Oxidative DNA Damage is Concurrently Repaired by BER and APE1-initiated NHEJ in Cortical Neurons.
Oxidative DNA Damage is Concurrently Repaired by BER and APE1-initiated NHEJ in Cortical Neurons.
复制标题
皮层神经元中 BER 和 APE1 启动的 NHEJ 同时修复氧化 DNA 损伤。
DOI：
10.1111/nan.12584
复制
发表时间：
2019
影响因子：
5
通讯作者：
Shang
中科院分区：
文献类型：
--
作者：
Jenq;Wei‐Yu Chen;S. Mukda;Yun;Shuhui Sun;Shang
旨在
越来越多的研究表明，碱基切除修复（BER）是神经元DNA氧化损伤的主要修复途径，而神经元缺乏其他DNA修复途径，包括核苷酸切除修复和同源重组修复。然而，一些研究表明，神经元可以有效地修复谷氨酸和甲萘醌诱导的双链断裂（DSB），这表明DSB修复机制可能与神经元的健康有关。在这项研究中，我们假设BER和非同源末端连接（NHEJ）共同修复神经元中的氧化DNA损伤。
方法
采用免疫组化和共聚焦显微镜观察AP内切酶1（APE 1）、组蛋白变体2AX（γH2AX）和磷酸化p53结合蛋白（53BP1）的共定位。分别应用APE 1抑制剂和shRNA抑制APE 1活性和蛋白表达，以确定APE 1与DSB形成的相关性。中性彗星试验用于确定和定量DSB的形成。
结果
在甲萘醌诱导的大鼠皮层神经元氧化损伤中，γH2AX和53BP1均上调，并与APE 1共定位。磷酸化53BP1灶被有效消除，但γH2AX灶在APE 1活性抑制后持续存在。彗星试验表明，抑制APE 1减少DSB的形成。
结论
我们的研究结果表明，APE 1可以参与NHEJ机制在神经元中的氧化DNA损伤的修复。这些发现提供了深入了解的机制，有效修复氧化DNA损伤的神经元，尽管高氧化负荷。
AIMS
Accumulating studies have suggested that base excision repair (BER) is the major repair pathway of oxidative DNA damage in neurons, and neurons are deficient in other DNA repair pathways, including nucleotide excision repair and homologous recombination repair. However, some studies have demonstrated that neurons could efficiently repair glutamate- and menadione-induced double-strand breaks (DSBs), suggesting that the DSB repair mechanisms might be implicated in neuronal health. In this study, we hypothesised that BER and non-homologous end join (NHEJ) work together to repair oxidative DNA damage in neurons.
METHODS
Immunohistochemistry and confocal microscopy were employed to examine the co-localization of AP endonuclease 1 (APE1), histone variant 2AX (γH2AX), and phosphorylated p53-binding protein (53BP1). APE1 inhibitor and shRNA were respectively applied to suppress APE1 activity and protein expression to determine the correlation of APE1 and DSB formation. The neutral comet assay was used to determine and quantitate the formation of DSB.
RESULTS
Both γH2AX and 53BP1 were upregulated and co-localized with APE1 in the nuclei of rat cortical neurons subjected to menadione-induced oxidative insults. Phospho53BP1 foci were efficiently abolished, but γH2AX foci persisted following the suppression of APE1 activity. Comet assays demonstrated that inhibition of APE1 decreased the DSB formation.
CONCLUSIONS
Our results indicate that APE1 can engage the NHEJ mechanism in the repair of oxidative DNA damage in neurons. These findings provide insights into the mechanisms underlying the efficient repair of oxidative DNA damage in neurons despite the high oxidative burden.