Down-regulation Of Orai1 Expression Inhibits Autophagy Through Calcineurin/TFEB Pathway In The Pathogenesis Of Parkinson’s Disease

Objective:Parkinson’s disease（PD）is a common neurodegenerative disease characterized by progressive loss of dopaminergic neurons in the substantia nigra（SN）.Calcium homeostasis imbalance and abnormal autophagy are involved in the pathogenesis of PD,and cytoplasmic Ca²⁺is closely related to autophagy.In PD models in vitro,it was found that the function of Orai1,a Ca²⁺channel protein located in cytomembrane,was damaged,which was involved in the death of dopaminergic neurons,but whether Orai1 could regulate autophagy remained unknown.Transcription factor EB（TFEB）is a transcription factor for autophagy-lysosomal pathway（ALP）-related proteins,which promote autophagy activity in PD.In addition,Orai1-mediated Ca²⁺influx could activate calcineurin.Calcineurin,a Ca²⁺-dependent serine/threonine protein phosphatase,could promote TFEB dephosphorylation and translocation into the nucleus to exert transcriptional activity,thus enhancing autophagy,which has not been confirmed in PD yet.This study aimed to verify whether Orai1 was involved in the pathogenesis of PD,and to explore the detailed mechanisms by which Orai1 promoted the occurrence of PD.Methods:C57BL/6 mice were randomly divided into control group,MPTP group,Orai1inhibitor group（GSK7975A+MPTP）and calcineurin inhibitor group（GSK7975A+FK506+MPTP）.PD model was established by intraperitoneal injection of MPTP.On the basis of MPTP,Orai1 function was inhibited by injection of GSK7975A,followed by injection of FK506 to further inhibit calcineurin activity.After administration,the motor function of mice was evaluated by pole test,suspension test and gait test.Dopaminergic neurons were observed and counted by immunohistochemical staining.Western blot was used to detect the expression of Orai1,calcineurin,TFEB and autophagy-related proteins in the SN.Results:1.Down-regulation of Orai1 was involved in PD pathogenesisCompared with the control group,the expression of Orai1 in the SN of the MPTP group decreased,suggesting that the down-regulation of Orai1 might be involved in PD.When Orai1 was inhibited with GSK7975A,compared with the MPTP group,the expression of Orai1 in the GSK7975A+MPTP group decreased,indicating that Orai1 was inhibited successfully（P<0.05）.Compared with the control group,the TH（+）neurons in the SN and motor coordination ability decreased in the MPTP group,which proved that the PD mice models were made successfully.When Orai1 was inhibited,compared with MPTP group,the TH（+）neurons of GSK7975A+MPTP group reduced significantly,the crawling time from the top to the bottom of the pole was longer in pole test,and the drop times score was lower in suspension test for GSK7975A+MPTP group.In gait test,compared with MPTP group,only the run speed and swing speed were slower,while run duration,standing time and stride length did not show statistically significant differences for GSK7975A+MPTP group（P<0.05）.2.Down-regulation of Orai1 was involved in PD pathogenesis by inhibiting autophagyCompared with the control group,LC3II decreased and p62 increased in MPTP group.When Orai1 was inhibited,LC3II was further decreased and p62 was further increased compared with MPTP group.At the same time,as mentioned above,the TH（+）neurons and the motor coordination ability of GSK7975A+MPTP group further deteriorated（P<0.05）.3.Down-regulation of Orai1 was involved in PD pathogenesis by inhibiting autophagy through the Calcineurin/TFEB pathwayCompared with the control group,the expression of calcineurin decreased and p-TFEB increased in MPTP group,indicating that the function of calcineurin and TFEB were inhibited in PD.After inhibiting Orai1,the expression of calcineurin was further decreased,while p-TFEB was further increased in GSK7975A+MPTP group（P<0.05）.When calcineurin was inhibited with FK506 on the basis of GSK7975A+MPTP group,it was observed that LC3II was further decreased,while p62 and p-TFEB were further increased in GSK7975A+FK506+MPTP group,which suggested that calcineurin could affect p-TFEB and autophagy activity.Meanwhile,the TH（+）neurons were further reduced,and the motor symptoms deteriorated in GSK7975A+FK506+MPTP group（P<0.05）.Conclusions:1.Down-regulation of Orai1 was involved in the pathogenesis of PD;2.Down-regulation of Orai1 was involved in PD by inhibiting the expression of autophagy-related proteins;3.Down-regulation of Orai1 inhibited autophagy by inhibiting the Calcineurin/TFEB pathway,accelerated the death of dopaminergic neurons,and ultimately involved in the occurrence of PD.