α-synuclein和tau相互作用参与锰诱导的神经毒性机制及染料木素的保护作用
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摘要
【背景】
锰是机体必需的一种微量元素，但摄入过量的锰则会对机体产生毒性作用。锰的毒性作用主要累及中枢神经系统，尤其是基底核。慢性锰中毒主要表现为锥体外系功能障碍，与帕金森病（Parkinson’s disease, PD）的临床表现类似。
α-共核蛋白(α-synuclein，α-syn)是由140个氨基酸组成的可溶性非折叠蛋白,它是帕金森病(Parkinson’s disease, PD)特征性病理结构——路易氏小体（Lewy’s body）的主要组成部分。体内和体外实验均表明高表达α-syn可促进神经元的损伤，诱导PD相关毒性症状的发生。因此越来越多的实验试图揭示α-syn的生理功能及其在神经退行性疾病中的作用机制。但是关于α-syn的生理功能目前还不十分清楚，有研究表明α-syn参与调节突触传递和神经元的可塑性。另有研究提示它可能在信号转导过程中发挥重要作用。近年来，许多研究证实α-syn在Lewy小体病和帕金森病的发生机制中起到了关键的作用,但是其在锰神经毒性作用中的机制尚不清楚。
tau蛋白是一种微管相关蛋白，在神经系统的发育和维持微管功能等方面具有重要作用。生理和病理条件下，tau蛋白的许多苏氨酸和丝氨酸残基可以被多种激酶磷酸化。tau蛋白病理条件下的过度磷酸化将影响其正常功能，使其促微管聚合功能丧失，神经元微管解体，tau蛋白聚集成双螺旋丝（paired helical filaments，PHF）以及形成神经原纤维缠结（neurofibrillary tangle，NFT）结构，并转变成为毒性分子，最终引起神经元变性损伤。
作为存在于细胞质内的可溶性蛋白质，α-syn与tau蛋白之间存在着相互作用。研究表明α-syn与tau蛋白在细胞内除了细胞核外大部分是共定位的，在靠近细胞膜的细胞质边缘，两者的相互作用最强烈。新近研究表明tau蛋白突变可抑制α-syn与tau蛋白的相互作用；α-syn突变也可抑制α-syn与tau蛋白的相互作用。此外，截去羧基端的tau蛋白与α-syn之间的相互作用也被抑制。
染料木素（genistein，Gen）又称三羟异黄酮，主要是从大豆等豆科植物中提取出来的活性成分，为异黄酮类物质中活性功能最高的一种。Gen对神经系统具有显著的保护作用，其作用机制是多方面的。包括抗氧化、调节细胞内的钙离子浓度、与细胞内信号转导途径相互作用、增强胆碱能神经功能、抑制凋亡等途径，从而提高神经细胞的存活能力。
【目的】
在动物和细胞水平探讨锰对α-syn与tau蛋白相互作用的影响，以及这种影响在锰神经毒性中的作用。并进一步探讨染料木素对锰神经毒性可能的抑制效应，以及这种抑制效应与α-syn-tau的相关性。为进一步阐明锰神经毒性的分子机制以及寻找有效的防护措施提供新的思路和实验依据。
【方法】
1．利用灌胃的方法建立慢性锰暴露的动物模型，采用免疫荧光染色、Western blot和免疫共沉淀检测多巴胺能神经元的损伤以及α-syn与tau蛋白相互作用的变化。
2．利用高分化型PC12细胞建立锰毒性的细胞模型，MTT、LDH和TUNEL检测细胞毒性；siRNA转染、Western blot、免疫荧光染色和免疫共沉淀检测α-syn与tau蛋白相互作用的变化。
3．MTT、TUNEL和氧化应激试剂盒检测染料木素对锰毒性的保护作用。同时，利用Western blot、免疫荧光染色检测染料木素对锰诱导的α-syn蛋白表达、tau磷酸化以及两者共定位的影响。
【结果】
1．锰对大鼠黑质多巴胺能神经元损伤和α-syn与tau蛋白相互作用的影响
通过灌胃方法建立慢性锰暴露的动物模型，免疫荧光染色和Western blot结果显示大鼠黑质TH染色阳性细胞数明显减少，同时黑质TH表达水平显著降低，提示锰诱导了多巴胺能神经元损伤；Western blot结果显示大鼠黑质α-syn和tau表达水平均升高；免疫荧光染色和免疫共沉淀结果显示大鼠黑质α-syn和tau蛋白共定位增强，同时这两种蛋白在黑质的相互作用增强。
2．锰对PC12细胞的损伤以及对α-syn与tau蛋白相互作用的影响
MTT结果显示，随着染锰浓度的增加和染锰时间的延长，PC12细胞活力显著下降；TUNEL结果显示，随着锰浓度的增加，PC12细胞凋亡水平逐渐增加；Western blot结果显示锰可使PC12细胞α-syn与tau磷酸化水平增加；免疫荧光染色和免疫共沉淀结果显示锰可使α-syn与tau蛋白在PC12细胞内共定位增强，同时α-syn和tau蛋白相互作用增强。
3．siRNA α-syn和LiCl对锰诱导的PC12细胞α-syn与tau蛋白相互作用的影响
MTT结果显示，干涉α-syn的表达或者加入LiCl可以明显改善锰对PC12细胞的活力抑制；Western blot结果显示，干涉α-syn的表达可以使锰诱导的高p-tau396水平降低；免疫荧光染色结果显示，siRNAα-syn和LiCl均可抑制锰诱导的PC12细胞α-syn与tau蛋白的共定位；免疫共沉淀结果进一步显示LiCl可抑制锰诱导的PC12细胞α-syn与tau蛋白的相互作用。
4．染料木素对锰诱导的PC12细胞毒性的保护作用
MTT结果显示，染料木素可以明显抑制锰诱导的PC12细胞活力下降； TUNEL结果显示，染料木素可以抑制细胞凋亡；氧化应激试剂盒检测结果提示，染料木素可以抑制锰诱导的PC12细胞氧化应激水平的增高。此外，Western blot实验结果显示，染料木素可抑制锰诱导的α-syn蛋白表达水平的增高以及tau蛋白的磷酸化。免疫荧光染色结果表明染料木素可抑制锰诱导的PC12细胞内α-syn与tau蛋白共定位的增强。
【结论】
1．体内外实验均表明，锰可以引起多巴胺能神经元的损伤，诱导α-syn与p-Tau396蛋白表达水平增加；使α-syn与p-tau396蛋白相互作用增强，并且其共定位增强。
2．体外实验表明，分别降低α-syn或p-Tau396蛋白的表达水平，可在抑制锰毒性的同时一定程度上抑制锰诱导的两种蛋白相互作用的增强。表明α-syn和p-Tau396蛋白的相互作用可能在锰诱导的PC12细胞损伤中发挥一定的作用。
3．染料木素可抑制锰诱导的PC12细胞毒性，其机制可能与其抑制锰诱导的氧化应激，α-syn蛋白表达水平增高，tau蛋白的磷酸化以及α-syn与tau的相互作用有关。
综上所述，我们的研究初步阐明锰诱导的多巴胺能神经元的损伤作用中存在α-syn和p-Tau396蛋白的相互作用，这种作用可能参与了锰诱导的损伤过程，并可作为锰损伤防护（如染料木素）的一个潜在作用靶点。我们的结果将为进一步阐明锰神经毒性的分子机制以及寻找有效的防护措施提供新的思路和实验依据。
Background
Manganese is an essential ubiquitous trace element that is required for normal growth,development, and cellular homeostasis. But excessive exposure to manganese results inneurotoxicity. The main target of manganese-induced toxic effects is the central nervoussystem, especial the basal ganglia. The clinical symptoms of chronic manganism arecharacterized by extrapyramidal dysfunction, same as those of Parkinson’s disease.
α-synuclein(α-syn) is a140-amino acid heat stable protein that was identified as theprecursor of the non-Aβ protein in amyloid plaques of human Alzheimer's disease. It ishighly conservative among vertebrates. Since the discovery of its relationship with thepathological processes of neurodegenerative disorders, more and more studies have triedto reveal the physiological functiosn of α-syn and its corelation with other proteins. Although the physiological functions of α-syn are still poorly understood, some studieshave shown that it is associated with neuronal synaptic plasticity. Other studies have foundthat it may be important in the processes of signal transduction. Recently many studieshave confirmed that α-syn plays a key role in the pathogenesis of Lewy body disease andParkinson’s disease. However the mechanism of α-syn in manganese neurotoxicity is stillnot yet clear.
Tau is one of the member of microtubule-associated proteins, which plays animportant role in the development of nervous system and maintain of microtubule stability.Specific characteristic pathological changes associated with tau have been found in manyneurodegenerative diseases. Hyperphosphorylation of tau may affect its normal functionand cause a series of pathological lesions. Hyperphosphorylated tau would lose itsfunction to promote the microtubule polymerization and result in neuronal microtubuledispolymerization, which is easy to aggregate and form paired helical filaments andeventrally neurofibrillary tangles. Thus tau turns to be toxic and can bind with normalmicrotubule-associated proteins, leading to microtubule dispolymerization and neuronaldegeneration.
As two soluble proteins in cytosol, there is an interaction between α-syn and tau.Studies have showed that α-syn and tau are co-localized in cells except nucleus, and theinteraction between them is most intense on the edge of the cytoplasm near the cellmembrane. Recent studies have shown that tau mutation can inhibit the interactionbetween α-syn and tau, and α-syn mutation can inhibit that too. Furthermore, C-terminaltruncated tau can also inhibit its interaction with α-syn.
Genistein (Gen), also called trihydroxyisoflavone, is an active component mainlyextracted from soybean and other leguminous plants. It is one of the most activeisoflavones. Genistein plays a prominent role in protecting the nervous system throughmultiple machanisms, including anti-oxydation, modulation of cellular calciumhomeostasis, interaction with cellular signaling pathways, strengthening the function ofcholinergic neurons and inhibition of apoptosis, etc, and improvement of neuronalviability.
Aims
We investigated the effect of manganese on interaction between α-syn and tau inanimal and cell models, and the role of this effect in manganese neurotoxicity. We furtherinvestigated the possible inhibitive effects on manganese neurotoxicity by genistein, andthe association with α-syn-tau interaction. Our results may provide new ideas andexperimental bases to further our understanding of manganese-induced neurotoxicity andeffective protective measures.
Methods
1. The animal model of chronic manganese exposure was established by lavagemethod; immunofluorescence, western blot, and co-immunoprecipitation were used todetermine dopaminergic neuron injury and interaction between α-syn and tau.
2. Highly differentiated PC12cell line was used to establish the cell model ofmanganism, and the cytotoxic effect was analyzed through MTT assay, LDH assay, andTUNEL; siRNA transfection, western blot, immunofluorescence, andco-immunoprecipitation were performed to determine the change of α-syn-tau interaction
3. The neuroprotective effect of genistein was analyzed by MTT, TUNEL, andoxidative stress kit. Western blot, co-immunoprecipitation and immunofluorescence wereperformed to determine the effect of genistein on manganese-induced α-syn expression,tau phosphorylation, and their interaction.
Results
1. The effect of manganese on dopaminergic neuron injury and interaction betweenα-syn and tau
We established the animal model of chronic manganese exposure by lavage method,and the results of immunofluorescence staining and western blot showed that aftermanganese administration, there was an obviously loss of TH positive neurons and andecreased expression of TH in the substantia nigra, suggesting that manganese induced dopaminergic neural injury. Meanwhile, western blot also showed that there was anincreased expression of α-syn and tau. The results of immunofluorescence andco-immunoprecipitation showed that the colocalization of those two proteins wasenhanced and the interaction between them was also increased.
2. The effect of manganese on PC12cells injury and interaction between α-syn andtau
The results of MTT assay showed that manganese inhibited cell viability of PC12cells in a concentration-dependent and time-dependent manner. The increased level ofapoptosis with the increase of manganese concerntion were detected by TUNEL. Westernblot results showed that manganese enhanced the expression level of α-syn and tauphosphorylation in PC12cells. As shown by immunofluorescence andco-immunoprecipitation, the colocalization of those two proteins were enhanced and theinteraction between them was also increased.
3. The effect of siRNA α-syn and LiCl on manganese-induced interaction betweenα-syn and tau in PC12cells
MTT showed that both siRNA α-syn and LiCl reversed the inhibition ofmanganese-induced PC12cell viability. Western blot results showed that after siRNAtransfection, the increased expression of p-tau396induced by manganese was inhibited.Immunofluorescence showed that both siRNA α-syn and LiCl inhibitedmanganese-induced α-syn-tau colocalization. And co-immunoprecipitation showed thatLiCl inhibited manganese-induced α-syn-tau interaction.
4. The neuroprotective effect of genistein on manganese-induced PC12cytotoxicity
MTT assay showed that genistein could markedly inhibit the decrease of PC12cellviability induced by manganese. TUNEL assay showed that genistein inhibitedmanganese-induced apoptosis. The results of oxygenic stress kit indicated that genisteincould inhibit the increased oxygenic stress level induced by manganese. Furthermore,Western blot results showed that genistein inhibited manganese-induced α-synoverexpression and tau phosphorylation. Immunofluorescence showed that genistein inhibited manganese-induced α-syn-tau colocalization.
Conclusion
1. Both in vivo and vitro studies showed that manganese may cause dopaminergicneurons injury; induce increased level of α-synuclein and phosphorylated tau expression;promote the interaction between α-syn and tau; and enhance the colocalization of thosetwo proteins.
2. In vitro research showed that when the expression of α-syn and phosphorylated tauwere decreased respectively, the increased interaction between them was inhibited,suggesting that the interaction between α-syn and phosphorylated tau may play a role inmanganese-induced cytotoxicity.
3. Genistein may inhibit manganese-induced PC12cells cytotoxicity, which can beconnected with its inhibitory effects on manganese-induced oxidative stress, α-synoverexpression, tau hyperphosphorylatation, and the interaction between α-syn and tau.
In summary, our research preliminarily verified that there was an interaction betweenα-syn and tau during manganese-induced dopaminergic neural injury, which may beinvolved in manganese-induced neural injuries and can be a new potential therapeutictarget against manganism. Genistein could protect against manganese neurotoxicity tosome extent, which may be connected with its effects on interaction between α-syn and tau.Our results may provide new ideas and experimental bases to further our understanding ofmanganese-induced neurotoxicity and effective protective measures.
锰是机体必需的一种微量元素，但摄入过量的锰则会对机体产生毒性作用。锰的毒性作用主要累及中枢神经系统，尤其是基底核。慢性锰中毒主要表现为锥体外系功能障碍，与帕金森病（Parkinson’s disease, PD）的临床表现类似。
α-共核蛋白(α-synuclein，α-syn)是由140个氨基酸组成的可溶性非折叠蛋白,它是帕金森病(Parkinson’s disease, PD)特征性病理结构——路易氏小体（Lewy’s body）的主要组成部分。体内和体外实验均表明高表达α-syn可促进神经元的损伤，诱导PD相关毒性症状的发生。因此越来越多的实验试图揭示α-syn的生理功能及其在神经退行性疾病中的作用机制。但是关于α-syn的生理功能目前还不十分清楚，有研究表明α-syn参与调节突触传递和神经元的可塑性。另有研究提示它可能在信号转导过程中发挥重要作用。近年来，许多研究证实α-syn在Lewy小体病和帕金森病的发生机制中起到了关键的作用,但是其在锰神经毒性作用中的机制尚不清楚。
tau蛋白是一种微管相关蛋白，在神经系统的发育和维持微管功能等方面具有重要作用。生理和病理条件下，tau蛋白的许多苏氨酸和丝氨酸残基可以被多种激酶磷酸化。tau蛋白病理条件下的过度磷酸化将影响其正常功能，使其促微管聚合功能丧失，神经元微管解体，tau蛋白聚集成双螺旋丝（paired helical filaments，PHF）以及形成神经原纤维缠结（neurofibrillary tangle，NFT）结构，并转变成为毒性分子，最终引起神经元变性损伤。
作为存在于细胞质内的可溶性蛋白质，α-syn与tau蛋白之间存在着相互作用。研究表明α-syn与tau蛋白在细胞内除了细胞核外大部分是共定位的，在靠近细胞膜的细胞质边缘，两者的相互作用最强烈。新近研究表明tau蛋白突变可抑制α-syn与tau蛋白的相互作用；α-syn突变也可抑制α-syn与tau蛋白的相互作用。此外，截去羧基端的tau蛋白与α-syn之间的相互作用也被抑制。
染料木素（genistein，Gen）又称三羟异黄酮，主要是从大豆等豆科植物中提取出来的活性成分，为异黄酮类物质中活性功能最高的一种。Gen对神经系统具有显著的保护作用，其作用机制是多方面的。包括抗氧化、调节细胞内的钙离子浓度、与细胞内信号转导途径相互作用、增强胆碱能神经功能、抑制凋亡等途径，从而提高神经细胞的存活能力。
【目的】
在动物和细胞水平探讨锰对α-syn与tau蛋白相互作用的影响，以及这种影响在锰神经毒性中的作用。并进一步探讨染料木素对锰神经毒性可能的抑制效应，以及这种抑制效应与α-syn-tau的相关性。为进一步阐明锰神经毒性的分子机制以及寻找有效的防护措施提供新的思路和实验依据。
【方法】
1．利用灌胃的方法建立慢性锰暴露的动物模型，采用免疫荧光染色、Western blot和免疫共沉淀检测多巴胺能神经元的损伤以及α-syn与tau蛋白相互作用的变化。
2．利用高分化型PC12细胞建立锰毒性的细胞模型，MTT、LDH和TUNEL检测细胞毒性；siRNA转染、Western blot、免疫荧光染色和免疫共沉淀检测α-syn与tau蛋白相互作用的变化。
3．MTT、TUNEL和氧化应激试剂盒检测染料木素对锰毒性的保护作用。同时，利用Western blot、免疫荧光染色检测染料木素对锰诱导的α-syn蛋白表达、tau磷酸化以及两者共定位的影响。
【结果】
1．锰对大鼠黑质多巴胺能神经元损伤和α-syn与tau蛋白相互作用的影响
通过灌胃方法建立慢性锰暴露的动物模型，免疫荧光染色和Western blot结果显示大鼠黑质TH染色阳性细胞数明显减少，同时黑质TH表达水平显著降低，提示锰诱导了多巴胺能神经元损伤；Western blot结果显示大鼠黑质α-syn和tau表达水平均升高；免疫荧光染色和免疫共沉淀结果显示大鼠黑质α-syn和tau蛋白共定位增强，同时这两种蛋白在黑质的相互作用增强。
2．锰对PC12细胞的损伤以及对α-syn与tau蛋白相互作用的影响
MTT结果显示，随着染锰浓度的增加和染锰时间的延长，PC12细胞活力显著下降；TUNEL结果显示，随着锰浓度的增加，PC12细胞凋亡水平逐渐增加；Western blot结果显示锰可使PC12细胞α-syn与tau磷酸化水平增加；免疫荧光染色和免疫共沉淀结果显示锰可使α-syn与tau蛋白在PC12细胞内共定位增强，同时α-syn和tau蛋白相互作用增强。
3．siRNA α-syn和LiCl对锰诱导的PC12细胞α-syn与tau蛋白相互作用的影响
MTT结果显示，干涉α-syn的表达或者加入LiCl可以明显改善锰对PC12细胞的活力抑制；Western blot结果显示，干涉α-syn的表达可以使锰诱导的高p-tau396水平降低；免疫荧光染色结果显示，siRNAα-syn和LiCl均可抑制锰诱导的PC12细胞α-syn与tau蛋白的共定位；免疫共沉淀结果进一步显示LiCl可抑制锰诱导的PC12细胞α-syn与tau蛋白的相互作用。
4．染料木素对锰诱导的PC12细胞毒性的保护作用
MTT结果显示，染料木素可以明显抑制锰诱导的PC12细胞活力下降； TUNEL结果显示，染料木素可以抑制细胞凋亡；氧化应激试剂盒检测结果提示，染料木素可以抑制锰诱导的PC12细胞氧化应激水平的增高。此外，Western blot实验结果显示，染料木素可抑制锰诱导的α-syn蛋白表达水平的增高以及tau蛋白的磷酸化。免疫荧光染色结果表明染料木素可抑制锰诱导的PC12细胞内α-syn与tau蛋白共定位的增强。
【结论】
1．体内外实验均表明，锰可以引起多巴胺能神经元的损伤，诱导α-syn与p-Tau396蛋白表达水平增加；使α-syn与p-tau396蛋白相互作用增强，并且其共定位增强。
2．体外实验表明，分别降低α-syn或p-Tau396蛋白的表达水平，可在抑制锰毒性的同时一定程度上抑制锰诱导的两种蛋白相互作用的增强。表明α-syn和p-Tau396蛋白的相互作用可能在锰诱导的PC12细胞损伤中发挥一定的作用。
3．染料木素可抑制锰诱导的PC12细胞毒性，其机制可能与其抑制锰诱导的氧化应激，α-syn蛋白表达水平增高，tau蛋白的磷酸化以及α-syn与tau的相互作用有关。
综上所述，我们的研究初步阐明锰诱导的多巴胺能神经元的损伤作用中存在α-syn和p-Tau396蛋白的相互作用，这种作用可能参与了锰诱导的损伤过程，并可作为锰损伤防护（如染料木素）的一个潜在作用靶点。我们的结果将为进一步阐明锰神经毒性的分子机制以及寻找有效的防护措施提供新的思路和实验依据。
Background
Manganese is an essential ubiquitous trace element that is required for normal growth,development, and cellular homeostasis. But excessive exposure to manganese results inneurotoxicity. The main target of manganese-induced toxic effects is the central nervoussystem, especial the basal ganglia. The clinical symptoms of chronic manganism arecharacterized by extrapyramidal dysfunction, same as those of Parkinson’s disease.
α-synuclein(α-syn) is a140-amino acid heat stable protein that was identified as theprecursor of the non-Aβ protein in amyloid plaques of human Alzheimer's disease. It ishighly conservative among vertebrates. Since the discovery of its relationship with thepathological processes of neurodegenerative disorders, more and more studies have triedto reveal the physiological functiosn of α-syn and its corelation with other proteins. Although the physiological functions of α-syn are still poorly understood, some studieshave shown that it is associated with neuronal synaptic plasticity. Other studies have foundthat it may be important in the processes of signal transduction. Recently many studieshave confirmed that α-syn plays a key role in the pathogenesis of Lewy body disease andParkinson’s disease. However the mechanism of α-syn in manganese neurotoxicity is stillnot yet clear.
Tau is one of the member of microtubule-associated proteins, which plays animportant role in the development of nervous system and maintain of microtubule stability.Specific characteristic pathological changes associated with tau have been found in manyneurodegenerative diseases. Hyperphosphorylation of tau may affect its normal functionand cause a series of pathological lesions. Hyperphosphorylated tau would lose itsfunction to promote the microtubule polymerization and result in neuronal microtubuledispolymerization, which is easy to aggregate and form paired helical filaments andeventrally neurofibrillary tangles. Thus tau turns to be toxic and can bind with normalmicrotubule-associated proteins, leading to microtubule dispolymerization and neuronaldegeneration.
As two soluble proteins in cytosol, there is an interaction between α-syn and tau.Studies have showed that α-syn and tau are co-localized in cells except nucleus, and theinteraction between them is most intense on the edge of the cytoplasm near the cellmembrane. Recent studies have shown that tau mutation can inhibit the interactionbetween α-syn and tau, and α-syn mutation can inhibit that too. Furthermore, C-terminaltruncated tau can also inhibit its interaction with α-syn.
Genistein (Gen), also called trihydroxyisoflavone, is an active component mainlyextracted from soybean and other leguminous plants. It is one of the most activeisoflavones. Genistein plays a prominent role in protecting the nervous system throughmultiple machanisms, including anti-oxydation, modulation of cellular calciumhomeostasis, interaction with cellular signaling pathways, strengthening the function ofcholinergic neurons and inhibition of apoptosis, etc, and improvement of neuronalviability.
Aims
We investigated the effect of manganese on interaction between α-syn and tau inanimal and cell models, and the role of this effect in manganese neurotoxicity. We furtherinvestigated the possible inhibitive effects on manganese neurotoxicity by genistein, andthe association with α-syn-tau interaction. Our results may provide new ideas andexperimental bases to further our understanding of manganese-induced neurotoxicity andeffective protective measures.
Methods
1. The animal model of chronic manganese exposure was established by lavagemethod; immunofluorescence, western blot, and co-immunoprecipitation were used todetermine dopaminergic neuron injury and interaction between α-syn and tau.
2. Highly differentiated PC12cell line was used to establish the cell model ofmanganism, and the cytotoxic effect was analyzed through MTT assay, LDH assay, andTUNEL; siRNA transfection, western blot, immunofluorescence, andco-immunoprecipitation were performed to determine the change of α-syn-tau interaction
3. The neuroprotective effect of genistein was analyzed by MTT, TUNEL, andoxidative stress kit. Western blot, co-immunoprecipitation and immunofluorescence wereperformed to determine the effect of genistein on manganese-induced α-syn expression,tau phosphorylation, and their interaction.
Results
1. The effect of manganese on dopaminergic neuron injury and interaction betweenα-syn and tau
We established the animal model of chronic manganese exposure by lavage method,and the results of immunofluorescence staining and western blot showed that aftermanganese administration, there was an obviously loss of TH positive neurons and andecreased expression of TH in the substantia nigra, suggesting that manganese induced dopaminergic neural injury. Meanwhile, western blot also showed that there was anincreased expression of α-syn and tau. The results of immunofluorescence andco-immunoprecipitation showed that the colocalization of those two proteins wasenhanced and the interaction between them was also increased.
2. The effect of manganese on PC12cells injury and interaction between α-syn andtau
The results of MTT assay showed that manganese inhibited cell viability of PC12cells in a concentration-dependent and time-dependent manner. The increased level ofapoptosis with the increase of manganese concerntion were detected by TUNEL. Westernblot results showed that manganese enhanced the expression level of α-syn and tauphosphorylation in PC12cells. As shown by immunofluorescence andco-immunoprecipitation, the colocalization of those two proteins were enhanced and theinteraction between them was also increased.
3. The effect of siRNA α-syn and LiCl on manganese-induced interaction betweenα-syn and tau in PC12cells
MTT showed that both siRNA α-syn and LiCl reversed the inhibition ofmanganese-induced PC12cell viability. Western blot results showed that after siRNAtransfection, the increased expression of p-tau396induced by manganese was inhibited.Immunofluorescence showed that both siRNA α-syn and LiCl inhibitedmanganese-induced α-syn-tau colocalization. And co-immunoprecipitation showed thatLiCl inhibited manganese-induced α-syn-tau interaction.
4. The neuroprotective effect of genistein on manganese-induced PC12cytotoxicity
MTT assay showed that genistein could markedly inhibit the decrease of PC12cellviability induced by manganese. TUNEL assay showed that genistein inhibitedmanganese-induced apoptosis. The results of oxygenic stress kit indicated that genisteincould inhibit the increased oxygenic stress level induced by manganese. Furthermore,Western blot results showed that genistein inhibited manganese-induced α-synoverexpression and tau phosphorylation. Immunofluorescence showed that genistein inhibited manganese-induced α-syn-tau colocalization.
Conclusion
1. Both in vivo and vitro studies showed that manganese may cause dopaminergicneurons injury; induce increased level of α-synuclein and phosphorylated tau expression;promote the interaction between α-syn and tau; and enhance the colocalization of thosetwo proteins.
2. In vitro research showed that when the expression of α-syn and phosphorylated tauwere decreased respectively, the increased interaction between them was inhibited,suggesting that the interaction between α-syn and phosphorylated tau may play a role inmanganese-induced cytotoxicity.
3. Genistein may inhibit manganese-induced PC12cells cytotoxicity, which can beconnected with its inhibitory effects on manganese-induced oxidative stress, α-synoverexpression, tau hyperphosphorylatation, and the interaction between α-syn and tau.
In summary, our research preliminarily verified that there was an interaction betweenα-syn and tau during manganese-induced dopaminergic neural injury, which may beinvolved in manganese-induced neural injuries and can be a new potential therapeutictarget against manganism. Genistein could protect against manganese neurotoxicity tosome extent, which may be connected with its effects on interaction between α-syn and tau.Our results may provide new ideas and experimental bases to further our understanding ofmanganese-induced neurotoxicity and effective protective measures.
引文
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