Clk1缺失通过AMPK/mTORC1信号通路抑制自噬增加多巴胺能神经元死亡
首发时间:2017-04-12
摘要:目的:探究在帕金森病模型中,Clk基因缺失对多巴胺能神经元存活的调控作用及其作用机制。方法:用病毒沉默Clk1,Q-PCR检测mRNA水平,MTT法检测细胞活力。Western blot检测自噬相关蛋白LC3, P62, LAMP1的表达情况。免疫荧光检测自噬体的数量。Western blot 检测AMPK/mTORC1信号通路的蛋白水平。免疫荧光检测TFEB的核转录调控。结果:MPP+会降低Clk蛋白表达,Clk1缺失的神经元活力降低。Clk1缺失通过AMPK/mTORC1信号通路减少TFEB的核转录抑制细胞自噬,增加神经元细胞的死亡。用Metformin处理可以一定程度保护多巴胺能神经元。结论:Clk1通过AMPK/mTORC1/TFEB信号通路调节多巴胺能神经元细胞自噬,增加多巴胺能神经元死亡。
关键词: 药理学 Clk1 多巴胺能神经元 自噬 AMPK/mTORC1, Metformin
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Clk1 deficiency inhibits autophagy through the AMPK/mTORC1 pathway that sensitized dopaminergic neuronal death
Abstract: Aim:To investigate the functional role of Clk1 in the regulation of dopaminergic neurons survival. Methods: Virus was used to knockdown Clk1 in MN9D cells, the expression of Clk1 was analyzed with Western blot and Q-PCR. MTT assay was used to detect cell viability. The expression of LC3, P62 and LAMP1 was analyzed with Western blot. The amount of autophagosome was detect by immunofluorescence. The expression of AMPK and mTORC1 was analyzed with Western blot. The nuclear transcription of TFEB was detect by immunofluorescence. Results: The expression of Clk1 was decreased after MN9D cells were treated with MPP+, and this repression was time and dose dependent. Clk1 deficency is related to dopaminergic neurons survival. Clk1 deficiency inhibited autophagy through AMPK/mTORC1 pathway and the nuclear translocation of TFEB was decreased. Clk1 deficiency influenced dopaminergic neurons death. Importantly, Metformin (AMPK agonist) treatment protected dopaminergic neurons in Clk1-deficient PD models. Conclusion: Clk1 deficiency inhibited autophagy through AMPK/mTORC1/TFEB pathway and influenced dopaminergic neurons death.
Keywords: Pharmacology, Clk1, dopaminergic neurons, autophagy, AMPK/mTOR, Metformin?
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Clk1缺失通过AMPK/mTORC1信号通路抑制自噬增加多巴胺能神经元死亡
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