Study On The Molecular Mechanism Of Manganese-Induced Snitrosylation Of Autophagy Regulatory Proteins Disturbing Autophagic Activation

Objective:Manganese(Mn)is an essential trace element that is naturally found in the environment and is necessary as a cofactor for many enzymes and is important in several physiological processes that support development,growth,and neuronal function.However,overexposure to Mn may induce neurotoxicity and lead to manganism or Parkinson's-like disease.Autophagy plays a pivotal role in Mn induced neuron toxicity.Because neural function and survival are mainly dependent on the efficient elimination of cytotoxic proteins by autophagic degradation.Autophagosome formation is controlled by the ATG12–ATG5-ATG16L1/2 protein complex,which acts partly as an E3 ligase for microtubule associated protein 1 light chain 3,known autophagosomal marker.In most cases,autophagy activation ensures cell survival during starvation or stressful conditions,whereas,autophagy dysfunction leads to cell damage.It has recently been found that autophagy-lysosomal dysfunction was caused by lysosomal failure induced by excessive Mn accumulation,destroying the structure and normal function of organelles.However,our previous study found that Mn could activate autophagy to scavenge alpha-synuclein oligomers in mice.Therefore,elucidation of the mechanisms involved in Mn-induced autophagy activation is necessary for the better understanding of its role in Mn neurotoxicity.It has recently been found that JNK/Bcl-2/Beclin1 and IKK?/AMPK/mTORC1 are mainly signaling pathways in autophagy regulation.The activation of JNK results in Bcl-2 phosphorylation and release of its inhibitory interaction with Beclin1,which accelerates in the formation of autophagosomes.In the same situation,the activation of IKK?causes phosphorylation of AMPK,which inactivates mTORC1 in an AMPK dependent manner and then activates the autophagy pathway.Nitric oxide(NO)is a bioactive molecule and plays an important role on the signal transmission in the brain.S-nitrosylation of cysteine residues consists of the covalent addition of a nitric oxide moiety to a reactive sulfhydryl called posttranslational modification resulting in the formation of S-nitrosylated proteins,and disturbs signal transduction cascades.On the basis of this observation,we hypothesized that JNK,Bcl-2,and IKK?proteins may be affected by Mn-induced NO disturbing autophagy activation.To verify the above speculation,C57BL/6J mice were used to establish an animal model to study the effects of manganese exposure on neurotoxicity and autophagy in mice.Then,SH-SY5Y cell line was established to study the effect of manganese-induced S-nitrosylation on JNK/Bcl-2/Beclin1 and IKK?/AMPK/mTORC1 autophagy signal transduction pathways.Finally,specific inducible nitric oxide synthase inhibitor1400W was employed as the intervention agent to further verify the role of S-nitrosylation of autophagy regulatory protein in autophagy dysfunction.Methods:1.To study the effects of manganese exposure on neurotoxicity and autophagy in mice:Twenty-four mice were randomly assigned to four groups(N=6;equal numbers of male and female):The control group mice were intraperitoneally(i.p.)injected with physiological saline(group 1).MnCl₂-treated mice were respectively i.p.injected with 100,200 and 300?mol/kg MnCl₂ diluted in sterile distilled water(group2,3 and 4).The volume of injection was 2 ml/kg body weight,5 times/week for continuously six weeks.After six weeks,we use murine neurobehavior tests to evaluate the effect of exposure to Mn.Then,we detect the level of manganese in striatum,apoptosis and autophagy in nerve cells.At last,the expression of Beclin1,LC3 and p62was measured by western blot.2.To study the effect of manganese-induced S-nitrosylation on JNK/Bcl-2/Beclin1and IKK?/AMPK/mTORC1 autophagy signal transduction pathways:SH-SY5Y cells were divided into four groups.Respectively,the control group(group culture),low manganese group(50?M MnCl₂),medium manganese group(100?M MnCl₂),high manganese group(200?M MnCl₂).First,cells were treated with different doses of 0,50,100,and 200?M MnCl₂ for 0,6,12,18,and 24h,respectively,to detect CCK8 and lactate dehydrogenase(LDH)release,for cell cytotoxicity analysis.According to the above experimental results,the time point 24h was selected to detect the changes of apoptosis and autophagy in nerve cells treated with different doses of MnCl₂.At last,the expression of S-nitrosylated JNK,Bcl-2 and IKK?and their phosphorylation was measured by biotin switch assay and western blot.3.To further verify the role of S-nitrosylation of autophagy regulatory protein in autophagy dysfunction:SH-SY5Y cells were divided into six groups.Respectively,the control group(group culture),high manganese group(200?M MnCl₂),1400W alone pretreatment group(20?M 1400W),low dose 1400W intervention group(5?M1400W+200?M MnCl₂),medium dose 1400W intervention group(10?M1400W+200?M MnCl₂),high dose 1400W intervention group(20?M 1400W+200?M MnCl₂).After 1400W pretreatment for 12h,nerve cells were treated with 200?M MnCl₂for 24h to detect the intracellular NO production,the level of iNOS protein activity,m RNA and protein expression.At last,the expression of S-nitrosylated JNK,Bcl-2 and IKK?and their phosphorylation was measured by biotin switch assay and western blot.Results:1.In vivo experiments:Compared to control group,after exposure to300?mol/kg MnCl₂ for 42 days,the open-field test results shown that the mice were less active and spent less time in the inner central;In the rotarod performance test,grip strength and weight-bearing test,the mice showed a clear decreased locomotor activity during these testing.To detect subtler differences in the gait of mice after the exposure to Mn,their footprints were automatically detected with the Catwalk gait analysis system,and various gait parameters were analyzed.The changes in gait parameters indicated an increased instability in the group treated with 300?mol/kg of MnCl₂,including measures of stance,step and duty cycle,the base of support,and initial and terminal dual stance.Compared to control group,swing speed and stride length were significantly decreased in the group treated with 300?mol/kg of MnCl₂.With the increasing manganese dose,the early apoptosis rate increased in a dose-dependent manner.These data suggest that Mn could impair motor coordination,balance and grasping strength in mice,and further indicate that our mice were a successful model of manganism.After 0-300?mol/kg MnCl₂ exposure,the average fluorescence intensity of MDC positive cells gradually increased with increased Mn dosage.At the highest Mn dose,the number or average fluorescence intensity of MDC-positive cells in mice increased significantly compared to the 200?mol/kg group.Similar results were observed for the protein expression level.Mn treatment resulted in a dose-dependent increase in Beclin1 both in vivo.However,LC3II/LC3I ratio decreased,and p62 protein level increased in the mice treated with the highest Mn dose.2.In vitro experiments:With the increase of concentration and time of manganese treatment,cytotoxicity increased.After different dose of MnCl_2-treated 24h,the early apoptosis rate increased in a dose-dependent manner.Compared to the control group,Mn treatment resulted in a dose-dependent increase in Beclin1 both in vitro,LC3II/LC3I ratio increased,and p62 protein level decreased,autophagy activated in the100?M Mn-treated group.On the contrary,compared to the 100?M Mn-treated group,LC3II/LC3I ratio decreased,and p62 protein level increased,autophagy inhibited in the200?M Mn-treated group.In the JNK/Bcl-2/Beclin1 autophagy pathway,compared to the control group,phosphorylation levels of JNK and Bcl-2 protein were significantly increased in the 100?M MnCl₂ treated group.Compared to the group treated with100?mol/kg MnCl₂,the expression of S-nitrosylated JNK and Bcl-2 were significantly increased in the 200?M MnCl₂ group,and their phosphorylation were decreased.On the other hand,in the IKK?/AMPK/mTORC1 autophagy pathway.The results showed that 4EBP1 and s6k1,as downstream proteins of mTORC1,are involved in autophagy regulation.After different dose of MnCl_2-treated 24h,compared to the control group,the intracellular phosphorylation levels of IKK?and AMPK were significantly increased in the 100?M Mn-treated group.Compared to 100?M Mn-treated group,the level of S-nitrosylated IKK?increased and the phosphorylation decreased significantly in 200?M Mn-treated group.The phosphorylation level of AMPK decreased,while the4EBP1 and s6k1 were increased.3.After the cells pretreated with different concentrations of 1400W for 12h and200?M MnCl₂ for 24h,compared to the control group,the level of intracellular NO production,iNOS protein activity,m RNA expression and protein expression increased significantly in the 200?M Mn-treated group.Compared to the group treated with200?M MnCl₂,the level of intracellular NO production and iNOS protein activity were significantly decreased in 20?M1400W intervention group,however the level of iNOS m RNA expression and protein expression were not significantly changed.1400W could reduce the NO production in cells by inhibiting the activity of iNOS protein.In the JNK/Bcl-2/Beclin1 autophagy pathway,compared to 200?M Mn-treated group,the level of S-nitrosylated JNK and Bcl-2 decreased and the phosphorylation level increased significantly.In the IKK?/AMPK/mTORC1 autophagy pathway,the level of S-nitrosylated IKK?decreased and the phosphorylation level increased significantly.The phosphorylation level of AMPK increased,while the 4EBP1 and s6k1 were significantly decreased.Conclusion:In summary,the outcomes of this study demonstrate that Mn could induce nitrosative stress through activating iNOS and subsequently S-nitrosylated JNK,Bcl-2,and IKK?.S-nitrosylated Bcl-2 increased the affinity of Bcl-2/Beclin1 complex due to the lack of phospho-Bcl-2,which led to autophagy dysregulation.Moreover,S-nitrosylated IKK?reduced the phosphorylation of AMPK,which affected the regulation of autophagy through the mTORC1 signaling pathway.