泛素连接酶RNF220在中枢去甲肾上腺素能神经元发育中的功能研究

In the vertebrate central neural system, noradrenergic neurons is mainly found in a distict metencephalic nucleus---the locus coeruleus---and a series of loosely organized cell groups that are distributed thoroughout the hindbrain. Noradrenaline transmission is associated with a wide range of physical and behaviroal process. Dysfunction of the noradrenaline system leads to a variety of neurological disorders, such as depression, epilepsy and Parkinson’s disease. Athough the central noradrenergic neurons development is mainly controlled by BMP-Mash1-Phox2a-Phox2b-TH/DBH/AADC pathway, the precise molecular mechanism is largely unknown. The E3 ligase RNF220 is a new and function unknown gene which strongly and specially expresses in the midbrain, hind-brain and ventral spinal cord in the mouse developing embryos. Through phenotyping on the RNF220 knockout mice, we found that the RNF220 is requried for the central noradrenergic neurons development and works upstream of Phox2a. And also, we found that RNF220 enhances cannoical Wnt singaling through USP7 mediated deubiquitination of β-catenin in in vitro cultured mammalian cell lines. Based on the previous findings, we will analyze the roles and molecular mechanism of RNF220 during the central noradrenergic neurons development and try to raise the central noradrenergic neurons specifically defected mouse model in this project. With this project, we hope to reveal new mechanisms controlling the vertebrate central noradrenergic neurons development and raise the mouse model with dysfunctioned central noradrenergic neurons.

中枢去甲肾上腺素（Noradrenaline，NE）能神经元主要位于后脑的蓝斑核及更尾侧的少量神经元核团，参与动物机体众多生理机能及躯体行为的调控，但目前关于其发育的调控机制仍很不清楚。RNF220基因编码一种泛素连接酶，特异表达于小鼠胚胎的中后脑及脊髓的腹侧区域。通过基因敲除小鼠模型的分析，我们发现RNF220是中枢NE能神经元发育所必需的，且在其发育调控网络中的Phox2a的上游发挥功能。体外实验发现，RNF220与USP7共同调控β-catenin蛋白稳定性，从而正调控经典Wnt信号通路。基于前期发现，本项目拟开展以下工作：1）分析RNF220在中枢NE能神经元发育调控网络中的功能。2）筛选其靶蛋白，解析其参与中枢NE能神经元发育的分子机制。3） 建立中枢NE能神经元特异缺陷的小鼠模型。本研究有望揭示调控中枢NE能神经元发育的新的分子机制，并建立中枢NE能神经元缺陷的小鼠模型。

中枢去甲肾上腺素能神经元的发育分化由BMP-Mash1-Phox2a/2b-TH/DBH通路介导完成。原位杂交和免疫荧光的分析发现，泛素连接酶RNF220在发育中的中枢去甲肾上腺素能神经元中特异高表达。通过对RNF220基因敲除小鼠的表型分析发现，RNF220基因敲除小鼠中枢去甲肾上腺素能神经元不能正常分化发育。进一步的实验分析发现，RNF220敲除小鼠中，Phox2a/2b可以正常起始表达，但其表达不能维持，导致其下游的TH/DBH不能正常表达，从而导致中枢去甲肾上腺素能神经元发育分化的缺陷。体外生化实验分析发现，RNF220可以与Phox2a/2b相互作用，且可以促进Phox2a/2b的单泛素化修饰。通过一些列的突变分析发现，RNF220对Phox2a/2b的单泛素化修饰发生在Phox2a的K178以及Phox2b的K185位点上。通过对下游靶基因表达水平的分析发现，RNF220介导的Phox2a/2b的泛素化修饰可以增强其转录活性。因此，上述结果揭示了泛素连接酶RNF220在中枢去甲肾上腺素能神经元分化发育中的功能和作用机制。