| Mitochondrial quality control(MQC)and Mitochondrial-associated membrane(MAM)are thought to be the hubs of many cellular functions,closely related to neurodegenerative diseases such as Parkinson's disease(PD)and Alzheimer's disease(AD),but the role of post-translational modifications(PTMs)of target proteins remains to be elucidated.Mitofusin 2(Mfn2)is a key molecule regulating mitochondrial fusion,and its PTMs are critical for the regulation of mitochondrial homeostasis.Protein glutathionization is a reversible PTMs induced by redox,similar to acetylation and phosphorylation affecting a variety of biological processes,such as mitochondrial morphology,calcium homeostasis,cytoskeletal remodeling,protein folding,etc.It plays an important role in the neurotoxicity induced by environmental toxic substances.Cadmium(Cd)and cadmium compounds(Cd2+)are one of the most common environmental heavy metal pollutants,mainly through occupational(mining,electric welding)and non-occupational exposure(contact with cadmium-contaminated food,drinking water and smoking)in the body.The accumulation of cadmium in the body mainly causes kidney,liver and neurotoxicity.Previous studies in our laboratory found that(1)cadmium is involved in renal damage through the endoplasmic reticulum stress eIF2a-ATF4 signaling pathway,and mitochondrial dynamic disorder.However,the role of cadmium exposure in cytotoxic damage and the regulation mechanism of glutathionylation of Mfn2 in MQC process remains to be studied.Objectives:CdCl2-induced Mfn2 glutathionization modification(Mfn2-SSG)is involved in mitochondrial dynamic disorder and MAM structure and calcium transport by cadmium chloride(CdCl2).Mfn2-SSG and MAM structure and calcium transport disorder involved in CdCl2 neurontoxicity.The results provide potential intervention targets and strategies for CdCl2-induced neurological damage.Methods:(1)In vivo test:Evaluation of cadmium chloride(CdCl2)exposure-induced brain injury:Adult male BALB/c mice(6-8 weeks old)of SPF grade were used.Mice were randomized into 4 groups(n=10),including control group(ddH2O),CdC12 exposure group(50 mg/L),NAC exposure group(1 g/L)and combined exposure group(CdCl2 50 mg/L +NAC 1 g/L).The drug is exposed to drinking water for 8 weeks.In the whole animal,nerve tissue and blood samples,the following tests were carried out:Morris water maze test to detect changes in learning and memory function of mice.Inductively coupled plasma mass spectrometry to detect the content of cadmium ions in liver,kidney,hippocampus and plasma;qRT-PCR was used to detect the content of metal lothionein in liver,kidney and hippocampus;color reaction method was used to detect malondialdehyde(MDA).Detection of reduced glutathione(GSH)and oxidized glutathione(GSSG)by DNTB rate colorimetry;detection of Grx1,Grx1,SOD2,a-syn,TH,Mfn2,VCP and expression of necroptosis-related protein.IHC observed the expressiorn and distribution of a-syn,TH and necroptosis protein p-MLKL in hippocampus.HE staining to observe the pathological changes of hippocampus.In vitro test:Human neuroblastoma cell SH-SY5Y was used to treat CdC12,and relevant indicators for toxicity damage model were established.Western blot(WB)detection of cell MQC-related protein,mitochondrial redox-related protein,necroptosis-related protein and neurological function-related protein levels;laser confocal microscopy(IF)technology to detect Mfn2 and PSSG colocalization of mitochondria;co-immunoprecipitation(Co-IP)to detect Mfn2-SSG modification level;PLA test to detect the proximity of p-MLKL and a-syn protein spatial location.The Mfn2,a-syn and necroptosis proteins were co-distribution in MAM;TEM observation the MAM structure and spacing;high-content cellular analysis(HCA)to detect Ca2+transport in MAM;modified expression of GLRXl to verify the role of Mfn2-SSG in CdCl2-induced neurotoxicity;mitochondrial antioxidant(Mito-TEMPO)and antioxidant(NAC)treatment to establish a ROS inhibition model to verify mitochondrial ROS targeted intervention in CdC12 induced neurontoxicity.Results:(1)In vivo test:Compared with the control group,the Morris water maze test showed that the number of platform crossings in the CdC12^treatment group was significantly reduced(P<0.05)and the target residence time was reduced,suggesting that the mice were induced to learn memory impairment.ICP-MS showed that the cadmium ion content in li'ver tissue,kidney tissue and hippocampus of CdCl2 treatment group was significantly increased,suggesting that CdCl2 not only accumulates in the liver and kidney but also accumulates in the nervous system through drinking water exposure;qRT-PCR experiment was used to detected metallothionein 1 and 2(Mil and Mt2)in the liver and kidney tissues,and the specific metallothionein 3(Mt3)in the hippocampus was detected.TBA method and DNTB rate colorimetric assay for detected the MDA,GSSG and GSH levels,CdC12 treatment group MDA and GSSG levels increased(P<0.05),GSH levels decreased;and in the NAC intervention group,MDA levels decreased(P<0.05)and GSSG levels decreased(P<0.05),WB results display the levels of necroptotic proteins RIPK3,p-MLKL and ca-syn in CdCl2 treatment group were increased,while the levels of Mfn2 and TH were decreased.In the NAC intervention group,the necroptosis protein RIPK3,p-MLKL,a-syn protein levels decreased,and Mfn2 and TH levels increased;IHC results were consistent with WB results,demonstrating that CdCl2 induced neuronal necroptosis.(2)In vitro test:Compared with control group,the levels of RIPK3 and p-MLKL proteins in the CdC12 treatment group were increased,the expression levels of VCP and Grxl protein were increased,and the protein glutathionylation was increased.The level of mitochondrial dynamics related protein Mfn2 protein decreased,the level of SOD2 was decreased.IF experimental observation showed that the average fluorescence intensity of cadmium ion probe in CdC12 treatment group increased,mitochondrial lipid peroxidation detection showed CdC12 treatment the fluorescence intensity of C11-BODIPY was increased,IF experiment showed that the colocalization of Mfn2 and PSSG with Mito-Tracker was enhanced in the CdC12 treatment group,the Co-IP experiment showed that the glutathionylated protein complex contained Mfn2 protein.It suggested that Mfn2 could undergo glutathionylation modification;PLA results showed that the fluorescence signal of p-MLKL-a-syn was enhanced in CdC12 group.This results suggesting that p-MLKL and a-syn to be adjacent induced by CdC12.Isolated MAM fraction,WB results showed that p-MLKL and a-syn were co-distributed on the MAM and increased in the CdC12 treated group.TEM showed CdC12 exposed group.the structure of MAM was farther away.HCA results showed that the average fluorescence levels of mitochondrial Ca2+a and cytosolic Ca2+ increased in CdCl2 treatment group,and the average fluorescence level of endoplasmic reticulum Ca2+decreased,suggesting that CdCl2 induced MAM Ca2+transport function changes.Knockdown of GLRXI increased Mfn2-SSG levels and p-MLKL and RIPK3 compared to the CdCIl,treated group;whereas overexpression of GLRXI reduced Mfn2-SSG levels and p-MLKL and RIPK3,suggesting that Mfn2-SSG is involved in CdC12 inducted necroptosis.In the Mito-TEMPO and NAC intervention models,compared with the CdC1,treatment group,the levels of necroptotic proteins p-MLKL and RIPK3 were decreased and cell viability was increased(P<0.05),suggesting that the elimination of mitochondrial ROS may rescue the neurotoxicity induced by CdCl2.Conclusions:CdCl2 exposure induces mitochondrial redox balance disorder in brain tissue and neuronal cells,glutathionization of mitochondrial fusion key protein Mfn2,structural and functional changes in MAM,and necroptosis;Mfn2-SSG participate in the regulation of ER-Mito contact and necroptosis induced by CdCIh,overexpression of GLRXI and antioxidant interventions inhibited neurotoxic effects induced by CdCl2.This study suggests that Mfn2-SSG may be a potential intervention target in CdC12-induced neurotoxicity... |
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