A Cellular Model To Monitor Proteasome Dysfunction by α-Synuclein

Abstract

Impairment of the ubiquitin-proteasome degradation system has recently been suggested to be related to the onset of neurodegenerative disorders such as Alzheimer′s disease and Parkinson's disease. In this study, we investigated whether intracellular α-synuclein affects proteasome activity in SH-SY5Y cells. To monitor intracellular proteasome activity, we used a reporter consisting of a short peptide degron fused to the carboxyl-terminus of green fluorescent protein (GFP-CL1), which is known to be degraded by proteasome. The level of intact GFP-CL1 was dramatically increased by coexpression of GFP-CL1 and α-synuclein, as judged by confocal microscopic and immunoblot analyses. Expression of two pathogenic mutants of α-synuclein, A30P and A53T, and phosphomimetic S129D mutant increased the intensities of GFP more effectively than did wild-type α-synuclein. GFP fluorescence in cells transfected with Δ73-83 mutant or β-synuclein, which does not assemble into filaments <i>in vitro</i>, was not changed as compared with that in cells expressing GFP-CL1 alone. Thus, the ability of α-synuclein to inhibit proteasome activity is related to its propensity to assemble into filaments. Furthermore, we observed that some compounds inhibiting α-synuclein filament formation <i>in vitro</i> prevented the α-synuclein-mediated proteasome dysfunction in cells transfected with both GFP-CL1 and α-synuclein. The cellular model expressing both GFP-CL1 and α-synuclein may be a useful tool to screen compounds protecting neurons from α-synuclein-mediated proteasome dysfunction