p150glued通过其微管结合域调控COPII介导的内质网输出在运动神经元变性死亡中的作用及机制研究

p150glued参与微管依赖的物质运输，其微管结合域功能缺失型突变G59S引起家族性运动神经元病。我们之前创建的p150glued微管结合域缺失的转基因小鼠表现出特异性的脊髓运动神经元变性死亡，但机制不明。本项目预实验发现p150glued微管结合域缺失：①改变COPII亚基的含量、分布及与微管正端结合蛋白的相互作用；②上调转运至细胞膜的AMPA和NMDA受体亚基的含量及神经元对谷氨酸兴奋性毒性的易感性；③激活未折叠蛋白反应信号通路。我们推测p150glued微管结合域缺失通过影响COPII介导的内质网输出而增强谷氨酸受体的膜运输及兴奋性毒性效应、并引发内质网应激，从而导致脊髓运动神经元损伤和变性。我们将使用形态学、电生理、细胞与分子生物学技术，阐明p150glued通过其微管结合域调控COPII介导的内质网输出在运动神经元变性死亡中的作用及机制，为运动神经元病的治疗提供新的靶点和依据。

Dynactin subunit p150glued plays a key role in dynein/dynactin-driven microtubule minus end-directed intracellular transport. G59S mutation within the microtubule binding domain (MTBD) of p150glued disrupts its microtubule-binding function, causing familial motor neuron disease with selective degeneration of lower motor neurons. In our previous work, by using Cre-LoxP strategy we generated a new line of transgenic mouse which only express MTBD-truncated p150glued. The transgenic mouse exhibited selective degeneration of spinal motor neurons, but the pathogenic mechanism is still elusive. Our preliminary study showed that loss of MTBD in p150glued changed protein level and distribution of COPII subunits, and altered the interaction between COPII subunit Sec23 and microtubule plus-end binding protein EB1. Moreover, loss of MTBD in p150glued increased the total and neuronal surface protein level of AMPA and NMDA-type glutamate receptor subunits, and exacerbated glutamate-induced neuronal excitotoxicity. In addition, loss of MTBD in p150glued activated the endoplasmic reticulum unfolded protein response signaling pathway. We hypothesize that loss of MTBD in p150glued up-regulates the surface delivery and excitotoxic effect of ionotropic glutamate receptors and increases endoplasmic reticulum stress level through modulation of COPII-mediated endoplasmic reticulum export, and thus results in damage and degeneration of spinal motor neurons. By working on the transgenic mice and cultured cells and using experimental techniques of morphology, electrophysiology, cellular and molecular biology, we will investigate the role and mechanism of p150glued's regulation of COPII-mediated endoplasmic reticulum export via its MTBD in the pathogenesis of motor neuron degeneration. The results of our study will provide new clues for the treatment of patients with motor neuron disease.