E3泛素连接酶NEDD4调控神经元细胞周期重返及凋亡的机制研究

Apoptosis represents one major common mechanism of neuronal death both in the nervous system injury and in neurodegenerative diseases. Aberrant cell cycle re-entry (CCE) has been found to be one mechanism leading to apoptosis in neurons. Because of its high specificity in protein degradation, ubiquitination is emerging as an important novel mechanism in cell cycle regulation. We found that the E3 ligase NEDD4 mediates the ubiquitination of both IGF-1R and PTEN, resulting in suppression and activation of PI3K/Akt signaling, respectively. Due to the important roles of PI3K/Akt signaling pathway in neuronal cell survival, we hypothesize that NEDD4 plays a role in neuronal cell cycle regulation. To test this hypothesis, we will use primary neurons to investigate multiple cell cycle associated molecules that may be regulated by NEDD4 (e.g., over-expressing NEDD4 using wt-NEDD4 or silencing NEDD4 with siRNA). Furthermore, we will investigate these changes under various neuronal stressed conditions such as oxygen-glucose deprivation, oxidation mediated by H2O2 and nitric oxide (NO) and Zn2+ homeostasis disturbance induced by ZnSO4; in particular, the downstream signaling and the sub-cellular localization of IGF-1R and PTEN will be studied in the context of neuronal cell cycle control and apoptosis. Lastly, we will validate our findings in an in vivo model, spinal cord contusion injury in mice (since IGF-1 and PI3K/Akt signaling has been found to be particularly important to the survival of motor neurons and cell cycle events have been associated with motor neuron death). We will try to elucidate the mechanism underlying neuronal cell cycle re-entry and designate potential targets for neuroprotection drugs.

凋亡是神经系统外伤和退行性疾病中神经元死亡的主要共同机制，异常的细胞周期重返（cell cycle re-entry）是凋亡的诱发因素之一，但调控机制尚不清楚。PI3K/Akt通路参与细胞周期调控并显著影响细胞的存活，IGF-1R和PTEN分别激活和抑制该通路。神经元E3泛素连接酶NEDD4同时介导IGF-1R和PTEN的泛素化，可能经PI3K/Akt通路调控神经元细胞周期重返和凋亡。为验证该假设1）建立NEDD4表达上调或下调细胞模型，检测可能受NEDD4调控的细胞周期蛋白；2）以培养脊髓神经元为离体模型，在氧糖剥夺等应激条件下检测NEDD4相关分子，把IGF-1R与PTEN的泛素化、亚细胞定位、下游信号与细胞周期调控和凋亡联系起来；3）以小鼠脊髓损伤为模型检测NEDD4相关信号通路，在体验证该假设。通过以上研究探讨NEDD4介导泛素化对神经元细胞周期重返的调控机制，发现神经保护新靶点。

异常的细胞周期重返是诱发神经元死亡的因素之一，但调控机制尚不清楚。PI3K/AKT通路在细胞存活和凋亡中起重要调控作用，胰岛素、血管内皮细胞生长因子（VEGF）等促生长和存活的因子都可以激活该信号途径，而抑癌基因PTEN则能够负调控该通路。泛素化是清除损伤或陈旧蛋白质的关键机制，神经元内主要的泛素连接酶NEDD4能介导PTEN的多泛素化和水解，激活PI3K/AKT通路，但NEDD4是否在细胞周期调控中发挥作用尚不清楚。.根据前期的结果和最新的进展，我们以VEGF作为生长因子增强NEDD4功能，并以MG132抑制蛋白酶体功能，考虑到炎症在脊髓损伤急性期的重要作用，同时也引入MIF、ISO-1等干预因素，观察NEDD4、PTEN对神经元细胞周期相关分子及信号通路的调控情况。主要结果显示，1）脊髓损伤后应激引起PI3K/AKT信号通路的短时间激活，同时NEDD4-1表达上调和PTEN磷酸化增强都引起PI3K/AKT通路的激活，有利于脊髓损伤后细胞的存活，但该激活仅能维持很短时间；2）虽然损伤初期细胞周期调控分子CDK4和CDK5的转录和翻译水平协同变化；3）在观测期（损伤后急性期）内，损伤引起急性的炎症反应，促炎因子MIF表达显著增强，MIF阳性细胞在损伤局部大量聚集；4）损伤后在MIF拮抗剂ISO-1作用下，NEDD4-1显著上调，AKT磷酸化持续升高，总PTEN增加但磷酸化水平没有显著改变，CDK4和CDK5的蛋白水平和转录水平均上调；5）意外的发现是MIF也能升高NEDD4-1水平，短时间增强AKT磷酸化，升高总PTEN水平，且在ISO-1作用下MIF也能维持高水平。研究结果提示，脊髓损伤后急性期内的炎症反应可能与NEDD4-1和PTEN的表达及相互作用密切相关，参与伤后细胞重返G1期的启动调控。.MIF是巨噬细胞迁移抑制因子，也发挥促炎效应，与PI3K/AKT等多条信号途径相关，能够调控细胞增殖、迁移、分化等。我们发现抑制MIF能进一步能增强NEDD4-1的表达和提高AKT的磷酸化水平，这为阐释NEDD4-1和PTEN复杂作用关系提供了新的切入点。同时，脊髓损伤后早期的炎症反应能够促进受损组织碎片和死亡细胞的清除，但过度的炎症反应对损伤后细胞存活和再生是有害的。在观测的损伤后急性期（3-5天），从MIF的角度进行调控可能找到促进脊髓损伤后再生的新途径。