树突高尔基前哨运动的分子机制与功能

The structure and function of the Golgi complex are closely related to the physiological and pathological mechanisms of the nervous system. The Golgi complex in a neuron is present in both the soma and dendrites (dendritic Golgi Outposts, referred to as GOs). The previous studies show that the GOs involved in protein transport, dendritic development and synaptic plasticity, but the mechanism is still unclear. The applicant’s published work on the sensory neurons in larvae of Drosophila found that GOs has both single and multi-compartments structure; and further disclosed the molecular mechanism of GOs structure and the function on dendritic development, by using multi-color fluorescence to label different cisternae, combining with high resolution in vivo imaging. Meanwhile, the preliminary results show that the movement of the multi-compartments is closely related to the neuronal activities. The goal of the project is to reveal the molecular mechanism of the movement of the single/multiple compartments of GOs based on the unique bidirectional movement characteristics and then clarify the physiological functions of GOs. In the project, we should first characterize the motion of the single / multi-compartments, and establish the motion model of the single/multi compartments. The movement related binding proteins were identified by RNA interference screening, and the fusion protein and colocalization analysis were used to determine the specificity of protein localization. Finally, the studies will reveal the physiological function of GOs movement by using in vivo imaging on dendritic protein transport and dendritic development, and the regulation of touch sensory circuit. The elucidation of this mechanism will provide a new model for the non-canonical secretory pathway and sheds new insights into the mechanism of the neural diseases.

高尔基体与神经系统的生理病理机制密切相关。神经元的高尔基体同时存在于胞体和树突（树突高尔基前哨，简称前哨）。研究表明前哨参与蛋白转运，树突发育和突触可塑性，但机制仍未明。申请人前期以果蝇幼虫感觉神经元为对象，用膜囊特异的多色荧光标记，结合高分辨成像揭示前哨的单层和多层膜囊结构及分子机制。同时预实验发现，多膜囊前哨的运动与神经元活性密切相关。本研究将以前哨的双向运动特性为基础，揭示单／多膜囊型前哨运动的分子机制，用于阐述其生理功能。项目拟描述单／多膜囊型前哨的运动特征，建立单／多膜囊型前哨的运动模型。通过RNA干扰筛选发现运动相关的衔接蛋白；构建融合蛋白和共定位分析确定蛋白定位特异性；并使用突变与遗传拯救实验来验证充要性。最后，在体成像研究树突蛋白转运和树突发育，及触觉感觉的调控揭示前哨运动的生理功能。本机制的阐明将为非经典的蛋白分泌途径提供新模型，并为相关神经疾病研究提供新思路。

运动是树突高尔基体前哨（简称前哨）区别于胞体高尔基体的独特点之一。在本基金项目的支持下，我们以前哨的双向运动特性为基础，结合前哨独特的单/多膜囊的结构特点，从三方面开展了工作：前哨运动的动力学机制研究，老年痴呆症中前哨的运动改变机制研究，和前哨运动对神经元活动调控机制的研究。在动力学机制研究中，通过量化前哨运动行为，我们重点关注了前哨运动的两大特征：运动前哨的比例和顺逆向运动前哨的占比。进而，我们筛选出了马达蛋白和衔接蛋白，提出了动力蛋白亚基Dhc和驱动蛋白亚基Khc协同调控多膜囊前哨的顺逆向运动的模型。这些发现为后续机制和功能研究奠定了基础。在老年痴呆症机制研究中，我们研究了淀粉样蛋白前体蛋白(APP)导致前哨运动异常的分子机制。结果发现，APP通过调节动力蛋白亚基Dhc和衔接蛋白Syd改变了多膜囊前哨的顺逆向运动的占比，从而导致了树突退化。这为揭示老年痴呆症中神经元树突缺陷提供了新思路。在前哨运动对神经元活动调控机制的研究中，我们发现多膜囊前哨运动比例是神经元的内在兴奋性变化的细胞学指标，运动的多膜囊前哨通过调节神经元内在兴奋性，进而操控了神经元的活动。这一研究，首次揭示了前哨相关的生理功能，同时也为神经可塑性的发生机制开拓了新方向。总之，本项目系统性的研究了前哨运动特征，建立了前哨运动功能与疾病及神经活动的关联，填补了前哨病理和生理功能的研究空白。