DDX3/DDX5在ALS发病中对Wnt信号通路的调控作用及分子机制

Amyotrophic lateral sclerosis (ALS) is a chronic neurodegenerative disorder characterized by progressive degeneration and death of motor neurons. The precise mechanisms have not been fully understood and there are no effective treatments available. Our previous study showed that the Wnt/β-catenin signaling pathway was activated in the pathogenesis of ALS. We aslo found that both DDX3 and DDX5 mRNA were up-regulated during the onset of ALS. Studies have confirmed that DDX3 and DDX5 can trigger the Wnt signaling or be associated with the activation of its transcription. But it is still not clear whether DDX3 and DDX5 are involved in the development and progression of ALS by regulating the Wnt signaling pathway. We will examine the expression pattern of DDX3 and DDX5 in the pathogenesis of ALS, using SOD1G93A ALS transgenic mice animal model, NSC34 cell model with SOD1G93A mutation and primary cultured astrocytes model from SOD1G93A transgenic mice by a variety of studies in vitro and in vivo, so as to define the regulatory role of DDX3 and DDX5 for the Wnt signaling pathway. We will focus on the role and molecular mechanism of DDX3 and DDX5 by regulating the Wnt signaling pathway in the motor neuron degeneration of ALS, which may provide a new target for the effective treatment of ALS.

肌萎缩侧索硬化症(ALS)是一种以运动神经元进行性变性、死亡为主要特征的慢性神经退行性疾病。目前，其发病机制尚不清楚，也无有效的治疗方法。我们前期研究发现，ALS发病过程中Wnt/β-catenin信号通路被激活。预实验结果显示，DDX3和DDX5 mRNA在ALS发病期均上调。研究报道，DDX3和DDX5与Wnt信号启动或转录激活有关，但DDX3/DDX5是否通过调控Wnt信号通路参与ALS发生和发展尚不明确。本项目拟采用SOD1G93AALS转基因鼠动物模型、SOD1G93A突变的NSC34运动神经元以及原代培养的星形胶质细胞模型，通过多种在体和离体实验方法，体内体外实验相结合，研究DDX3/DDX5在ALS发生发展中的表达规律，明确DDX3/DDX5对Wnt信号通路的调控作用，阐明DDX3/DDX5调控Wnt信号通路在ALS运动神经元退变中的作用及分子机制，为ALS临床治疗提供新靶点

肌萎缩侧索硬化症(ALS)是一种以运动神经元进行性变性、死亡为主要特征的慢性神经退行性疾病，发病机制不明确，无有效治疗方法。在前期发现ALS发病期Wnt/β-catenin信号通路被激活的基础上，本研究选取SOD1-G93A突变的ALS转基因鼠动物模型、SOD1-G93A突变的NSC34运动神经元以及原代培养的星形胶质细胞模型，通过RT-PCR、Western blot、免疫荧光染色、激光共聚焦技术、流式细胞术、MTS、EdU等多种技术方法，体内体外实验相结合，深入探讨了DDX3/DDX5在ALS发病中对Wnt信号通路的调控作用及机制，结果显示：（1）与野生型鼠相比，DDX3、DDX5以及Wnt信号分子CK1ε和TCF12等在ALS转基因鼠发病期脊髓、脑干、海马、纹状体、大脑皮层等多种部位表达异常，DDX3和CK1ε与神经元共表达，DDX5和TCF12与神经元和星形胶质细胞均共表达，DDX5/GFAP、TCF12/GFAP双阳性细胞在ALS鼠脊髓和脑干明显增多，表明DDX3和CK1ε表达改变与ALS运动神经元退变密切相关，而DDX5和TCF12表达改变与ALS运动神经元退变和星形胶质细胞增生均密切相关。（2）选取ALS转基因鼠脊髓组织，通过免疫共沉淀实验证实DDX3和CK1ε存在相互作用，DDX5与TCF12、Cyclin D1均存在相互作用。（3）SOD1-G93A突变型NSC34细胞中应用小干扰RNA分别沉默DDX3和DDX5，发现DDX3通过调控CK1ε蛋白抑制SOD1突变型NSC34细胞的增殖、促进细胞凋亡，影响神经突起的生长。DDX5通过调节TCF12和Cyclin D1抑制SOD1突变NSC34细胞的增殖、促进细胞凋亡，但不影响神经突起的生长。（4）DDX5和TCF12在原代培养的SOD1-G93A突变型星形胶质细胞中表达高于原代培养的野生型星形胶质细胞。应用基因沉默慢病毒感染SOD1突变型星形胶质细胞，发现沉默DDX5抑制了星形胶质细胞的增殖，降低了TCF12和Cyclin D1的表达，表明在ALS发病过程中，DDX5通过调节Wnt信号分子TCF12 和Cyclin D1影响星形胶质细胞增生。本研究明确了DDX3和DDX5通过调控Wnt信号通路参与ALS发生和发展，为诠释ALS发病机制提供了新的视角，为ALS治疗靶点筛选和新药开发提供了实验依据。