Iduna调控PTEN泛素化在辐射后脊髓神经元凋亡中的作用和机制

Ubiquitin ligase Iduna acts a critical role in the endogenous neuroprotection. Previously researchers focused on Iduna regulating PARP1-pathways to alleviate oxidative damage of neurons, instead of its effects on apoptosis. We found that Iduna was down-regulated and an anti-oncogene PTEN was up-regulated in rat spinal cords with irradiation. Recently, a report showed that Iduna could induce PTEN ubiquitination and degradation in breast tumor. Is Iduna involved in PTEN up-regulation in RSCI rats? What relationship between spinal cord neuron apoptosis and PTEN upregulation after radiation? This project intends to identify the relationship between Iduna and PTEN in neuron apoposis in vivo and in vitro using immunofluorescence combined with Tunel staining. In two neuron models with up-regulated or down-regulated Iduna by virus infection, Western blot is performed to analyze PTEN expression and its phosphorylation level. The regulating mechanism of Iduna on PTEN is clarified by the Co-IP and ubiquitin detection. In vitro neuron and in vivo with Iduna null mice, the effect of Iduna regulating PTEN on neuron apoptosis with irradiation is confirmed. All of these jobs will clarify the mechanism of Iduna regulating PTEN in RSCI.

泛素连接酶Iduna在内源性神经保护中具有重要作用。既往多关注Iduna调控PARP1通路对神经元氧化损伤的影响，而对其与凋亡的关系研究甚少。我们发现辐射后大鼠脊髓中存在Iduna下调、抑癌基因PTEN上调及神经元数量减少的现象。新近发现Iduna能诱导PTEN泛素化降解而调控肿瘤生长。那么辐射后大鼠脊髓中PTEN上调是否与Iduna有关？PTEN与辐射后脊髓神经元凋亡的关系如何？本项目拟通过免疫荧光结合Tunel染色明确辐射后小鼠脊髓及离体细胞Iduna、PTEN表达与神经元凋亡的关系；在神经元中通过RNAi及病毒感染调控Iduna表达，免疫印迹分析PTEN表达及磷酸化变化规律；利用Co-IP及泛素化检测了解Iduna调控PTEN的机制；通过细胞实验及基因敲除鼠模型分析Iduna抑制PTEN的调控方式对辐射后神经元凋亡的影响，阐明Iduna对PTEN的调控异常在脊髓辐射损伤中的分子机制。

放射性脊髓损伤（Radiation spinal cord injury，RSCI）是继发于放疗后以神经元损伤为主要病理表现的神经系统不可逆并发症。我们前期发现具有E3泛素连接酶活性的神经保护分子Iduna（其编码基因为RNF146）能够通过抑制PARP1依赖的、非caspase死亡通路（Parthanatos），对辐射后脊髓神经元的存活发挥保护作用。然而PARP1抑制剂联合caspase抑制剂的神经保护效果显著高于单独PARP1抑制剂。其中抑癌基因PTEN在辐射脊髓组织中表达显著上调，与此同时伴有脊髓神经元凋亡增加。深入研究中我们发现PTEN是Iduna的下游效应分子，Iduna通过调控PTEN的泛素化降解来控制脊髓神经元的凋亡速率。大剂量电离辐射正是通过引起脊髓神经元中Iduna表达下调，导致其对PTEN的阻遏调控失效，因而出现了神经元凋亡增加的现象。神经元中上调Iduna的表达，能够有效促进PTEN的泛素化修饰进而诱导其在蛋白水平发生降解。我们的工作丰富了Iduna在脊髓神经元保护方面的相关机制，为下一步开发基于Iduna的神经保护靶点提供了理论支撑。