Gao J, Kang X-Y, Sun S, Li L, Zhang B-L, Li Y-Q, Gao D-S
Department of Anatomy and Histology, The Fourth Military Medical University, Xian 710003, Shanxi, China.
Department of Neurobiology and Anatomy, Xuzhou Key Laboratory of Neurobiology, Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, Xuzhou Medical College, Xuzhou 221004, Jiangsu, China.
Cell Death Dis. 2016 May 5;7(5):e2217. doi: 10.1038/cddis.2016.120.
Glial cell line-derived neurotrophic factor (GDNF) has strong neuroprotective and neurorestorative effects on dopaminergic (DA) neurons in the substantia nigra (SN); however, the underlying molecular mechanisms remain to be fully elucidated. In this study, we found that the expression level of transcription factor Six2 was increased in damaged DA neurons after GDNF rescue in vivo and in vitro. Knockdown of Six2 resulted in decreased cell viability and increased the apoptosis of damaged DA neurons after GDNF treatment in vitro. In contrast, Six2 overexpression increased cell viability and decreased cell apoptosis. Furthermore, genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) indicated that Six2 directly bound to the promoter CAGCTG sequence of smad ubiquitylation regulatory factor 1 (Smurf1). ChIP-quantitative polymerase chain reaction (qPCR) analysis showed that Smurf1 expression was significantly upregulated after GDNF rescue. Moreover, knockdown of Six2 decreased Smurf1 expression, whereas overexpression of Six2 increased Smurf1 expression in damaged DA neurons after GDNF rescue. Meanwhile, knockdown and overexpression of Smurf1 increased and decreased p53 expression, respectively. Taken together, our results from in vitro and in vivo analysis indicate that Six2 mediates the protective effects of GDNF on damaged DA neurons by regulating Smurf1 expression, which could be useful in identifying potential drug targets for injured DA neurons.
胶质细胞系源性神经营养因子（GDNF）对黑质（SN）中的多巴胺能（DA）神经元具有强大的神经保护和神经修复作用；然而，其潜在的分子机制仍有待充分阐明。在本研究中，我们发现，在体内和体外GDNF挽救受损的DA神经元后，转录因子Six2的表达水平升高。在体外，敲低Six2会导致细胞活力下降，并增加GDNF处理后受损DA神经元的凋亡。相反，Six2过表达会增加细胞活力并减少细胞凋亡。此外，全基因组染色质免疫沉淀测序（ChIP-seq）表明，Six2直接与Smad泛素化调节因子1（Smurf1）的启动子CAGCTG序列结合。ChIP定量聚合酶链反应（qPCR）分析表明，GDNF挽救后Smurf1表达显著上调。此外，在GDNF挽救后，敲低Six2会降低Smurf1表达，而Six2过表达则会增加受损DA神经元中Smurf1的表达。同时，敲低和过表达Smurf1分别增加和降低p53表达。综上所述，我们的体外和体内分析结果表明，Six2通过调节Smurf1表达介导GDNF对受损DA神经元的保护作用，这可能有助于确定受损DA神经元的潜在药物靶点。
