Role Of α-synuclein-tau Interaction In Manganese-induced Neurotoxicity And Protective Effect Of Genistein

BackgroundManganese 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. AimsWe 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.Methods1. 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 interaction3. 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.Results1. The effect of manganese on dopaminergic neuron injury and interaction betweenα-syn and tauWe 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 andtauThe 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 PC12cellsMTT 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 PC12cytotoxicityMTT 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.Conclusion1. 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.