Study On Manganese Induces Glu And GABA Release Disorder By Disrupting SNARE Complex-mediated Synaptic Vesicle Fusion

Objective:Manganese（Mn）is an essential trace element in human body that is required for maintaining proper function and regulationof numerous biochemical and cellular reactions.Despite itsessentiality,at excessive levels Mn is toxic to the central nervous system through brain-blood barriers,and accumulated in striatumand the substantianigra（SN）.Overexposure to Mn from environmental sources can result in a condition known as manganism.Although dopamine depletion,mitochondrial dysfunction,abnormal release of neurotransmitter and oxidative stress have been actively investigated as neurotoxic mechanisms of Mn,the underlying mechanisms are still unclear.A previous study demonstrated that manganese released into the synaptic cleft may influence synaptic neurotransmission in neuron,and disturbance of neurotransmitter release is also one of the important cellular and molecular correlates of neurodegenerative diseasesresulting from chronic Mn exposure.SNAREs（soluble N-ethylmaleimidesensitive fusion protein attachment protein receptors）and associatedproteins play critical roles in mediating synaptic vesicle fusion.The SNARE complex is bridging vesicles tothe plasma membrane during regulated exocytosis and consists of three compartmentally defined proteins:Syntaxin 1,VAMP-2 and SNAP-25.SNAP-25 combined with synaptotagmin is essential for theexocytosis of the contents of the synaptic vesicle in the nerve terminal.Munc18bound to syntaxin-1 appeared to prevent syntaxin-1 from engaginginto SNARE complexes,and led to the suggestion that Munc18 is an inhibitor of fusioninstead of a component of the fusion machine.Besides being a member of the SNARE complex VAMP-2also occurs in a complex with Synaptophysin.The VAMP-2-Synaptophysin complex represents a reserve pool forVAMP-2 enabling synaptic vesicles to adjust to an increaseddemand for synaptic efficiency.Calpainscould cleave several cytosolic,membrane or cytoskeleton-associated proteins,seems to play an importantrole in synaptic plasticity.Proteolysis by calpains is likely to change the integrity,localization,and/or activityof endogenous proteins,and results in either the activation or the inhibition of substrate functions.Recent datasuggested that SNAP-25 was substrates of members of the calpains familyin neurons.Alphy-Synuclein（α-Syn）localizes to synaptic termini,existing in equilibrium between the cytosol and synaptic membrane.The regulatory or modulatory functions ofα-Syn havebeen proved for several cellular processes,including a role in the regulation of synaptic functions and vesicletraffic,plasticity,neurotransmitter release,monoamineneurotransmitter homeostasis,and modulating of proteasomal activity.There is evidence thatα-Syn directly binds to VAMP2,and promotes SNARE-complex assembly and synaptic vesicles fusion.At both low micromolarand overexpressed levels,α-Syn became cytotoxicity.In addition,the over-expression ofα-Syn might disrupt synaptic function,resulting in cognitive disturbance.α-Syn may be an important protein in the development of neurodegeneration.The current study employed primary cultured neuron,SH-SY5Y cell line and adult mouse to investigate whether the protein expression and their interaction of SNARE complex associated proteins were the media between Mn exposure and synaptic vesicle fusion disorders;and we employed calpeptin,an inhibitor of calpains,and lentivirus vector ofα-Syn shRNA to examine whether specific SNAP-25 cleavage andα-Syn overexpression were involved in influencing the formation of SNARE complex by SH-SY5Y cells.Methods:1、Primary cultures yielded more than 90%neurons,as determined by neuron-specific enolase（NSE）immunostaining.The cytotoxicity of Mn was observed by MTT and LDH assay after exposed to Mn（0-200μM）for（0-24h）.According to the experimental results,Mn exposures insubsequent experiments were restricted to concentrations of100μM Mn for 0,6,12,18,24 h.At this level and time,the mRNA and protein expressions of syntaxin-1,VAMP-2,SNAP-25,synaptophysin,synaptotagmin 1,Munc 18,the combination of syntaxin-1 and Munc 18,synaptophysin and VAMP-2,the assembling of SNARE complex,and the synaptic vesicles fusion were determined.2、RNA interference lentiviral vector was tranfected into SH-SY5Y cells.A scrambled shRNA was used as a negative control.After confirming that the transfection efficiency and the silencing efficiency were more than 90%,the following assays were performed after exposed to 100μM Mn for 24h.At this level and time,CCK-8and LDH release asssy for determine the cytotoxicity of Mn and lentiviral vector.After that,the mRNA and protein expressions of syntaxin-1,VAMP-2,SNAP-25,synaptophysin,the combination ofα-Syn and VAMP-2,synaptophysin and VAMP-2,the assembling of SNARE complex,and the synaptic vesicles fusion were determined.3、A total of 24 male and 24 female adult Kunming mice weighing 25±2 g were obtainedfrom the Laboratory Animal Center of China Medicine University（SPF grade,Certificate No.SCXK 2013–0001）.The mice were randomly divided into eight groups of six mice（three male and three female）:controlgroup,MnCl₂-exposed groups（25,50,100μmol/kg）,calpeptin control group,and calpeptin pre-treated groups（25,50,100μg/kg）.The volume of injection was 2 ml/kg body weight,once every day,continuous injection for 24 days.8,16,24 days after the administration,each mouse was placed individuallyinto the open field for test.After mice were treated for 24 days,all the mice were sacrificed by decollation after anesthetized.Firstly,one mouse in each group were perfused with buffered 4%paraformaldehyde solution,and then the basal nuclei was removed for pathological observation.Secondly,two mice in each group prepared the single cell suspension of cerebral cortex for[Ca²⁺]_iand calpain activity determination,and prepared synaptosomes for synaptic vesicle fusion,Glu and GABA concentration determination.Thirdly,three mice in each group were sacrificed and dissected the basal nuclei to determine Mn levels,the mRNA and protein expressions of syntaxin-1,VAMP-2,SNAP-25.Results:1、Cells treated with Mn induced significant celldamages in a concentration-dependent manner,at 200μM Mntreatment for 24h,the viability of neurons were significantly decreased.According to the results,Mn exposures in subsequent experiments were restricted to concentrations of 100μM for 0,6,12,18,24h.Mn was found to down-regulate the expression of SNAP-25 and up-regulate the expression of VAMP-2 in cultured neurons.Moreover,the interaction of Munc 18 and Syntaxin increased significantly in response to Mn treatment for 18-24h,and the interaction of VAMP-2 and Synaptophysin increased first and then decreased.FM1-43-labeled synaptic vesicles also provided evidence that the treatment with Mn resulted in neurotransmitter vesicle fusion increasing first and then decreasing,which was consistent with the 80 kDa protein levels of SNARE complexes.2、After normal cells were treated with Mn for 24h,Mn was found to up-regulate the expression ofα-Synand VAMP-2.The interaction of VAMP-2 andsynaptophysin decrease,but the interaction of VAMP-2 andα-Syn increased significantly in response to 100μM Mn treatment normal cells.The formation of SNARE complexes and FM1-43-labeled synaptic vesicle fusionwere decrease as VAMP-2.After LV-α-Syn shRNA cellswere treated with Mn for 24h,expression ofα-Syn decreased compared with normal cells treated with 100μM Mn,The interaction of VAMP-2andsynaptophysin increase,and the interaction of VAMP-2 andα-Syndecreased significantly in response to normal cells treated with 100μM Mn.The formation of SNARE complexes and FM1-43-labeled synaptic vesicle fusionwere increased as the interaction of VAMP-2 andsynaptophysin.3、Mn deposition increased significantly in basal nuclei in Mn-treated and calpeptin pre-treated groups.Behaviorally,less time spent in the center of the area and decreased average velocity significantly in an open field test after 24 days of Mn exposure.With the increase in MnCl₂ dosage,[Ca²⁺]_iand calpain activity increased significantly,but pretreatment with calpeptin caused a dose-dependent decrease in calpains activity.There were fragments of N-terminal of SNAP-25 protein appearance in Mn-treated groups,but it is decreased with pretreatment of calpeptin.FM1-43-labeled synaptic vesicles also provided evidence that the treatment with Mn resulted in increasing first and then decreasing,which was consistent with Glu release and the 80 kDa protein levels of SNARE complexes.Conclusion:In conclusion,our findings clearly demonstrated that Mninduced the disorders of neurotransmitter vesicle release via disturbing the protein expression and their interaction ofSNARE complex associated proteins.The probably mechanism might be that Mn increase the combination of VAMP-2 andα-Syn by over-expressingα-Syn,and over-expression ofα-Syn has multiple binding sites for VAMP-2 on the v-vesicle,they might sequester most of the VAMP-2 on the vesicle to make less VAMP-2 available for SNARE complex formation.Furthermore,Mn could increase concentration of intracellular Ca²⁺and disturbed SNARE-mediated synaptic vesicle fusion via cleaving SNAP-25 by excessive activation of calpains,disrupt Glu and GABA release,result in neurodegenerative disease.Together,these results give insight into the neurochemical alterations that take place in nerve cells during elevated Mn exposure.