Chakrabarty Koushik, Serchov Tsvetan, Mann Stefan A, Dietzel Irmgard D, Heumann Rolf
Department of Molecular Neurobiochemistry, NC7/174, Ruhr University, 44780 Bochum, Germany.
Eur J Neurosci. 2007 Apr;25(7):1971-81. doi: 10.1111/j.1460-9568.2007.05457.x.
The poor differentiation and survival of dopaminergic neurones are practical constraints in their therapeutic applications. Here we explored the role of neuronally activated Ras in ventral mesencephalon-derived neurospheres generated from synRas mouse embryos. The expression of Val12 Ha-Ras transgene and enhanced Ras activity was evident after differentiation of the neurospheres with a corresponding activating phosphorylation of mitogen-activated protein kinase. Phosphorylation of Akt/PKB, the target kinase of phosphoinositide 3-kinase, along with phosphorylation of Bad and CREB were enhanced in synRas-derived differentiated neurosphere cultures. Furthermore, increased Nurr1 expression was associated with elevated numbers of dopaminergic neurones in synRas-derived cultures compared with the wild-type. Correspondingly, tyrosine hydroxylase promoter assays revealed enhanced transcriptional activation of the promoter in synRas-derived cultures. synRas-derived dopaminergic neurones were greatly resistant to degeneration induced by various noxious stimuli. Consistently, the transgenic expression of activated Ras attenuated the adverse 6-hydroxydopamine effects on dopaminergic neurones. Dopaminergic neurones derived from both wild-type and synRas cultures expressed voltage-gated potassium and sodium currents, fired action potentials and exhibited electrical network activity. Thus, expression of the transgene promotes survival and enhances differentiation towards a dopaminergic cell fate without altering their basic electrical properties. Our results suggest that intracellular cell therapy mimicking trophic signalling may offer potential benefit in models of human disease associated with dopamine neurone dysfunction.
多巴胺能神经元的低分化和低存活率是其治疗应用中的实际限制因素。在此，我们探讨了神经元激活的Ras在源自synRas小鼠胚胎的腹侧中脑来源神经球中的作用。在用丝裂原活化蛋白激酶进行相应的激活磷酸化后，神经球分化后Val12 Ha-Ras转基因的表达和增强的Ras活性明显可见。在源自synRas的分化神经球培养物中，磷酸肌醇3激酶的靶激酶Akt/PKB的磷酸化以及Bad和CREB的磷酸化均增强。此外，与野生型相比，源自synRas的培养物中Nurr1表达增加与多巴胺能神经元数量增加相关。相应地，酪氨酸羟化酶启动子分析显示源自synRas的培养物中启动子的转录激活增强。源自synRas的多巴胺能神经元对各种有害刺激诱导的变性具有高度抗性。一致地，活化Ras的转基因表达减弱了6-羟基多巴胺对多巴胺能神经元的不良影响。源自野生型和synRas培养物的多巴胺能神经元均表达电压门控钾电流和钠电流，发放动作电位并表现出电网络活动。因此，转基因的表达促进存活并增强向多巴胺能细胞命运的分化，而不改变其基本电特性。我们的结果表明，模拟营养信号的细胞内细胞疗法可能在与多巴胺神经元功能障碍相关的人类疾病模型中提供潜在益处。
