Effects Of α-synuclein On The Regulation Of Ferroportin And Hepcidin

ObjectiveParkinson’s disease(PD)is one of the most common neurodegenerative diseases.The main pathological features are the loss of dopaminergic neurons in the substantia nigra pars compacta(SNpc)and the formation of Lewy bodies(LBs).The main component of LBs is aggregated α-synuclein(α-Syn).Previous studies have shown that α-Syn released from neurons can be taken up by surrounding cells,but its complicated effects on the recipient cells have not been fully elucidated.A number of studies have shown that iron deposition is closely related to α-Syn aggregation.Ferroportin(FPN)is the only iron efflux channel located on the cell membrane.Hepcidin can bind to the extracellular domain of FPN,induce ubiquitination and degradation,and then down-regulate the expression of FPN protein.Therefore,FPN and hepcidin are important targets for the regulation of intracellular iron metabolism.The effects of extracellular α-Syn on FPN and hepcidin remain unclear This study aims to investigate the effects of α-Syn on the expression of FPN and hepcidin,and to explore the possible mechanisms.MethodsIn C56BL/6 mice with α-Syn preformed fibrils(PFF)injection into the substania nigra,gait changes were detected by Cat Walk gait analysis system,and motor behavior changes were detected by open field test,pole test,and rotation rod test.The expression of FPN,iron regulatory protein 1(IRP1)and ferritin was detected by western blotting in the substantia nigra or striatum with α-Syn PFF injection.In primary cultured ventral mesencephalic(VM)neurons with α-Syn PFF administration,western blotting was used to detect the expression of x-box binding protein 1(XBP1)and C/ EBP-Homologous Protein(CHOP),which were specific markers of endoplasmic reticulum stress,or FPN expression was detected in PC12 cells with over expression of α-Syn.The mRNA expression of hepcidin was detected by real-time quantitative PCR.The protein levels of α-Syn were detected by western blotting in HEK-293 T cells with α-Syn monomers or PFF.Results1.After injection of 1μg α-Syn PFF or 5μg α-Syn PFF into the substantia nigra of C57BL/6 mice,compared with normal saline injection group,there were no significant changes in the movement speed in open field,the time of turning head and descending rod while climbing the rod,and the sustained movement time on the rotating rod after 1 week,1 month,and 2 months.Three months later,Cat Walk gait analysis system was used to detect the movement behavior of the mice.The supporting time of left hind paw decreased by14.6%,the distance between heel of left hind paw and touchdown increased by 18.7%,and the number of single foot footprint increased by 171.4%.After injection of 5μg α-Syn PFF for 3 months,the expression of tyrosine hydroxylase(TH)decreased by 21.4% in the substania nigra of C57BL/6 mice,while did not change significantly in 1μg α-Syn PFF group.The results suggested that injection of 5μg α-Syn PFF into the substantia nigra could induce motor coordination dysfunction and dopaminergic neuron injury after 3 months.2.After nigral injection of 5μg α-Syn PFF for 3 months,the expression of FPN protein in substania nigra decreased by 21.4% in C57BL/6 mice,accompanied by a 74.6% increase of ferritin expression.After striatal injection of 5μg α-Syn monomer and 5μg α-Syn PFF for 24 h,the expression of FPN protein in the striatum in 5μg α-Syn monomer group did not change significantly,and the expression of FPN protein in 5μg α-Syn PFF group decreased by 21.2% in the striatum of C57BL/6 mice.These results suggest that α-Syn PFF can induce down-regulation of FPN protein expression.3.After treatment with 1 μg/m L α-Syn PFF for 24 h,the protein expression of FPN and IRP1,as well as hepcidin mRNA levels in primary cultured VM neurons did not change.After treatment with 5 μg/m L α-Syn PFF for 24 h,the protein levels of FPN in primary cultured VM neurons were significantly down-regulated,and the mRNA levels of hepcidin were not changed at 6h and 12 h,however,significantly increased by 81.3% at 24 h.Ferric ammonium citrate co-treatment of 6 h prior to the end of 5 μg/m L α-Syn PFF treatment induced a 50% upregulation of ferritin.These results suggest α-Syn PFF can induce downregulation of FPN and up-regulation of hepcidin,as well as iron accumulation.4.After treatment with 5 μg/m L α-Syn PFF for 24 h,the expression of endoplasmic reticulum stress specific markers XBP-1 and CHOP protein did not change significantly.These results suggest that endoplasmic reticulum stress may not be involved in the upregulation of α-Syn PFF on hepcidin mRNA levels.5.After doxycycline treatment with 2 μg/m L for 24 h,the expression of α-Syn was significantly increased by 187.8%.The mRNA level of hepcidin increased by 38.0% in these cells,while the protein expressions of FPN and IRP1 did not change significantly.When PC12 cells were treated with 5μg/m L α-syn PFF for 24 h,the level of FPN was also significantly down-regulated.The results suggested that α-Syn overexpression could upregulate the expression of hepcidin,but not down-regulate the expression of FPN.6.After 24 h treatment with α-Syn monomer or PFF,α-Syn protein levels were significantly increased in HEK-293 T cells in both α-Syn monomer group and α-Syn PFF group.An increase of 55.6% was observed in α-Syn monomer group compared with α-Syn PFF group.The results suggested that α-Syn PFF might be less taken up by HEK-293 T cells compared to α-Syn monomer.Conclusion1.α-Syn PFF could induce dopaminergic neuron damages and motor coordination dysfunctions in mice.2.α-Syn PFF could down-regulate FPN protein levels and up-regulate hepcidin mRNA levels.3.The up-regulation of hepcidin might be independent of endoplasmic reticulum stress,the down-regulation of FPN might be associated with its effects on cell membrane.