摘要
在原代海马神经元上建立通过外加Aβ寡聚体所导致的突触毒性模型,并使用蛋白印迹、激酶活性、激光共聚焦显微成像和电生理等技术检测PKC激酶的非选择性激动剂Bryostatin-1是否可以保护Aβ寡聚体导致的突触毒性。结果显示,在Aβ毒性模型中,Bryostatin-1可以改善树突棘的形态,并促进树突棘成熟。此外,电生理结果显示Bryostatin-1可以显著改善突触后的微型兴奋性突触后电流频率,AMPA受体与NMDA受体的比例并没有发生显著改变。蛋白印迹结果显示Bryostatin-1可以上调突触生物标记物水平。蛋白印迹试验结果表明,Bryostatin-1可以上调mTOR-S6K1信号通路的活性。通过在神经元中转染GFP-RFP-LC3质粒并进行激光共聚焦显微观察,结果提示Aβ寡聚体可以抑制神经元内自噬通量,而Bryostatin-1可以提高被Aβ寡聚体所抑制的自噬通量。结果提示,Bryostatin-1激活mTOR-S6K1信号通路,并随后调节细胞自噬功能可能是其在Aβ寡聚体毒性环境下保护神经元突触功能的分子机制。
By using Western Blot,kinase activity assay,confocolmicroscopy,and electrophysiology recording methods,the synapse toxicity induced by Bryostatin-1 protects Aβoligomers was studied and its potential mechanisms was investigated.Results showed that in the present of Aβoligomers,Bryostatin-1 could significantly protect the morphology of dendritic spines,and promote the maturation of dendritic spines.Furthermore,electrophysiology recording result also showed that Aβoligomers could inhibit mEPSC and Bryosta-tin-1 could rescue it.The AMPA/NMDA ratio did not change significantly among each group.Western Blot results also showed higher pre-and post-synaptic markers in the present of Bryostatin-1.The mechanisms underlying the protective effect of Bryostatin-1 was in-vestigated.Western Blot results showed that Bryostatin-1 could activate mTOR-S6K1 signal pathway.At last,GFP-RFP-LC3 plasmid was transfected into primary hippocampus neuron culture.Confocolmicroscopy results showed that Aβoligomers could inhibit the au-tophagic flux,and Bryostatin-1could promote the autophagic flux in the present of Aβoligomers.In summary,the present study showed that Bryostatin-1 could activate mTOR-S6K1 signal pathway and then promote autophagic flux in the present of Aβoligomers,which may be the mechanisms underlying the protective effect of PKC against Aβoligomers induced synaptic toxicity.
作者
潘文阳
陈鹏
孙邈
包小峰
PAN Wenyang;CHEN Peng;SUN Miao;BAO Xiaofeng(Department of Pharmacology,Nantong University,Nantong 226019,China)
出处
《生物学杂志》
CAS
CSCD
北大核心
2024年第3期85-91,共7页
Journal of Biology
基金
国家自然科学基金项目(No.81801080,No.82071174)
南通市科学技术基金项目(No.JC22022043)
南通市第六期江海英才市级培养专项。
