Effects Of Iron And Extracellular α-synuclein On Cultured Astrocytes

Parkinson’s disease(PD)is a neurodegenerative disorder characterized in its late phase by the sustained loss of dopaminergic neurons in the substantia nigra pars compacta(SNpc).PD is clinically characterized by rest tremor,bradykinesia,rigidity and postural instability.The mechanisms underlying PD pathogenesis have not been revealed yet.Multiple factors might be involved,such as genetic mutation,environmental factors,aging,et al.Neuropathological hallmarks of PD include the degeneration and loss of dopaminergic neurons in the substantia nigra(SN)and the formation of Lewy body(LB).α-Synuclein is the main component of LB and abnormal aggregation of α-synuclein in neurons can alter cell membrane potential,cause mitochondria dysfunction,induce endoplasmic reticulum(ER)stress,in which ER stress is one of the core toxic mechanisms of α-synuclein.α-Synuclein can be released by neurons and then be transported between cells by endocytosis and exocytosis.Iron deposition is another important factor in the etiology of PD.Post-mortem examinations revealed that individual dopaminergic neuron in substantia nigra shows raised iron levels in PD and iron levels in substantia nigra are associated with the progression of PD.Previous studies have confirmed that iron can promote the aggregation of α-synuclein and enhance the cytotoxicity of α-synuclein,but the relationship between iron deposition and extracellularα-synuclein has not been reported.Specific damaged neurons were much concerned in the field of neurodegenerative diseases in the past.Astrocytes account for 20%-50% of the brain’s volume and play an important role in the development of the nervous system,synaptic transmission and ion balance.Therefore,astrocytes contribution in neurodegenerative diseases has been attracting more attentions.Astrocytes themselves express low levels of α-synuclein and can also take extracellular α-synuclein.The accumulation of α-synuclein into astrocytes promotes the release of proinflammatory and chemical factors,leading to neuronal damage and subsequent activation of microglia.In the present study,using C6 glioma cells,primary cultured ventral mesencephalon(VM)astrocytes and VM neurons as cell models,we used western blot,immunofluorescence,real-time PCR,fluorometric assays to investigate the effects of extracellular α-synuclein as an insult on the expression of proinflammatory cytokines in VM astrocytes and whether and how iron modulate this process,and evaluate whether ER stress was involved in the toxic effects of α-synuclein,as well as the VM neurons injuries.The results were shown asfollows:1.IL-1β and TNF-α mRNA levels were increased in 100 nmol/L and 300 nmol/Lα-synuclein monomers-treated C6 glioma cells for 24 h.This increase was to a larger extent in the 300 nmol/L α-synuclein group when compare with the 100 nmol/Lα-synuclein group.IL-1β mRNA levels were up-regulated to 1.83 folds and 2.69 folds when compared with the control group.TNF-α mRNA levels were up-regulated to 1.35 folds and 1.74 folds when compared with the control group.These results indicatedα-synuclein monomers up-regulated the proinflammatory cytokines expressions in C6 glioma cells in a concentration-dependent manner.2.IL-1β and TNF-α mRNA levels were increased in 100 nmol/L and 300 nmol/Lα-synuclein monomers-treated VM astrocytes for 24 h.This increase was to a larger extent in the 300 nmol/L α-synuclein group when compared with the 100 nmol/Lα-synuclein group.IL-1β mRNA levels were up-regulated to 9.58 folds and 21.7 folds when compared with the control group.TNF-α mRNA levels were up-regulated to 4.80 folds and 6.17 folds when compared with the control group.The results indicated thatα-synuclein monomers can up-regulated the proinflammatory cytokines expressions in VM astrocytes in a concentration-dependent manner.3.100 μmol/L α-synuclein monomers were incubated at 37 oC for 3 d to prepareα-synuclein fibrils.IL-1β mRNA levels were up-regulated to 3.22 folds and 7.91 folds,TNF-α mRNA levels were up-regulated to 3.09 folds and 4.33 folds in 100 nmol/L and300 nmol/L α-synuclein fibrils-treated VM astrocytes when compared with the control group.The results indicated that α-synuclein fibrils can also up-regulated the proinflammatory cytokines expressions in VM astrocytes in a concentration-dependent manner and α-synuclein monomers were more toxic than α-synuclein fibrils.4.CHOP protein levels were up-regulated to 1.33 folds and 1.50 folds in 100 nmol/L and300 nmol/L α-synuclein monomers-treated C6 glioma cells for 24 h when compared with the control group.ATF-4 protein levels were up-regulated to 2.05 folds and 2.10 folds when compared with the control group.XBP-1s protein levels were up-regulated to 1.24 folds and 1.25 folds when compared with the control group.These results indicated that extracellular α-synuclein could induce ER stress in C6 glioma cells.The expression of CHOP and ATF-4 were increased in 20 μmol/L MG132 and 40 μmol/L chloroquine-treated C6 glioma cells for 24 h when compared with the control group, suggesting that inhibition of ubiquitin-proteasome(UPS)and autophagy-lysosome pathway(ALP)can induce ER stress.And there was no significant difference in 100nmol/L α-synuclein/chloroquine group when compared with the chloroquine group,suggesting that extracellular α-synuclein could be degraded by UPS.However,CHOP and ATF-4 expressions were significantly decreased in α-synuclein/MG132 group when compared with the MG132 group.These results indicated that extracellular α-synuclein can trigger ER stress in C6 glioma cells and α-synuclein can be degraded by both UPS and ALP,ALP was more likely to be activated by extracellular α-synucleinin in the case of UPS suppression.5.CHOP protein levels were up-regulated to 1.96 folds and 2.02 folds in 100 nmol/L and300 nmol/L α-synuclein monomers-treated VM astrocytes for 24 h when compared with the control group.ATF-4 protein levels were up-regulated to 2.23 folds and 2.32 folds when compared with the control group.XBP-1s protein levels were up-regulated to 1.29 folds and 1.48 folds when compared with the control group.These results indicated that extracellular α-synuclein could induce ER stress in VM astrocytes6.IL-1β mRNA and TNF-α mRNA levels were unchanged in 10 μmol/L ferric ammonium citrate(FAC)-treated C6 glioma cells for 24 h when compared with the control group.IL-1β mRNA and TNF-α mRNA levels were increased in 100 μmol/L FAC group when compared with control group.And there was no statistical changes inα-synuclein/FAC group when compared with the α-synuclein group.These results indicated that iron has no significant effect on α-synuclein-treated C6 glioma cells.7.IL-1β mRNA and TNF-α mRNA levels were increased in 10 μmol/L FAC-treated VM astrocytes for 24 h,but not statistically significant when compared with the control group.But the levels were increased in 10 μmol/L FAC/α-synuclein grouop.IL-1β mRNA and TNF-α mRNA levels were up-regulated to 11.92 folds and 4.95 folds when compared with the control group and 1.26 folds and 1.18 folds when compared with the α-synuclein group.IL-1β mRNA and TNF-α mRNA levels were also increased in 100 μmol/L FAC-treated VM astrocytes,and the increase of TNF-α mRNA was statistically significant when compared with the control group.IL-1β mRNA and TNF-α mRNA levels were up-regulated to 11.59 folds and 6.05 folds in 100 μmol/L FAC/α-synuclein group when compared with the control group and 1.22 folds and 1.44 folds when compared with the α-synuclein group.These results indicated that iron could increase the toxicity of α-synuclein in VM astrocytes.8.The expression of CHOP 、 ATF-4 、 XBP-1s was increased in 100 μmol/L FAC/α-synuclein-treated VM astrocytes for 24 h when compared with the control group.The expression of CHOP、ATF-4、XBP-1s was up-regulated to 1.61、1.93、2.26 folds when compared with the control group and 1.24、1.25、1.76 folds when compared with theα-synuclein group.The above results indicated that elevated levels of iron in primary cultured VM astrocytes could enhance ER stress induced by extracellular α-synuclein.9.Mitochondrial transmembrane potential(ΔΨm)is decreased in 100 nmol/L and 300nmol/L α-synuclein-treated VM neurons for 24 h.ΔΨm was decreased by 12% and 15%when compared with the control group.CHOP protein levels were up-regulated to 1.62 folds and 1.33 folds in 100 nmol/L and 300 nmol/L α-synuclein monomers-treated VM neurons when compared with the control group.ATF-4 protein levels were up-regulated to 1.26 folds and 1.25 folds when compared with the control group.XBP-1s protein levels were up-regulated to 1.21 folds and 1.28 folds when compared with the control group.These results indicated that extracellular α-synuclein could induce damage and ER stress in VM neurons.The above results indicate that extracellular α-synuclein monomers and fibrils can induce the activation of VM astrocytes,and α-synuclein monomers are more toxic thanα-synuclein fibrils.Extracellular α-synuclein can trigger endoplasmic reticulum stress in C6 glioma cells and α-synuclein can be degraded by both UPS and ALP,and ALP was more likely to be enhanced by extracellular α-synuclein in the case of UPS suppression.Extracellular α-synuclein could induce the expression of ER stress marker CHOP,ATF-4,XBP-1s in C6 glioma cells and VM astrocytes,indicating α-synuclein can induce ER stress in C6 glioma cells and VM astrocytes.Intracellular high iron levels can increase the expression of proinflammatory cytokines and enhance ER stress induced by extracellularα-synuclein in VM astrocytes.In this study,we investigated the relationship between iron deposition and extracellular α-synuclein in VM astrocytes.These results provide new experimental basis for further exploring the mechanisms of interaction between iron andα-synuclein and intervention strategies in PD.