Figure 1

Over-expression of Cathepsin D lowers α-synuclein in dopaminergic cells. (A) Western blot analyses under reducing conditions of MES23.5 cells [38] expressing untagged, wild-type human SNCA (hSNCA WT) or vector (vec) plasmid DNA following transfection with increasing amounts of CTSD cDNA. Lysates collected 24 hours after transfection show an inverse decrease in the level of full-length (FL) α-synuclein (aSyn), as shown with polyclonal antibodies (Ab), 7071AP [46], hSA-2 [39, 40], and monoclonal Ab Syn-1 (not shown); anti-β-actin was used as loading control. Note, no low molecular fragments of aSyn are detected under these conditions. (B) ELISA [hSA2/Syn1-B] and statistical analyses of MES-aSyn cells transfected with increasing amounts of CTSD cDNA, demonstrating the quantitation of the aSyn-lowering effect by ectopic Cathepsin D (**, P < 0.01; ***, P < 0.001). Note, the addition of Pepstatin A to the cell lysis buffer did not change the observed reduction of the intracellular aSyn concentration (up to < 80 per cent). (C) ELISA [hSA2/Syn1-B] and statistical analyses of MES23.5 cells demonstrating the quantitation of ectopic Cathepsin D expression (5 μg per dish) on mutant aSyn proteins and wild-type rat aSyn (see text for details). Data in (B) and (C) are expressed as mean [± standard deviation] of the intracellular concentration of aSyn (in pg/per μg total protein) in MES23.5 cell lysates from each dish and graphically displayed in per cent numbers of vector control without ectopic CTSD expression. Graphs reflect six independent experiments.