Regulatory mechanism of interferon regulatory factor 1 by α-synuclein in mouse Parkinson's disease model

Abstract

To investigate the molecular mechanism by which α-synuclein (α-Syn) regulates interferon regulatory factor 1 (IRF-1) expression. SH-SY5Y cells overexpressing α-Syn and transgenic mouse model carrying human α-Syn gene with A53T mutation (3 and 6 months old) were examined for IRF-1 mRNA and protein expressions using real-time PCR and Western blotting, respectively. The subcellular localization of IRF-1 was determined with immunofluorescence staining and cytoplasmic/nuclear protein isolation. The optimal concentrations of the proteasome inhibitor MG132 (0.01-2.0 μmol/L) and lysosomal inhibitor chloroquine (5-200 μmol/L) for treatment of SH-SY5Y cells for 24 h were determined by examining the cell viability. SH-SY5Y cells were treated with 0.2 μmol/L MG132 and 30 μmol/L chloroquine for 24 h (the maximum dose that did not cause cell damage), and the changes of IRF-1 protein expressions was analyzed. The effects of α-Syn on MDM2 protein expression and IRF-1 ubiquitylation were analyzed using Western blotting and ubiquitylation assay. α-Syn overexpression did not affect the mRNA level of IRF-1 but significantly increased its protein level (P < 0.01). In α-Synoverexpressing SH-SY5Y cells, IRF-1 translocation was observed from the cytoplasm to the nucleus (P < 0.001). Treatment of the cells with 0.2 μmol/L MG132 significantly aggravated α-Syn-induced increase of IRF-1 protein expression (P < 0.01) while 30 μmol/L chloroquine produced no significant changes in IRF-1 level. α-Syn overexpression caused an obvious decrease of MDM2 protein level and further inhibited the ubiquitylation of IRF-1 (P < 0.01). α-Syn blocks MDM2-mediated ubiquitylation of IRF-1 through ubiquitin proteasome pathway, thereby enhancing IRF-1 protein expression.