Cu~(2+)-Aβ复合物与Aβ单体诱导神经元H_2O_2释放作用的比较研究
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摘要
目的:比较不同摩尔比Cu~(2+)-Aβ复合物与Aβ单体诱导神经元H_2O_2释放作用的差异。方法:制备不同摩尔比(0.1-5)的Cu~(2+)-Aβ复合物,通过检测硫磺素T(Thioflavin T,Th T)荧光强度考察Cu~(2+)对Aβ纤丝形成的影响。利用原代培养的大鼠海马神经元细胞,分别以不同摩尔比Cu~(2+)-Aβ复合物,不同浓度Cu~(2+)-Aβ复合物(摩尔比为1),以及Aβ单体和Cu~(2+)处理细胞,检测培养上清中的H_2O_2含量;分离线粒体,分别检测不同浓度Cu~(2+)-Aβ复合物(摩尔比为1),以及Aβ单体和Cu~(2+)处理后H_2O_2的释放;观察不同摩尔比Cu~(2+)-Aβ复合物,不同浓度Cu~(2+)-Aβ复合物(摩尔比为1),以及Aβ单体和Cu~(2+)对神经元细胞活力的影响。结果:(1)Th T荧光试验结果表明,Cu~(2+)与Aβ(10μM)摩尔比为1~5范围内可明显抑制Aβ纤丝形成。(2)Cu~(2+)-Aβ复合物(摩尔比为1~5;Aβ浓度为10μM)以及摩尔比为1的Cu~(2+)-Aβ复合物(Aβ浓度分别为5,10μM)可显著诱导神经元释放H_2O_2;另外,摩尔比为1时,Cu~(2+)-Aβ复合物还可诱导神经元线粒体内H_2O_2释放;上述作用均强于Aβ单体或Cu~(2+)。(3)Cu~(2+)-Aβ复合物(摩尔比为1~5)可显著降低神经元细胞活力,该作用强于Aβ单体或Cu~(2+)。结论:与Aβ单体相比,Cu~(2+)-Aβ复合物诱发神经元细胞及其线粒体释放H_2O_2作用更强,并诱发更为明显的神经元毒性。提示Cu~(2+)与Aβ之间的配位结合可能增强其引发活性氧释放以及神经元毒性反应;Cu~(2+)-Aβ复合物引发的活性氧可能主要来自线粒体。
Objective: To compare the effects of Cu~(2+)-Aβ complex at different molar ratios with Aβ monomers on neuronal release of H_2O_2. Methods: Cu~(2+)-Aβ complex at different molar ratios(0.1-5) were prepared. Thioflavin T(Th T)-based fluorometric assay was used to examine the effect of Cu~(2+)on Aβ fibril formation. The primary hippocampal neurons were treated with Cu~(2+)-Aβ complex of different ratios, Cu~(2+)-Aβ complex of different concentrations(with a molar ratio of 1), Aβ monomers and Cu~(2+), respectively. H_2O_2 content in the culture medium was measured. The mitochondria were separated and treated with different concentrations of Cu~(2+)-A β complex(with a molar ratio of 1), A β monomers and Cu~(2+), respectively. H_2O_2 from mitochondria were also measured. Neuronal viability was detected using Cell Counting Kit-8(CCK-8) after the neurons were treated with Cu~(2+)-Aβ complex of different ratios, Cu~(2+)-Aβ complex of different concentrations(with a molar ratio of 1), Aβ monomers and Cu~(2+). Results:(1) Th T-based fluorometric assay demonstrated that Cu~(2+)inhibited Aβ fibril formation when the molar ratio of Cu~(2+)to Aβ(10 μM) was within 1~5.(2) Cu~(2+)-Aβ complex(the molar ratio was within 1~5; the concentration of Aβ was 10 μM) and Cu~(2+)-Aβ complex with molar ratio of 1(the concentrations of A were 5, 10 μM)significantly induced neuron releasing of H_2O_2; In addition, when the molar ratio was 1, Cu~(2+)-A β complex also induced neuronal mitochondria releasing of H_2O_2; All the above mentioned effects were stronger than that of Aβ monomer or Cu~(2+).(3) Cu~(2+)-Aβ complex(with a molar ratios within 1~5) significantly reduced the viability of neuron cells, which was stronger than that of Aβ monomers or Cu~(2+).Conclusions: Compared with A β monomers, Cu~(2+)-A β complex elicited a more robust H_2O_2 release from neuronal cells and their mitochondria. Moreover, it induced a more overt neuronal cell death. Our observations suggest that Cu~(2+)-Aβ interactions may enhance the ability of Aβ to induce reactive oxygen species release and neuronal toxicity; Cu~(2+)-Aβ complex-evokeded reactive oxygen species may primarily come from the mitochondria.
Objective: To compare the effects of Cu~(2+)-Aβ complex at different molar ratios with Aβ monomers on neuronal release of H_2O_2. Methods: Cu~(2+)-Aβ complex at different molar ratios(0.1-5) were prepared. Thioflavin T(Th T)-based fluorometric assay was used to examine the effect of Cu~(2+)on Aβ fibril formation. The primary hippocampal neurons were treated with Cu~(2+)-Aβ complex of different ratios, Cu~(2+)-Aβ complex of different concentrations(with a molar ratio of 1), Aβ monomers and Cu~(2+), respectively. H_2O_2 content in the culture medium was measured. The mitochondria were separated and treated with different concentrations of Cu~(2+)-A β complex(with a molar ratio of 1), A β monomers and Cu~(2+), respectively. H_2O_2 from mitochondria were also measured. Neuronal viability was detected using Cell Counting Kit-8(CCK-8) after the neurons were treated with Cu~(2+)-Aβ complex of different ratios, Cu~(2+)-Aβ complex of different concentrations(with a molar ratio of 1), Aβ monomers and Cu~(2+). Results:(1) Th T-based fluorometric assay demonstrated that Cu~(2+)inhibited Aβ fibril formation when the molar ratio of Cu~(2+)to Aβ(10 μM) was within 1~5.(2) Cu~(2+)-Aβ complex(the molar ratio was within 1~5; the concentration of Aβ was 10 μM) and Cu~(2+)-Aβ complex with molar ratio of 1(the concentrations of A were 5, 10 μM)significantly induced neuron releasing of H_2O_2; In addition, when the molar ratio was 1, Cu~(2+)-A β complex also induced neuronal mitochondria releasing of H_2O_2; All the above mentioned effects were stronger than that of Aβ monomer or Cu~(2+).(3) Cu~(2+)-Aβ complex(with a molar ratios within 1~5) significantly reduced the viability of neuron cells, which was stronger than that of Aβ monomers or Cu~(2+).Conclusions: Compared with A β monomers, Cu~(2+)-A β complex elicited a more robust H_2O_2 release from neuronal cells and their mitochondria. Moreover, it induced a more overt neuronal cell death. Our observations suggest that Cu~(2+)-Aβ interactions may enhance the ability of Aβ to induce reactive oxygen species release and neuronal toxicity; Cu~(2+)-Aβ complex-evokeded reactive oxygen species may primarily come from the mitochondria.
引文
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[20]Hu Z,Yu F,Gong P,et al.Subneurotoxic copper(Ⅱ)-induced NF-κB-dependent microglial activation is associated with mitochondrial ROS[J].Toxicol Appl Pharmacol,2014,276(2):95-103
[21]T?nnies E,Trushina E.Oxidative Stress,Synaptic Dysfunction,and Alzheimer's Disease[J].J Alzheimers Dis,2017,57(4):1105-1121
[22]Kim GH,Kim JE,Rhie SJ,et al.The Role of Oxidative Stress in Neurodegenerative Diseases[J].Exp Neurobiol,2015,24(4):325-340
[23]Nissanka N,Moraes CT.Mitochondrial DNA damage and reactive oxygen species in neurodegenerative disease[J].FEBS Lett,2017,Dec 27.[Epub ahead of print]
[2]Kocahan S,Dogan Z.Mechanisms of Alzheimer's Disease Pathogenesis and Prevention:The Brain,Neural Pathology,N-methyl-D-aspartate Receptors,Tau Protein and Other Risk Factors[J].Clin Psychopharmacol Neurosci,2017,15(1):1-8
[3]Karran E,De Strooper B.The amyloid cascade hypothesis:are we poised for success or failure[J]?J Neurochem,2016,139(Suppl 2):237-252
[4]Stewart KL,Radford SE.Amyloid plaques beyond Aβ:a survey of the diverse modulators amyloid aggregation[J].Biophys Rev,2017,9(4):405-419
[5]Tamano H,Takeda A.Is interaction of amyloidβ-peptides with metals involved in cognitive activity?[J].Metallomics,2015,7(8):1205-1212
[6]Huy PD,Vuong QV,La Penna G,et al.Impact of Cu(Ⅱ)Binding on Structures and Dynamics of Aβ42 Monomer and Dimer:Molecular Dynamics Study[J].ACS Chem Neurosci,2016,7(10):1348-1363
[7]Lin CJ,Huang HC,Jiang ZF.Cu(Ⅱ)interaction with amyloid-beta peptide:a review of neuroactive mechanisms in AD brains[J].Brain Res Bull,2010,82(5-6):235-142
[8]Tougu V,Tiiman A,Palumaa P.Interactions of Zn(Ⅱ)and Cu(Ⅱ)ions with Alzheimer's amyloid-peptide.Metal ion binding,contribution to fibrillization and toxicity[J].Metallomics,2011,3(3):250-261
[9]Mayes J,Tinker-Mill C,Kolosov O,et al.β-amyloid fibrils in Alzheimer disease are not inert when bound to copper ions but can degrade hydrogen peroxide and generate reactive oxygen species[J].J Biol Chem,2014,289(17):12052-12062
[10]Chassaing S,Collin F,Dorlet P,et al.Copper and heme-mediated Abeta toxicity:redox chemistry,A beta oxidations and anti-ROS compounds[J].Curr Top Med Chem,2012,12(22):2573-2595
[11]Yu F,Gong P,Hu Z,et al.Cu(Ⅱ)enhances the effect of Alzheime's amyloid-βpeptide on microglial activation[J].J Neuroinflammation,2015,12:122
[12]Sharma AK,Pavlova ST,Kim J,et al.The effect of Cu2+ and Zn2+ on the Aβ42 peptide aggregation and cellular toxicity[J].Metallomics,2013,5(11):1529-1536
[13]Atwood CS,Moir RD,Huang X,et al.Dramatic aggregation of Alzheimer A by Cu(Ⅱ)is induced by conditions representing physiological acidosis[J].J Biol Chem,1998,273(21):12817-12826
[14]Goch W,Bal W.Numerical Simulations Reveal Randomness of Cu(Ⅱ)Induced AβPeptide Dimerization under Conditions Present in Glutamatergic Synapses[J].PLoS One,2017,12(1):e0170749
[15]Chen WT,Liao YH,Yu HM,et al.Distinct effects of Zn2+,Cu2+,Fe3+,and Al3+ on amyloid-beta stability,oligomerization,and aggregation:amyloid-beta destabilization promotes annular protofibril formation[J].J Biol Chem,2011,286(11):9646-9656
[16]Cheignon C,Jones M,Atrián-Blasco E,et al.Identification of key structural features of the elusive Cu-Aβcomplex that generates ROS in Alzheimer's disease[J].Chem Sci,2017,8(7):5107-5118
[17]Bolognin S,Zatta P,Lorenzetto E,et al.β-Amyloid-aluminum complex alters cytoskeletal stability and increases ROS production in cortical neurons[J].Neurochem Int,2013,62(5):566-574
[18]Sharma AK,Singh V,Gera R,et al.Zinc Oxide Nanoparticle Induces Microglial Death by NADPH-Oxidase-Independent Reactive Oxygen Species as well as Energy Depletion[J].Mol Neurobiol,2017,54(8):6273-6286
[19]Wong HS,Dighe PA,Mezera V,et al.Production of superoxide and hydrogen peroxide from specific mitochondrial sites under different bioenergetic conditions[J].J Biol Chem,2017,292(41):16804-16809
[20]Hu Z,Yu F,Gong P,et al.Subneurotoxic copper(Ⅱ)-induced NF-κB-dependent microglial activation is associated with mitochondrial ROS[J].Toxicol Appl Pharmacol,2014,276(2):95-103
[21]T?nnies E,Trushina E.Oxidative Stress,Synaptic Dysfunction,and Alzheimer's Disease[J].J Alzheimers Dis,2017,57(4):1105-1121
[22]Kim GH,Kim JE,Rhie SJ,et al.The Role of Oxidative Stress in Neurodegenerative Diseases[J].Exp Neurobiol,2015,24(4):325-340
[23]Nissanka N,Moraes CT.Mitochondrial DNA damage and reactive oxygen species in neurodegenerative disease[J].FEBS Lett,2017,Dec 27.[Epub ahead of print]