Protective Effect Of Thioredoxin Reductase 1 In Parkinson's Disease

Objective:Parkinson's disease(PD)is a neurodegeneration disease.Many factors includingneuroinflammation,neuronal excitotoxicity,genetic mutations and incorrect protein folding can explain the mechanism of PD.PD is characterized by the loss of dopaminergic neurons in the substantia nigra(SN).However,the precise mechanism for the decreased number of dopaminergic neurons is unknown.A growing body of research suggests that oxidative stress is a major factor in PD.Therefore,antioxidant therapy is an important approach for treating PD.The thioredoxin system is an important antioxidant system,which contains thioredoxin(Trx),thioredoxin reductase(TR)and nicotinamide adenine dinucleotide phosphate(NADPH).Thioredoxin reductase 1(TR1)is a major member of the thioredoxin system,so that the TR1 was overexpressed in the substantia nigra neurons.Then its protective effects are studied in the PD model.Methods:The overexpressed TR1 was constructed in PD mouse and cell models,also theprotective effect of overexpressed TR1 on PD was observed by transcriptome sequencing,immunofluorescence,immunohistochemistry,flow cytometry,antioxidant activity,western blotting,behavior and real-time quantitative PCR.Animal experiment:A53T mice(3 months old)were injected with 2?l lentivirus-TR1 in the substantia nigra pars compacta(SNc)on each side by intravenous injection.All mice were fed for ten months.Then,they were observed the changes in behavioral characteristics in the climbing pole experiment,the modified open field test and the olfactory function analysis in the WT,A53T,A53T-LV and A53T-TR1 groups of mice.Next,the mice were anaesthetized with 10%chloral hydrate and euthanized by beheading.The whole brains were removed and placed on a plate over crushed ice immediately.The midbrain was then dissected.The changes were detected in the genes level or the expression of proteins by RNA sequencing and bioinformatics analysis,quantitative real-time reverse transcription-PCR,immunohistochemistry,MDA,T-SOD and GSH production analysis,and?-galactosidase(?-gal)staining.The mice in the MPTP group were intraperitoneally injected with 20 mg/kg MPTP hydrochloride dissolved in saline,with four injections at 3 h intervals over a period of one day.WT indicates the normal C57BL/6 mice;we detected the suspension time of the mice,which was in the stick,after being injected with MPTP or saline(WT),and the levels of GSH,MDA and the activity of T-SOD were also examined in the midbrain and striatum.Cellular experiment:N2a or PC12 cells transferred with lentivirus-TR1 or plasmid-TR1 for 24h were then treated with MPP⁺(1 m M)for 48 h.CCK-8,MTT,immunofluorescence,PCR,western blotting analysis and flow cytometry analysis were used to detect changes in gene and protein expression.Results:Based on KEGG enrichment analysis,the results of gene ontology classification prove that antioxidant activity,mitochondrion-associated adherens complex,cell damage,cell growth,reproduction,signaling,enzyme regulator activity,cerebral cortex regionalization and neuron development are affected in A53T mice compared with WT mice.According to transcriptome analysis:antioxidant activity,enzyme regulator activity,cell damage,cell growth,reproduction and signaling are related in A53T mice compared with A53T-TR1 mice.Seven months after the injection of lentivirus-TR1,the mice were euthanized,and the tissues were collected for subsequent analyses,the cytokine,IL-15 and IL-7 level increased,but the C3,NADPH,CD14,m KATP and L-type Ca²⁺levels decreased in A53T-TR1 mice compared with A53T mice,the symptoms are recovered to some extent in PD model.The data shows that oxidative stress increased,TR1 and TH decreased in the SNc,and cognitive competence,olfactory function and muscular tension decreased in models of PD compared with the WT mice,and TR1 emerged as an important antioxidation agent in dopaminergic neurons.Therefore,TR1 was overexpressed in the MPP⁺-induced cellular model and in the A53T transgenic mouse model of PD,showing that overexpression of TR1 in neuronal cells decreased apoptosis and increased viability,decreased DNA damage,cell senescence,MDA and ROS generation,and increased T-SOD activation and GSH production,TH expression,learning and memory in the PD model.Autophagosomes appeared and the mitochondrial membrane potential was damaged in the V group compared with the NS group in the NS,V,V-LV and V-TR1 groups according to electron microscopy and immunofluorescence.After the overexpression of TR1,autophagy decreased and mitochondrial membrane potential increased in the V-TR1 group compared with the V group in the NS,V,V-LV and V-TR1 groups.This study demonstrates that the overexpression of TR1 could be developed as a novel neuroprotective agent for PD and that reduction of the expression of GSK-3?and NF-?B could be a promising therapeutic strategy for PD.This research suggests a new direction to treat PD.NS(N2a cells cultivated in medium);V(N2a or PC12 cells treated with MPP⁺(1 m M)for 48 h);V-LV(N2a cells transferred with lentivirus for 24 h then treated with MPP⁺(1 m M)for 48 h)and V-TR1(N2a or PC12 cells transfected with lentivirus-TR1 for 24 h then treated with MPP⁺(1 m M)for 48 h).WT(normal C57BL/6 mice);A53T(the C57BL/6 mice which were transfected with mutant human alpha-synuclein);A53T-LV(A53T mice which were injected with lentivirus 2?l in SNc on each side);A53T-TR1(A53T mice which were injected with lentivirus-TR1 2?l in SNc on each side).Conclusion:The protection mediated by the overexpression of TR1 in PD.TR1 may become a targeted protein for the treatment of PD in the future.