| Parkinson’s disease(PD)is the second most common neurodegenerative disease following Alzheimer’s disease(AD).Although genetic vulnerability,environmental factors and aging play a role in the pathogenesis of PD,the exact mechanisms are not clear.The most important neuropathological feature of PD is the appearance of eosinophilic inclusion bodies in the dopaminergic neurons remaining in the substantia nigra,which is the Lewy body,whose main component is aggregated α-synuclein.Alpha-synuclein is a kind of Prion-like protein that is transmitted between cells by endocytosis and exocytosis.It can be released by neurons and then taken up by surrounding cells(including neurons,glial cells,etc.),therefore transmitted between cell and interconnected structures.Studies have shown that α-synuclein can be released by neurons through exosomes.The exosomes are 30-100 nm membranous vesicles that are released by fusion of intracellular multivesicular bodies(MVBs)and cell membranes.It is in turn taken up by the surrounding cells.At the same time,the exosomes can transport intracellular components such as RNA,DNA,and proteins to the outside of the cell,and act on adjacent or distant cells for information exchange between different cells under physiological as well as disease states.Exosomes can be found in biological samples such as blood,urine,and saliva,medium from cultured cells.The family of Rab protein plays an important role in the secretion of exosomes.Rab family is a small class of GTP-binding proteins consisting of approximately 200 amino acids with a molecular weight of approximately 21-25 kDa.As a molecular switch in cell vesicle transport,it is activated by guanine nucleotide exchange factor(GEF)and inactivated by GTP hydrolysis activator protein(GAP)to regulate membrane transport.The four main steps are: vesicle formation,vesicle transport,vesicle adhesion,and fusion of the vesicle membrane to the target.These different steps are achieved by binding of the Rab to the GTP and interacting with upstream and downstream effectors to bind to a particular membrane.Iron is the most abundant of all trace elements in the human body.The concentration of iron in the brain increases with age.Iron is abundant in the nucleus accumbens,deep cerebellar nucleus,red nucleus,part of the hippocampus and substantia nigra.The degeneration dopaminergic neurons in the nigrostriatal system caused by elevated iron is a hot issue for neuroscientists.Due to the cytotoxic effects of iron and its ability to promote the production of free radicals,its role in PD cannot be ignored.A large number of studies have suggested that iron accumulation exists in the substantia nigra of PD patients and animal models and is involved in the pathogenesis of PD.The 5’ untranslated region of α-synuclein mRNA contains an iron responsive element(IRE).In addition,α-synuclein exhibited iron reductase activity and was able to regulates the transferrin-mediated iron transport process.These evidences suggest that the relationship between α-synuclein and intracellular iron homeostasis is inextricably linked.The human SNCA gene(a gene encoding α-synuclein)A53T mutation promotes α-synuclein aggregation in the presence of iron and dopamine.Alpha-synuclein aggregates were involved in iron storage,and overexpression of α-synuclein leads to iron accumulation and iron redistribution in neurons.However,the effects of intracellular iron on exosome-mediated α-synuclein release is unclear.In the MES 23.5 dopaminergic cell line,this study investigated whether the intracellular iron statuse affects the release of α-synuclein and the iron content in exosomes by immunoblotting and iron assay.And we also explore the changes in Rab protein associated with the secretion of exosomes.The results were shown as follows: 1.MES 23.5 cells were treated with 100 μmol/L or 1 mmol/L ferric ammonium citrate(FAC)for 4 h,12 h,24 h and 48 h.Rab35 protein levels were significantly up-regulated in 4 h(54.99%)or 12 h(107.0%),compared with the control group(P<0.05,P<0.001).The expression of Rab35 protein returned to the basal level with treatment of 100 μmol/L and 1 mmol/L FAC for 24 h and 48 h.There were no changes observed in Rab5,Rab7,Rab11,and Rab27 a expression at various time points.2.MES 23.5 cells were treated with 0.5 mmol/L antioxidant N-acetyl-L-cysteine(NAC)and 100 μmol/L FAC for 4 h and 12 h.Rab35 protein levels in the NAC-treated group were significantly up-regulated by 1.24 folds at 4 h and 1.37 folds at 12 h(P<0.001).Compared with the control group,Rab 35 protein levels were up-regulated by 89.21% and 1 fold,respectively,in FAC-treated and NAC/FAC co-treated groups for 4 h.There was no difference between FAC-treated and NAC/FAC co-treated groups.Compared with the control group,Rab 35 protein levels were 1.41 folds and 1.17 folds,respectively in FAC-treated and NAC/FAC co-treated groups for 12 h(P< 0.001).There was no difference between FAC-treated and NAC/FAC co-treated groups.The results suggest that the antioxidant NAC does not block iron-induced upregulation of Rab35 protein.3.MES 23.5 cells were treated with the ferroptosis inhibitor Liproxstatin-1 for 12 h,and Rab35 protein level expression did not change.MES 23.5 cells were pretreated with 10 μmol/L Liproxstatin-1 for 30 min,then MES 23.5 cells were co-treated with 10 μmol/L Liproxstatin-1 and 100 μmol/L FAC for 12 h.Compared with the control group,Rab35 protein levels was up-regulated by 22.10% in the FAC-treated group(P<0.01)and 22.73% in the FAC/Liproxstatin-1 co-treated group(P<0.01).There is no difference between FAC/Liproxstatin-1 co-treated group and FAC treated group.After treatment with ferrostatin-1 for 12 h,the expression of Rab35 was up-regulated by 36.15%,and the difference was statistically significant(P<0.01).After pretreatment with 10 μmol/L Ferrostatin-1 for 30 min,MES 23.5 cells were co-treated with 100μmol/L FAC for 12 h,and the expression of Rab35 was not significantly changed.The results suggest that the ferroptosis inhibitor Liproxstatin-1 could not block iron-induced Rab35 protein up-regulation.And the ferroptosis inhibitor Ferrostatin-1 may could block iron-induced Rab35 protein up-regulation.4.The exosomes were collected by ultracentrifugation,and the expression of Alix was detected by immunoblotting,suggesting that exosomes were successfully prepared.MES 23.5 cells were treated with 100 μmol/L FAC for 24 h and 48 h.Compared with the control group,iron content in MES 23.5 cell exosomes increased significantly,96.01%(P< 0.01)and 2.21folds(P< 0.001),respectively at 24 h and 48 h.Iron content at 48 h was 69.20% higher,compared with the 24 h group(P<0.01).The above results suggested that intracellular high iron induced an increase in iron content in exosomes of MES 23.5 cells.5.MES 23.5 cells was transfected with α-synuclein overexpression vector for 24 h.The expression of α-synuclein in lysates,supernatant and exosomes increased significantly.In MES 23.5 cells over-expressing α-synuclein,α-synuclein was up-regulated by 67.47% with 100 μmol/L FAC for 24 h compared with the untreated group(P<0.01).6.There was no change in Rab35 expression in MES 23.5 cells overexpressing.α-synuclein,suggesting that α-synuclein itself does not affect Rab35 expression.There was no significant change of Rab35 in MES 23.5 cells over-expressing α-synuclein with 100 μmol/L FAC treatment for 24 h,which was consistent with the above resultthat Rab35 protein returned to the basal level with FAC treatment for 24 h.The above results indicate that iron can induce the up-regulation of Rab35 protein expression in MES 23.5 cells.Antioxidant NAC and ferroptosis inhibitor Liproxstatin-1 could not block this process,indicating the effects might not be related to oxidative stress and ferroptosis.Iron induced α-synuclein upregulation in MES 23.5 cells and elevated iron content in exosomes.We reported the effect of iron on the exosomal release from MES 23.5 cells and investigated Rab35 might be involved in this process.α-synuclein itself has no effects on the expression of Rab35.This provides new experimental basis for further elucidation of the mechanisms underlying iron metabolism and α-synuclein interactions in the pathogenesis of PD. |
