| Parkinson’s disease(PD)is one of the most common neurodegenerative diseases,characterized by the progressive loss of dopaminergic neurons and the presence of Lewy bodies(LBs)mainly composed of aggregated α-synuclein(α-syn)in substantia nigra pars compacta(SNpc).Under physiological conditions,the structure of α-syn is usually stretchable and soluble.Alpha-syn aggregation may be induced by gene mutation(such as A53 T and E46K),α-syn post-translational modification(such as phosphorylation),increased concentration of α-syn and environmental factors(such as exposure to metal ions and pesticides).Alpha-syn aggregates affect the transport of synaptic vesicles and reduce the release of neurotransmitters,increase calcium uptake and promote mitochondrial dysfunction,induce endoplasmic reticulum stress and destroy endoplasmic reticulum-golgi transport,and lead to dysfunction of cell secretion.More importantly,α-syn can be released by and taken up by adjacent cells in the form of monomers,oligomers and fibrils,which plays a key role in the brain pathological transmission of α-syn and the progression of PD.In the post-mortem brain tissues of PD patients,there was amounts of redox active iron in the LBs of SNpc neurons,suggesting a relationship between α-syn aggregation and iron deposition.Our previous study found that intracellular α-syn can promote iron deposition in dopaminergic neurons,and iron deposition can increase the expression and aggregation of α-syn.However,the effect of intercellular α-syn on iron deposition is still not fully elucidated.We found that α-syn monomers can enter into the cells,and increase the protein levels of iron regulatory protein 1(IRP1)and the m RNA levels of hepcidin through endoplasmic reticulum stress,resulting in the decrease of ferroportin(FPN)protein levels in MES23.5 cells.In this study,primary ventral mesencephalic(VM)neurons,primary astrocytes and HEK293 T cells overexpressing FPN-Flag(HEK293T-FPN-Flag cells)were treated with α-syn PFF.Alpha-syn PFF was also injected into the SN and lateral ventricle regions of C57BL/6mice to mimics the intercellular transmission of α-syn PFF.Western blot was used to detect the protein levels of FPN,IRP1,heavy-chain ferritin(H-ferritin),light-chain ferritin(L-ferritin)and tyrosine hydroxylase(TH),real-time fluorescent quantitative PCR was used to detect hepcidin m RNA levels,immunofluorescence was used to detect FPN expression on the membrane,and co-immunoprecipitation was used to detect the interaction between α-syn and FPN.Open field test,pole test and rotarod test was used to detect the locomotor functions and olfactory discrimination task was used to detect the olfactory of C57BL/6 mice.This study aims to elucidate the regulation of extracellular α-syn PFF on FPN expression and explore the possible mechanisms.The results are as follows:1.α-syn PFF was successfully prepared.Alpha-syn PFF was prepared by dissolving 1 mg α-syn monomer in 200 μL 50 mM Tris HCl and 150 mM KCl buffer,shaking at 37 ℃ and1000 rpm for 7 days.The typical filaments of the fibrils were observed under transmission electron microscope.Compared with non-ultrasonicated α-syn PFF,the length of fibrils derived from 1 mg/mL α-syn PFF was shorter after water bath ultrasonication for 1 minute,while the fibrils derived from 0.1 mg/mL and 0.05 mg/mL α-syn PFF after ultrasonication was almost undetectable,only amorphous aggregates were observed.Amorphous aggregates rather than fibrils were obvious with the decrease in the concentration of α-synuclein.The results indicate that α-syn PFF can be decomposed by water bath ultrasonication,and ultrasonic treatment at low concentration can improve the ultrasonic efficiency was improved when the α-syn concentration is low in the solution.2.VM neurons,primary astrocytes and HEK293T-FPN-Flag cells were treated with 1 μg/mL or 5 μg/mL α-syn PFF for 24 hours.Compared with the control group,FPN protein levels were decreased by 28.85%,35.64% and 35.58% in 5 μg/mL α-syn PFF group of VM neurons,primary astrocytes and HEK293T-FPN-Flag cells(P<0.05),the difference was statistically significant;FPN protein levels were not significantly changed in 1 μg/mL α-syn PFF group of VM neurons and HEK293T-FPN-Flag cells.The α-syn protein levels were significantly up-regulated in MES23.5 cells transfected with FPN-Flag plasmid for24 hours,but FPN protein levels remained unchanged.These results suggest that extracellular α-syn PFF,rather than intracellular α-syn overexpression,can down-regulate FPN protein levels.3.VM neurons were treated with 1 μg/mL or 5 μg/mL α-syn PFF for 24 hours.Compared with the control group,hepcidin m RNA levels were increased by 91.55% in 5 μg/mL α-syn PFF group(P<0.001),the difference was statistically significant,the protein levels of IRP1 and heavy-chain ferritin(H-ferritin)were not significantly changed;hepcidin m RNA levels and the protein levels of IRP1 and H-ferritin were not significant changed in 1μg/mL α-syn PFF group.VM neurons were pretreated with toll-like receptor 4(TLR4)inhibitor resatorvid for 2 hours and then treated with 5 μg/mL α-syn PFF for 24 hours.Resatorvid completely blocked the up-regulation of hepcidin m RNA levels induced by α-syn PFF,while the down-regulation of FPN protein levels induced by α-syn PFF still existed.The results suggest that the down-regulation of FPN protein levels by α-syn PFF might not be related to hepcidin and IRP1.4.VM neurons and HEK293T-FPN-Flag cells were pretreated with the endocytosis inhibitor dynasore for 1 hours and then treated with 5 μg/mL α-syn PFF for 24 hours.Dynasore completely blocked the down-regulation of FPN protein levels induced by α-syn PFF.Immunofluorescence data showed that FPN was highly expressed on the cell membrane of HEK293T-FPN-Flag cells without triton X-100 treatment;after treatment with 5 μg/mLα-syn PFF for 24 hours,compared with the control group,FPN protein levels on the cell membrane were significantly decreased by 57.50%(P<0.01),the difference was statistically significant;HEK293T-FPN-Flag cells were pretreated with dynasore for 1hour and then treated with 5 μg/mL α-syn PFF for 24 hours.FPN protein levels on the membrane was significantly restored,similar to that in the control group.The results suggest that the down-regulation of FPN protein levels induced by α-syn PFF can be blocked by endocytosis inhibitors,suggesting FPN regulation by α-syn PFF might be related to the endocytosis process.5.HEK293T-FPN-Flag cells were pretreated with dynasore for 1 hour and then treated with5 μg/mL α-syn monomer or α-syn PFF for 24 hours.CO-IP detection showed that α-syn monomer and α-syn PFF could interact with FPN in the extracellular compartment.When MES23.5 cells were transfected with flag-α-syn plasmid for 24 hours,CO-IP detection showed that there was interaction between α-syn and FPN.The results suggest that α-syn interacts with FPN.6.5 μg α-syn PFF was injected into SN of C57BL/6 mice for 24 hours.There was no significant change in TH protein levels,suggesting that dopaminergic neurons were not damaged.Compared with the control group,the FPN protein levels of α-syn PFF group decreased by 26.87%(P<0.001),the difference was statistically significant,and there was no significant change in the protein levels of IRP1,H-ferritin and light-chain ferritin(Lferritin)in the SN.7.0.2 ng α-syn monomer,2 ng α-syn monomer,0.2 ng α-syn PFF or 2 ng α-syn PFF was injected into the lateral ventricle of C57BL/6 mice for consecutive 7 days.Compared with the control group,2 ng α-syn PFF group mice showed decreased velocity by 30.35% in the open field(P<0.01),longer time to turn and get off the pole by 78.29% and 39.71%(P<0.05),and decreased duration time on the rotating rod by 37.43%(P<0.001),the difference was statistically significant;0.2 ng α-syn monomer group mice decreased the velocity by 30.41% in the open field(P<0.01),the difference was statistically significant,there was no significant change in the time of turning,getting off the pole,and the duration on the rotating rod;2 ng α-syn monomer group mice showed longer time of turning on the pole by 70.52%(P<0.05),the difference was statistically significant,there was no significant change in the velocity in the open field,the time of getting off the pole,and the duration time on the rotating rod;0.2 ng α-syn PFF group mice was no significant change in the velocity in the open field,the time of turning and getting off the pole,and the duration time on the rotating rod.The results suggest that intracerebroventricular injection of 2 ng α-syn PFF for 7 days can cause motor behavior disorder in mice.8.0.2 ng α-syn monomer,2 ng α-syn monomer,0.2 ng α-syn PFF or 2 ng α-syn PFF was injected into the lateral ventricle of C57BL/6 mice for consecutive 7 days.There were no significant changes in the protein levels of α-syn,TH and glial fibrillary acidic protein(GFAP),suggesting that there was no α-syn aggregation,dopaminergic neuron damage and astrocytes activation in the SN.9.0.2 ng α-syn monomer,2 ng α-syn monomer,0.2 ng α-syn PFF or 2 ng α-syn PFF was injected into the lateral ventricle of C57BL/6 mice for consecutive 7 days.Compared with the control group,FPN protein levels were decreased by 26.11% and 25.28% in 0.2 ng α-syn PFF or 2 ng α-syn PFF group(P<0.05),the difference was statistically significant;IRP1 protein levels were decreased by 33.41% in 2 ng α-syn PFF group,the difference was statistically significant(P<0.05);and there was no significant change in the protein levels of H-ferritin and L-ferritin in the SN.10.0.2 ng α-syn monomer,2 ng α-syn monomer,0.2 ng α-syn PFF or 2 ng α-syn PFF was injected into the lateral ventricle of C57BL/6 mice for consecutive 7 days.There were no significant changes in the protein levels of α-syn,TH and GFAP,suggesting that there was no α-syn aggregation,dopaminergic neuron damage and astrocytes activation in the striatum.11.0.2 ng α-syn monomer,2 ng α-syn monomer,0.2 ng α-syn PFF or 2 ng α-syn PFF was injected into the lateral ventricle of C57BL/6 mice for consecutive 7 days.There were no significant changes in FPN,IRP1,H-ferritin and L-ferritin protein levels in the striatum.12.HEK293T-FPN-Flag cells were pretreated with proteasome inhibitor MG132 for 8 hours and then treated with 5 μg/mL α-syn PFF for 2 hours.There were no significant changes on ubiquitylation levels of FPN.The results showed that α-syn PFF treatment down-regulated FPN protein levels in VM neurons,primary astrocytes and HEK293T-FPN-Flag cells.TLR4 inhibitor restatorvid pretreatment can block the increase of hepcidin m RNA levels caused by α-syn PFF in VM neurons,but it can not block the decrease of FPN protein levels,and α-syn PFF does not change the expression of IRP1,suggesting that the down-regulation of FPN induced by α-syn PFF might not be mediated by hepcidin and IRP1.Endocytosis inhibitor dynasore pretreatment can block the decrease of FPN protein levels caused by α-syn PFF in VM neurons and HEK293T-FPN-Flag cells,and the interaction between α-syn PFF and FPN was detected in HEK293T-FPN-Flag cells.This suggested that α-syn PFF might bind to FPN extracellularly and cause its endocytosis,which leads to the down-regulation of FPN protein levels.C57BL/6mice injected with α-syn PFF for 24 h in the SN showed decreased FPN protein levels.C57BL/6 mice injected with α-syn PFF into the lateral ventricle for 7 days showed motor dysfunction and decreased FPN protein levels in SN.In this study,the regulation and mechanism of extracellular α-syn PFF on FPN were studied in VM neurons,primary astrocytes,HEK293 T cells and C57BL/6 mice.The results will help to clarify how extracellular α-syn PFF regulates FPN protein levels,which provides a new experimental basis for the study of interaction between α-syn aggregation and iron deposition. |
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