构建转基因斑马鱼模型探索轴突微管运输系统损伤致DA神经元逆行变性机制

The mechanism of the retrograde degeneration and death of dopaminergic (DA) neuron caused by the injury to axonal microtubule system is unknown. Recent investigations in vitro and our previous studies suggest that the dysfunciton of factor in axonal microtubule system is one of the probable initial reason of the degenerative process in DA neurons. Based on our previous work, this project will start with Dynactin, the key molecule affecting the retrograde transportation of axon. First, viviperception of GFP labeled DA neurons and its projection pathway between substantia nigra of midbrain and corpora striatum in transgenic zebrafish will be conducted using the confocal laser scanning microscope. The effect of Dynactin knock-down on the retrograde degeneration and death of DA neurons will be investigated. The degeneration and death of DA neurons caused by the dysfunction of retrograde transport factor will be verified. The rapid screening model for the lead compounds of microtubule stabilizing drugs will be established using a transgenic zebrafish. Then the environmental toxins induced degeneration of DA neurons mediated by the dysfunction of Dynactin will be verified. Microtubule stabilizing agents will be screened from a plant small molecular library using the transgene zebrafish model. Through experiments above, we try to demonstrate that the dysfunction of Dynactin in the axonal retrograde transport system is one of the critical factors that result in the retrograde death of DA neurons, and the environmental toxins can act on Dynactin to induce the retrograde degeneration and death of DA neurons. This project will provide experimental evidence for clinical treatment on PD and a screening model for natural molecules or compounds which have an effect on the stability of microtubule tranportation system.

轴突微管系统损伤致中脑黑质多巴胺（DA）神经元逆行变性死亡的机制尚不清楚。近年的体外研究和我们的前期实验提示，轴突微管运输系统分子的损伤很可能是DA神经元变性的重要原因。本项目从影响轴突逆行运输功能的关键分子-动力激活蛋白（dynactin）入手，构建GFP标记DA神经元的转基因斑马鱼，活体观察中脑黑质-纹状体DA神经元和投射纤维通路；研究dynactin敲降对DA神经元逆行变性和死亡的作用；证实轴突逆向运输分子受损导致DA神经元变性和死亡。同时证实环境毒素通过损伤dynactin诱发DA神经元变性；确定转基因斑马鱼作为微管稳定性药物先导化合物快速筛选模型并初步应用。目标：证实dynactin损伤是DA神经元逆行死亡的关键因素之一，dynactin也是环境毒素的重要作用靶点。该研究将为临床治疗PD提供实验依据，也为筛选稳定微管运输系统的天然分子或化合物提供模型。

轴突微管系统损伤致中脑黑质多巴胺（DA）神经元逆行变性死亡的机制尚不清楚。近年的体外研究和我们的前期实验提示，轴突微管运输系统分子的损伤很可能是DA神经元变性的重要原因。本项目从影响轴突逆行运输功能的关键分子—动力激活蛋白（dynactin）入手，构建GFP标记DA神经元的转基因斑马鱼，活体观察中脑黑质—纹状体DA神经元和投射纤维通路；斑马鱼间脑腹侧的7群细胞中，DC2和DC4细胞群的构成细胞主要为DA神经元细胞，而DC1、DC3、DC5和DC6等细胞群则可能不含有DA神经元或者数量较少；构成前顶盖区域和端脑区域的神经元主要为DA神经元，而构成蓝斑和和中缝核的神经元很可能并没有DA神经元。TPp区DA神经元的数量在发育过程中先增多后减少。本研究在较大程度上完善和补充了斑马鱼脑部DA神经元的定位和分布。为研究帕金森病发育机制和帕金森病药物筛选建立了良好的前期实验基础。.采用激光共聚焦显微镜首次观察并清晰记录了发育4days的Vmat2:GFP转基因斑马鱼间脑腹侧神经元投射，证明TPp区的DC2神经元轴突投射到端脑部位，而其他神经细胞群轴突投射的区域目前还不明确。与对照组相比用MPTP处理了发育3天的斑马鱼头部荧光强度（前顶盖及腹侧间脑）明显减弱;MPTP组与对照组斑马鱼形态存在显著差异，尾部向背侧弯曲，头部后仰，存在明显毒性反应。敲降dynactin对斑马鱼的形态发育及行为学都有明显影响，且导致多巴胺神经元减少。体外实验证实敲降dynactin，synuclein在突起积聚增加。在体内实验中证实Synuclein蛋白的积聚并不是由于synuclein来源增加造成。提示，在dynactin敲降的个体中synuclein蛋白的降解受阻。作为dynactin的特异性抑制剂MB使斑马鱼发育缓慢以及行为学的改变与dynactin MO具有相似的作用，二者可互为补充。证实了dynactin敲降对DA神经元逆行变性和死亡的作用；证实轴突逆向运输分子dynactin损伤是DA神经元逆行死亡的关键因素之一。该研究将为临床研究治疗PD提供实验依据。.另外，本项目还研究了MCM5 基因功能敲降对后脑面部运动神经元发育的作用及机制，以及dynactin亚基的磷酸化修饰在DA神经元退变中发挥重要的功能。