The Ginkgo biloba Extract EGb 761 Modulates Proteasome Activity and Polyglutamine Protein Aggregation
Figure 4
Effects of EGb 761 on degradation of polyQ proteins and polyQ aggregation. (a) d2GFP-HEK cells expressing htt_Q25 and (b–d) HEK293 cells expressing htt_Q103 were treated with EGb 761 or vehicle and subsequently chased with proteasome (MG132) or autophagy inhibitor (bafilomycin) to assess polyQ degradation and aggregation. (a) d2GFP-HEK cells expressing htt_Q25 were treated for 48 h with 150 μg/mL EGb 761 or vehicle. Then, cells were incubated for 4 h with or without increasing concentrations of MG132. Protein levels of whole cell extracts were analyzed by immunoblotting to their corresponding antibodies. Protein levels of short-lived, unstable proteins (polyubiquitin, d2GFP) accumulated with proteasome inhibition while levels of long-lived polyQ proteins were not significantly altered. Values of d2GFP or polyQ protein of vehicle-treated cells without MG132 were arbitrarily set to 1; . (b-c) HEK293 cells expressing htt_Q103 were treated for 48 h with 150 μg/mL EGb 761 or vehicle. Then, cells were subsequently incubated for 4 h with or without increasing concentrations of MG132. (b) Whole cell extracts were subjected to a filter retardation assay to assess polyQ aggregates, induced by pharmacologic proteasome inhibition. For the detection of aggregates of polyQ proteins trapped on nitrocellulose membrane an anti-eGFP antibody was used. Densitometric values of vehicle-treated cells without MG132 were arbitrarily set to 1; . (c) Protein levels of whole cell extracts (samples from b) were analyzed by immunoblotting to their corresponding antibodies. Protein levels of unstable, misfolded polyQ proteins accumulated with proteasome inhibition while levels of stable, soluble polyQ proteins were not altered. Values of soluble or insoluble polyQ proteins of vehicle-treated cells without MG132 were arbitrarily set to 1; . (d) HEK293 cells expressing htt_Q103 were treated for 48 h with 150 μg/mL EGb 761 or vehicle. Then, cells were subsequently incubated for 3 h with or without 1 μM of the lysosomal inhibitor bafilomycin A1 (denoted bafi.) to investigate the autophagic flux. Whole cell extracts were analyzed by immunoblotting or filter retardation assay. Cells treated with EGb 761 showed no significant alteration with bafilomycin treatment in protein levels of LC3-I to LC3-II, soluble and insoluble polyQ, indicating no direct effect on autophagy by EGb 761. Immunoblotting and filter retardation assay confirmed significant changes in aggregated polyQ proteins by EGb 761 from previous experiments (Figures 3(b)–3(d) and Figures 4(b)-4(c)). Densitometric values of vehicle-treated cells without bafilomycin were arbitrarily set to 1; . (a–d) All values are reported as mean ± S.D. and .
