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Abstract：
Injured neurons can initiate their own neurotoxin-induced repair mechanisms by expressing protective genes and activating specific intracellular signal transduction pathways. Although glial cell-derived neurotrophic factor (GDNF) plays a key role in the repair of dopaminergic (DA) neurons, whether there is high expression of GDNF in DA neurons at an early stage of injury has not yet been reported. In this study, neurotoxin-induced GDNF overexpression was detected for the first time in MES23.5 DA immortalized neuroblastoma (MES23.5 DA) cells soon after 6-hydroxydopamine (6-OHDA) treatment. We also observed that the phosphorylation of Akt1, a member of the protein kinase B family, was increased. Further studies showed that activated Akt1 increased the phosphorylation of the protein phosphatase Eya1, which is a member of the eyes absent (Eya) family of transcriptional cofactors. Then, activated Eya1 decreased the phosphorylation of the sine oculis-related homeobox 2 (Six2) transcription factor. In addition, chromatin immunoprecipitation coupled with quantitative polymerase chain reaction (ChIP-qPCR) revealed that Six2 promoted GDNF transcription in MES23.5 DA cells by directly binding to the GDNF promoter. Finally, we showed that inhibiting neurotoxin-induced GDNF overexpression increased MES23.5 DA cell death, while promoting GDNF expression via Six2 overexpression decreased DA neuronal death. These results suggest that MES23.5 DA cells with early 6-OHDA-induced injury can promote the overexpression of GDNF by activating the Akt1/Eya1/Six2 signaling pathway, and this overexpression of GDNF has protective effects on injured MES23.5 DA cells. Hence, this study highlights a new target for drug development for the treatment of Parkinson\'s disease.
摘要：
损伤的神经元可以通过表达保护基因和激活特定的细胞内信号转导途径来启动其自身的神经毒素诱导的修复机制。尽管神经胶质细胞源性神经营养因子（GDNF）在多巴胺能（DA）神经元的修复中起着关键作用，GDNF在损伤早期是否在DA神经元中高表达尚未见报道。在这项研究中,在6-羟基多巴胺(6-OHDA)治疗后不久,在MES23.5DA永生化神经母细胞瘤(MES23.5DA)细胞中首次检测到神经毒素诱导的GDNF过表达.我们还观察到蛋白激酶B家族成员Akt1的磷酸化，增加了。进一步的研究表明，激活的Akt1增加了蛋白磷酸酶Eya1的磷酸化，Eya1是眼睛缺失（Eya）转录辅因子家族的成员。然后，激活的Eya1降低了正弦眼相关同源盒2（Six2）转录因子的磷酸化。此外,染色质免疫沉淀与定量聚合酶链反应（ChIP-qPCR）偶联显示，Six2通过直接结合GDNF启动子来促进MES23.5DA细胞中GDNF的转录。最后,我们表明，抑制神经毒素诱导的GDNF过表达增加MES23.5DA细胞死亡，而通过Six2过表达促进GDNF表达则减少了DA神经元的死亡。这些结果表明，6-OHDA诱导的早期损伤的MES23.5DA细胞可以通过激活Akt1/Eya1/Six2信号通路促进GDNF的过表达，GDNF的过表达对损伤的MES23.5DA细胞具有保护作用。因此，这项研究为治疗帕金森病的药物开发提供了一个新的靶点。
