previous study showed that Trio controlled cerebellar granule cell axon development, including axon growth, axon branch, and axon guidance, indicating that Trio was required for cerebellar development. However, the molecular mechanism of Trio in controlling cerebellar development and cerebellar granule neuronal axon development is still unclear. In the present study, our preliminary data reveals that Trio null cerebellar granule cell (Trio-/-) is unresponsive to several neuronal signaling molecules, including Semaphorin6A, Slit2, Netrin1 and bFGF. Combining with other preliminary data, we propose that Trio may serve as a common molecule mediating mutiple neronal signaling pathways that control neuron growth, and the mechanism involved in Trio's effect on these pathways may be ralated to the dynamic regulation of membrane receptors. By using several gene knockout and transgene animal models and diverse exprimental techniques, such as in vivo neuronal tracing and live cell imaging, we will seek to clarify whether Trio controls the activity of different Rho GTPases in exquisitely precise spatial and temporal patterns via two GEF domains, thereby to coodinate the development of cerebellar granule neuronal axon. In addition, we will focus on two or three neuronal signaling pathways that Trio participates in to reveal Trio's function in these signaling pathways. Based on this study, we are expecting to propose a new molecular mechanism for regulating axon development.

Trio含有两个GEF结构域，是调节Rac1、Cdc42、RhoA的一个重要的分子。我们曾发现：Trio控制小脑颗粒细胞的轴突发育（包括生长，分支和导向），是小脑发育的必需分子，但其调控的分子机制还不清楚。本课题的前期研究发现：当Trio缺失时，小脑颗粒细胞的轴突生长对多种神经信号分子(如Semaphorin6A,Slit2,Netrin1,bFGF)的应答消失。结合其他初步结果，我们提出：Trio-GEF可能是控制轴突生长的神经信号通路通用的信号分子，其机制可能与膜受体蛋白的调控有关。为此，本课题将利用多种基因敲除和转基因的动物模型，神经示踪，活细胞观察等手段，首先从分子水平上阐明Trio是否通过双GEF功能域从时空上精确的调节不同的Rho GTPases，协调调控神经轴突发育，进而重点研究Trio在2-3个重要的神经信号通路中的作用方式，并提出新的关于神经细胞轴突发育的分子机制。

我们之前通过遗传学手段研究表明Trio是控制小脑正常发育的必须分子，其作用于小脑颗粒细胞的生长、分支和导向过程，但是其调控的分子机制并不清楚。为了阐明Trio鸟苷酸交换因子结构域介导的信号通路在小脑颗粒神经元发育中的机制，通过本课题的研究，我们分别建立了Trio两个鸟苷酸交换因子结构域的条件性敲除小鼠模型，但是这两个条件性敲除小鼠均表现为全长蛋白的缺失，TrioGEFD1敲除小鼠表型和之前的敲除小鼠表型一致，但是TrioGEFD2敲除小鼠表型却轻微许多，并且体外培养的TrioGEFD2结构域缺失的小脑颗粒神经元发育正常，这可能由于Trio8的互补功能。另一方面，为了研究Trio及其激活的下游蛋白对微管动力学的调节作用，我们建立了Tubb3-mCherry的BAC转基因小鼠。在该转基因品系中，红色荧光蛋白mCherry可以标记神经元中的微管蛋白，因而该转基因小鼠是研究神经元轴突生长的有用工具。