The Regulation Of α-Synuclein On Interferon Regulatory Factor 1 In Dopaminergic Neuron And Its Underlying Mechanism

Parkinson’s disease(PD)is a common neurodegenerative disease and characterized by a pathologically selective loss of dopaminergic(DA)neurons and accumulation of intracellular inclusions known as Lewy bodies(LBs).Under physiological conditions,α-synuclein(α-Syn)exerts no neurotoxicity,while in some cases,the misfolding of α-Syn aggregates and ultimately leads to the death of neurons.The first gene was found to be associated with PD is α-Syn(SNCA)gene.It is known that the initiation of PD may attribute partly to immune disorders.The immune-related protein interferon regulatory factor 1(IRF-1)is a 329 amino acids protein involved in the process of immune response,cell apoptosis,DNA damage and tumor suppression.IRF-1 can be rapidly synthesized in the cytoplasm and then transferred into the nucleus for transcription with stimulation.It has been reported that using RNA-Seq has found that IRF-1 is elevated in cells overexpressing α-Syn,but the mechanism has not clear.JAK-STAT pathway participates in the transcription of IRF-1.Protein degradation is decided by ubiquitin proteasome system and autophagy-lysosome pathway.Ubiquitination plays an important role in protein localization,metabolism,function and regulation.It has been reported that using RNA-Seq has found that IRF-1 is elevated in cells overexpressing α-Syn,but the mechanism has not clear.Therefore,the present study was designed to examine whether aggregation of α-Syn affected IRF-1 through ubiquitin proteasome system in PD and the possible mechanism.In the present study,the effect of α-Syn on IRF-1 were studied in WT α-Syn and A53 T α-Syn transfected SH-SY5 Y cells and human A53 T mutant α-Syn transgenic mice.Using immunofluorescence and molecular biological technology,the expression,location and signalling patheway of IRF-1 were detected in SH-SY5 Y cells transfected with α-Syn,respectively.The expression of IRF-1 was also measured after treated with proteasome inhibitor MG-132 and lysosomal inhibitor chloroquine.In addition,the ubiquitination of IRF-1 and the expression of IRF-1 E3 ubiquitin ligase MDM2 were detected by ubiquitination assay and Western blots,respectively.The results were as follows:1.Transfected with WT α-Syn and A53 T α-Syn for 24 hrs,the expression of SNCA increased 846 and 2593 times in SH-SY5 Y cells,respectively(P<0.001).The expression of α-Syn protein also increased by 73% and 106%,respectively(P<0.001).2.Compared with the control,the expression of IRF-1 protein in overexpressing WT α-Syn and A53 T α-Syn was increased by 57% and 118%(P< 0.001).In 3 and 6 months α-Syn A53T+/+ mice,the IRF-1 protein level of SN was also increased by 73% and 86%,respectively,compared with their WT littermates(P< 0.001).3.The immunofluorescence showed IRF-1 was mainly expressed in the nuclear of SH-SY5 Y cells transfected with α-Syn,increasing by 98% and 123%,respectively,compared with the control(P<0.001).In order to further verify the results,the cells were subjected to nucleoplasm separation.Western Blots showed the IRF-1 protein levels in nucleus also increased 1.9-fold and 2.3-fold higher,when compared with the control(P<0.001).4.Compared with the control group,there was no significant difference in the expression of IRF-1 in cells overexpressing WT α-Syn and A53 T α-Syn(P>0.05).The JAK-STAT pathway including JAK1,JAK2,STAT1α and STAT1β showed no obvious change when compared with the control(P>0.05).5.Treated with MG-132 for 24 hrs,expression of IRF-1 protein increased by 23% and 42% in SH-SY5 Y cell transfected with WT α-Syn and A53 T α-Syn,respectively(P<0.01),while no significant change was observed after treated with chloroquine(P>0.05).6.Myc-IRF-1/HA-empty vector/Flag-empty vector,Myc-IRF-1/HA-Ub/Flag-empty vector,Myc-IRF-1/HA-Ub/Flag-empty vector,Myc-IRF-1/HA-Ub/Flag-empty vector,Myc-IRF-1/HA-Ub/Flag-WT a-Syn and Myc-IRF-1/HA-Ub/Flag-A53T-a-Syn were transfected into HEK293 T cells.Compared with the Myc-IRF-1/HA-empty vector/Flag-empty vector group,the ubiquitination level of IRF-1 in the group with Myc-IRF-1/HA-Ub/Flag-WT α-Syn,Myc-IRF-1/HA-Ub/ Flag-A53 T α-Syn were significantly decreased.7.In SH-SY5 Y cells transfected with α-Syn,the protein levels of E3 ubiquitin ligase MDM2 was significantly lower than the control group(P<0.01).In 3 and 6 months α-Syn A53T+/+ mice,the protein level of SN was also decreased by 44% and 54%,respectively,compared with their WT littermates(P<0.001).In conclusion,the overexpression of α-Syn regulates IRF-1 not at the transcriptional level,but by down-regulating MDM2 levels,inhibiting the ubiquitin-proteasome pathway of IRF-1,leading to an increase in IRF-1 protein levels.However,the role of IRF-1 in PD remains to be further studied.This study provides new evidence for the regulation of α-Syn regulation of IRF-1 and its mechanism,and provides a certain experimental basis for further clarifying the involvement of IRF-1 in the pathogenesis of PD.