Molecular Mechanism Of Manganese-induced S-nitrosylation Of PINK1 Interference With Mitochondrial Homeostasis In Nerve Cell

Objective:Manganese（Mn）is a metal element widely existing in the earth’s crust and is also one of the essential trace elements in the human body.Occupational or environmental exposure to excess Mn can induce neurotoxicity with symptoms similar to Parkinson’s disease,known as manganism.Mitochondrial damage plays a major role in the neurotoxicity of Mn.Under physiological conditions,the structure,function,and number of mitochondria were maintained in a relatively stable state,which is called mitochondrial homeostasis.In order to maintain steady and dealing with mitochondria damage a series of serious consequences,cells have evolved multiple levels of mitochondrial quality control system.At the organelle level,mitochondrial homeostasis is maintained by two opposing mechanisms:the elimination of damaged mitochondria via mitophagy and the generation of new mitochondria via mitochondrial biogenesis.However,the effect of Mn on mitochondrial homeostasis and its molecular mechanism have not been fully elucidated.Our previous studies have shown that Mn can cause S-nitrosylation（SNO）of macroautophagy-related proteins and interfere with the activation of macroautophagy.Given that PTEN-induced putative kinase 1（PINK1）is also one of the target proteins of SNO modification,and its normal function is crucial to maintain mitochondrial homeostasis.Therefore,we speculate that Mn can interfere with mitochondrial homeostasis in nerve cells,leading to mitochondrial damage,and its regulation of mitochondrial homeostasis may be related to the S-nitrosylation of PINK1.In order to verify our conjecture,firstly,Wistar rats were used to establish an animal model of manganism to explore the effects of different doses of Mn exposure on the mitochondrial homeostasis of rat nerve cells.Secondly,the primary cultured neurons and the specific inhibitor 1400W of inducible nitric oxide synthase（i NOS/NOS2）were used to verify the role of PINK1 S-nitrosylation in Mn-induced neuron injury and mitochondrial damage.Last,we explored the specific molecular mechanism of PINK1 S-nitrosylation interfering with neuronal mitochondrial homeostasis from the perspectives of mitochondrial biogenesis and mitophagy.Methods:1.Sixty-four adult Wistar rats were randomly divided into 4 groups according to body weight,with 16 rats in each group（male and female in half）.Namely control group,low dose of Mn group,medium dose of Mn group and high dose of Mn group.The control group was intraperitoneally injected with normal saline,while the Mn-treated groups were intraperitoneally injected with 15,30 and 60 mg/kg Mn Cl₂for 6 weeks.We tested whether the animal model of manganism was successfully established by detecting the changes of neuroethology and the content of Mn in striatum of rats.We evaluated the damage of rat nerve cells by detecting the early apoptosis rate and ROS level,as well as the expression levels of mitochondrial apoptosis pathway associated proteins.We evaluated the mitochondrial damage of rat nerve cells by detecting the mitochondrial membrane potential,mitochondrial ROS（mt ROS）,ATP level,and the ultrastructure of mitochondria.We evaluated the mitophagy level of rat nerve cells by detecting the expression level of mitophagy related proteins and the co-localization of mitochondria and autophagy vesicles.We evaluated the mitochondrial biogenesis level of rat nerve cells by detecting the expression levels and m RNA levels of mitochondrial biogenesis related proteins,as well as mitochondrial DNA copy numbers.Last,we detected the S-nitrosylation level of PINK1and the phosphorylation level of its downstream proteins ZNF746 and Parkin.2.Primary neurons were treated with 0,50,100,and 200μM Mn Cl₂ for 0,6,12,18,and 24h to detect cell viability and LDH release.Combined with morphological changes of neurons,the doses of Mn in the further experiments were determined to be 100 and200μM,and the treatment time was 24h.To verify whether S-nitrosylation of PINK1 is involved in Mn-induced neuronal injury,we used NOS2 inhibitor 1400W as an intervention and grouped the cells as follows:Control group（culture medium）,low dose of Mn group（100μM Mn Cl₂）,high dose of Mn group（200μM Mn Cl₂）,1400W control group（20μM 1400W）,low dose of 1400W intervention group（10μM 1400W+200μM Mn Cl₂）,high dose of 1400W intervention group（20μM 1400W+200μM Mn Cl₂）.We explored the mechanism of S-nitrosylation of PINK1 after Mn treatment by detecting the activity of NOS2 and the level of NO.We evaluated the damage of primary neurons after Mn and 1400W pretreatment by detecting the early apoptosis rate,ROS level,and the expression levels of apoptosis related proteins in the mitochondrial pathway.We detected the mitochondrial membrane potential,mt ROS,ATP level and the mitochondrial network morphology to evaluate the damage of primary neuronal mitochondria after Mn and1400W pretreatment.3.To investigate the molecular mechanism of Mn induced S-nitrosylation of PINK1interfering with neuronal mitochondrial homeostasis,we examined the protein and m RNA expression levels of ZNF746,the ubiquitination and phosphorylation levels of ZNF746,the interaction between PINK1 and ZNF746 proteins,the nuclear translocation levels of ZNF746,the interaction between ZNF746 protein and PGC-1αpromoters,the expression levels and m RNA levels of mitochondrial biogenesis related proteins,and mitochondrial DNA copy numbers after Mn and 1400W pretreatment.The above indicators are used to evaluate the effect of Mn-induced S-nitrosylation of PINK1 on neuronal mitochondrial biogenesis.Next,we examined the interaction between PINK1 and Parkin protein,the phosphorylation level of Parkin,the translocation of p-Parkin to mitochondria,the co-localization level between mitochondria and autophagic vesicles,the co-localization level between mitochondria and lysosomes,and the expression level of mitophagy related proteins after Mn and 1400W pretreatment.The above indicators are used to evaluate the effect of Mn-induced S-nitrosylation of PINK1 on neuronal mitophagy.Results:1.The rats in Mn exposure group showed obvious neuroethology abnormalities,and the brain Mn content also increased significantly,which indicated that the rat model of manganism was successfully established.Compared with the controls,the nerve cells and its mitochondria in the Mn exposure group were significantly damaged.In addition,the level of mitochondrial biogenesis in rat nerve cells were significantly increased in the low-dose and medium-dose Mn exposure groups,but significantly decreased in the high-dose Mn group,indicating that low-dose Mn exposure can activate mitochondrial biogenesis,while high-dose Mn exposure inhibits mitochondrial biogenesis.Exposure to different doses of Mn can activate mitophagy.However,compared with the medium-dose Mn group,the mitophagy level was significantly decreased in high-dose Mn group,indicating that high-dose Mn exposure hindered the activation of mitophagy.Compared with the controls,the S-nitrosylation level of PINK1 was significantly increased in high-dose Mn group;Compared with the medium-dose Mn group,the phosphorylation levels of ZNF746 and Parkin both decreased to a certain extent.These results indicated that high-dose Mn exposure leads to the S-nitrosylation of PINK1,which interferes with its kinase activity and leads to a decrease in the phosphorylation level of its downstream proteins,namely ZNF746 and Parkin.2.Compared with the control group,the activity of NOS2,the expression of NOS2protein,the level of NO production and the level of SNO-PINK1 in the 200μM Mn-treated group were significantly increased.Compared with the 200μM Mn-treated group,the activity of NOS2,the level of NO production and the level of SNO-PINK1 in the 1400W intervention group were significantly decreased,indicating that Mn can activate NOS2 to produce a large amount of NO and cause the S-nitrosylation of PINK1.However,1400W can reduce the Mn-induced NO production by inhibiting the activity of NOS2,and then alleviate the S-nitrosylation of PINK1.Compared with the controls,the levels of ROS production,the early apoptosis rate,and the expression levels of apoptosis-related protein in the 100 and 200μM Mn-treated groups were significantly increased,indicating that Mn treatment caused primary neuron damage.In addition,mt ROS production,mitochondrial membrane potential and ATP levels,and the expression of apoptosis-related proteins in the mitochondrial pathway were significantly increased in the Mn-treated group,obvious mitochondrial network fragmentation also occurred,these results indicated that Mn treatment caused mitochondrial damage in primary neurons.Compared with 200μM Mn-treated group,the degree of neuronal injury and mitochondrial dysfunction in 1400W intervention group were significantly reduced,indicating that 1400W can alleviate the primary neuronal damage and mitochondrial dysfunction caused by Mn.3.Compared with the controls,mitochondrial biogenesis level was significantly increased in the 100μM Mn-treated group and significantly decreased in the 200μM Mn-treated group.The 200μM Mn-induced S-nitrosylation of PINK1 reduced the phosphorylation and ubiquitination levels of ZNF746,resulting in increased expression of ZNF746 protein in cells and its entry into the nucleus to inhibit the transcription of PGC-1αpromoter,thus affecting mitochondrial biogenesis.Mitophagy level was significantly increased in the Mn treatment groups,but 200μM Mn significantly inhibited the activation of mitophagy.The 200μM Mn-induced S-nitrosylation of PINK1 decreased the phosphorylation level of Parkin and its mitochondrial translocation,which affected the fusion of mitochondria with autophagy vesicles and mitochondria with lysosomes,thus hinding the activation of mitophagy.Compared with the 200μM Mn treatment group,the mitochondrial biogenesis level and mitophagy level in the 1400W intervention group was significantly increased.These results suggested that 1400W can alleviate the interference of high-dose Mn on mitochondrial homeostasis by decreasing the level of PINK1 S-nitrosylation.Conclusion:Mn-induced S-nitrosylation of PINK1 decreased the phosphorylation and ubiquitination levels of ZNF746,thereby inhibiting PGC-1α/NRF1/TFAM mediated mitochondrial biogenesis;On the other hand,S-nitrosylation of PINK1 reduces the phosphorylation level of Parkin and its mitochondrial translocation,thus hindering the activation of PINK1/Parkin mediated mitophagy.