The Regulation And Mechanisms Of NRF-1 On PINK1/Parkin Mediated Mitophagy

Objective Transcriptional regulation of PINK1(PARK6)and Parkin(PARK2)by nuclear respiratory factor 1(NRF-1)was studied to illustrate the role of NRF-1 in PINK1/Parkin mediated mitophagy and in mitochondrial turnover.Methods The promoter region on-line analysis software JASPER was used to analyze whether there are NRF-1 binding sites in the promoter region of PINK1 and Parkin.NRF-1 binding sites in the promoter region of PINK1 and Parkin were confirmed by Chromatin immunoprecipitation(ChIP).Luciferase reporter gene plasmids containing the PINK1 and Parkin promoter region on the basis of p GL3-Basic Vector plasmid were constructed and the effect of NRF-1 on the transcriptional activity of PINK1 and Parkin was detected by double luciferase reporter assay.NRF-1 expression was down-regulated by siRNA,shRNA,or partial knockout by CRISPR/Cas9.NRF-1expression plasmid was used to up-regulate NRF-1 expression in HEK293 T and SH-SY5 Y cell lines.The mRNA and protein levels of NRF-1,PINK1,Parkin were detected by reverse transcription-polymerase chain reaction and Western blotting.Mitotracker Deep Red(MTDR)fluorescent dye was utilized to measurethe number of mitochondria under different conditions by using flow cytometry.Mt-m-keima plasmid was used to analyze mitophagy qualitatively and quantitatively through laser confocal microscope.Mito-timer plasmid was used to detect mitochondrial turnover on Tet-on HEK293 T cells.Results JASPER online software analysis revealed some possible NRF-1 binding sites in the promoter region of human PINK1 and Parkin.ChIP experiments showed that NRF-1 had binding sites in the promoter region of PINK1 and Parkin.Double luciferase reporter assay demonstrated that NRF-1 could transcriptionally activate the expression of PINK1 and Parkin.Transient knockdown or partial knockout NRF-1expression down-regulated mRNA and protein levels of PINK1 and Parkin in HEK293 T cells.The mRNA and protein expression of PINK1 and Parkin on SH-SY5 Y and HEK293 T cells transfected with NRF-1 expression plasmid were up-regulated.The intensity ratio of Mt-m-keima under 555 nm and 488 nm laser performed decrease when HEK293 T cells co-transfected with Mt-m-keima plasmid and NRF-1interfering fragment,indicating the decreased levels of mitophagy.Immunofluorescence assay indicated that there was lower level of Parkin cluster in HEK293 T cells transfected with shRNA-NRF-1 plasmid.When Tet-on HEK293 T cells were co-transfected with Mito-timer plasmid and NRF-1 interfering fragment,the Red and Green intensity ratio of Mito-timer induced by doxycycline(DOX)increased,suggesting that mitochondrial turnover became slower.Conclusion PINK1 and Parkin could be transcriptional regulated by NRF-1 through directly acting on the promoters of PINK1 and Parkin.NRF-1 involved in mitochondrial quality control through regulating PINK1/Parkin-dependent mitophagy.