Amyloid-β peptide induces mitochondrial oxidative stress damage in SH-SY5Y neuroblastoma veils

来源 :中国神经科学学会第九届全国学术会议暨第五届会员代表大会 | 被引量 : 0次 | 上传用户:uuuu_uuu
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  Objective Alzheimers disease (AD) is one of the most common forms of neurodegenerative disease.Amyloid-β (Aβ) is considered as a centre molecule and plays a key role in AD pathological development.The accumulating aggregation of Aβ in neuritic plaques incurs neuronal oxidative damage, neurofibrillary tangles, and loss of hippocampal neurite, synapse and neuron.The elevated oxidative stress is thought to be associated with the age-related neurodegeneration.The elevated oxidative stress is also thought to be a key event on the Aβ-induced neurotoxicity.However, the pathways of Aβ-induced oxidative damages are under controversy.The processing of energy metabolism is a key way to generate reactive oxygen species (ROS), which are the free radicals damaging biomolecules.Cell energy is mainly generated in mitochondria, which plays an important role in cell energy metabolism.In this study, we investigated Aβ-induced oxidative damages on neurons and sub-cellular structures, especially on mitochondria, based on our work and on the recent research progress worldwide on the relations between oxidative stress and Alzheimers disease.Methods Cortical neurons or SH-SY5Y cells were cultured at 37 ℃ for 7 d.After the culture medium was removed, the cultured cortical neurons were incubated with fresh culture medium containing 5, 10 μrnol/L Aβ, at 37 ℃ in a humidified incubator.Morphological changes of ceils were detected using a microscope at differential interference contrast (DIC) model, and the chromatin condensation of cultured cells was detected by the staining assay of nucleus with Hoechst 33342 dye (a molecular probe).The hyperphosphorylation of Tau and GSK-3beta activity were detected by Western blot analysis.Cell viability was assessed by MTT, Hoechst 33342 assay and lactate dehydrogenase (LDH) assay, and the ROS levels were detected by ROS assay based on the ROS-mediated conversion of nonfluorescent 2,7-DCFH-DA into fluorescent DCFH.The free intracellular Ca2+ in cells was measured using the fluorescence Ca2+ indicator, fura-2/AM.Mitochondrial membrane potential was detected with JC-1 dye.Normal mitochondria (Red for J-aggregates) were recorded at Ex/Em 525/590 nm and depolarized mitochondria (Green for monomer) were recorded at Ex/Em 490/530 nm with 300 ms integration time.Cellular ATP levels were measured using a firefly luciferasebased ATP assay kit (Beyotime, China).Results The results indicate that AD-like symptoms are observed in cultured cortical neurons and SH-SY5Y cells, Aβ results in neuronal damage including cell viability and morphology, such as chromatin condensation, cell bodies, dendrites, and interaction between cells.Aβ induces hyperphosphorylation of Tau protein.GSK-3beta, as an important glycogen synthase, is involved in energy metabolism.GSK-3beta is involved in the pathway of tau hyperphosphorylation, Cell damage induced by Aβ may involve abnormity of cellular energy metabolism.To further understand the damage to energy metabolism, we detected the effects of Aβ on the mitochondria.The results indicated that Aβ results in the depolarization of mitochondriat membrane potential and the decrease of ATP generation but increase of intracellular ROS.As the same time, cellular oxidative stress is increased when cells were treated with Aβ; intracellular Ca2+ is increased less than 1 h after Aβ treatment, indicating that Ca2+ signaling is involved in the pathway of Aβ-induced neuronal damages.Conclusion Ale-induced tau hyperphosphorylation and neuronal damage are involved in mitochondrial dysfunction.Ca2+ influx is involved in cellular oxidative damage.
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