Yang Dehua, Peng Changgeng, Li Xuping, Fan Xiaolan, Li Liang, Ming Ming, Chen Sheng, Le Weidong
Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai, People's Republic of China.
J Neurosci Res. 2008 Nov 15;86(15):3393-400. doi: 10.1002/jnr.21774.
The transcription factor Pitx3 is crucial for the development and differentiation of dopamine (DA) neurons. Our previous work has shown the Pitx3 can up-regulate the expression of brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) in neuroblastoma cell line SH-SY5Y. Primary astrocytes are the major nonneuronal cells and can be easily modified genetically to deliver therapeutic molecules into the brain, so we investigated whether Pitx3 can increase the expression and secretion of BDNF and GDNF in primary astrocytes. We first transfected Pitx3 plasmid in purified rat astrocytes and collected the conditioned medium (CM) from the Pitx3-transfected cultures, and then we measured the BDNF and GDNF levels from the CM and tested the protective effect of the CM against rotenone-induced DA neuron injury in ventral mesencephalon (VM) cultures. We found that the BDNF and GDNF levels were 1.4-fold and 1.5-fold higher in the CM from Pitx3-transfected astrocytes than empty vectors-transfected controls. Incubation with the CM from Pitx3-transfected astrocytes significantly attenuated the rotenone-induced DA neuron injury, and such protection can be significantly blocked by preincubation with antibodies against either BDNF or GDNF, whereas preincubation with purified BDNF or GDNF replicated the neuroprotection against rotenone-induced injury in VM cultures. These results demonstrate that Pitx3-transfection in astrocytes can up-regulate BDNF and GDNF expression and produce protective benefit to DA neurons, which might be a potential therapeutic alternative for Parkinson's disease.
转录因子Pitx3对多巴胺（DA）神经元的发育和分化至关重要。我们之前的研究表明，Pitx3可以上调神经母细胞瘤细胞系SH-SY5Y中脑源性神经营养因子（BDNF）和胶质细胞系源性神经营养因子（GDNF）的表达。原代星形胶质细胞是主要的非神经元细胞，并且可以很容易地进行基因改造以将治疗性分子递送至大脑，因此我们研究了Pitx3是否可以增加原代星形胶质细胞中BDNF和GDNF的表达及分泌。我们首先在纯化的大鼠星形胶质细胞中转染Pitx3质粒，并从转染Pitx3的培养物中收集条件培养基（CM），然后我们测量CM中的BDNF和GDNF水平，并测试CM对中脑腹侧（VM）培养物中鱼藤酮诱导的DA神经元损伤的保护作用。我们发现，转染Pitx3的星形胶质细胞的CM中BDNF和GDNF水平分别比转染空载体的对照高1.4倍和1.5倍。用转染Pitx3的星形胶质细胞的CM孵育可显著减轻鱼藤酮诱导的DA神经元损伤，并且用抗BDNF或GDNF抗体预孵育可显著阻断这种保护作用，而用纯化的BDNF或GDNF预孵育可重现对VM培养物中鱼藤酮诱导损伤的神经保护作用。这些结果表明，星形胶质细胞中转染Pitx3可上调BDNF和GDNF表达并对DA神经元产生保护作用，这可能是帕金森病的一种潜在治疗选择。
