Objective: To investigate the effect of small ubiquitin-like modifier (SUMO-1) modification on the formation of Lewy body like inclusions in cytoplasm and apoptosis of HEK293 cell induced by overexpression and mutation of alpha-synuclein.
Methods: cDNA encoding the human alpha-synuclein without the stop codon was cloned into a pGEM T-easy vector. Restriction enzyme mapping and DNA sequencing were performed to analyze the plasmid, which was then subcloned into a pEGFP-N1 vector. The recombinant plasmid alpha-synuclein-pEGFP was transfected into HEK293 cells by lipofectamin method. Inclusions in the cultured cells were identified with HE staining. Apoptosis of the HEK293 cell was measured by Hoechst 33258 staining, MTT and Annexin V-PE flow cytometry.
Results: The Lewy-body like inclusions were found in cytoplasm of cultured cells. Hoechst staining showed that the nuclei of cells were enlarged in the wild-type and A53T mutation groups 48 h after transfection, chromatin were accumulated and appeared spot-like. The nucleus stain was equitable in the K96R and K96R-A53T groups. MTT assay showed that the viability of cells transfected with empty plasmid was 96.2%, but it dropped to 53.4% and 56.1% in cells transfected with wild-type alpha-synuclein-pEGFP and A53T mutant group, respectively. The viability was 72.3% and 69.8% in cells transfected with K96R and K96R-A53T, respectively (P<0.05). Forty eight hours after transfection, the apoptosis rate was 3.9% in empty plasmid group, 32.2% and 34.1% in cells transfected with wild-type and mutant alpha-synuclein-pEGFP, 19.4% and 20.3% in the K96R and K96R-A53T transfected cells. There was significant difference between the two groups (P<0.05).
Conclusion: SUMO-1 modification did not have influence on the Lewy body-like inclusions formation in cytoplasm of HEK293 cell in vitro, but had a toxic effect which could increase the apoptosis induced by wild type overexpression and mutation of alpha-synuclein.