Fig. 1: αSyn fragments are present in the conditioned medium of αSyn-overexpressing LUHMES neurons.

a Experimental design. LUHMES cells, grown in differentiation medium from plating onward, were transduced with GFP or αSyn adenoviral vectors (AV) two days after plating. Viruses were removed 24 h after transduction. Cells and conditioned medium (CM) were harvested at the indicated readout times. DIV: days in vitro. DPT: days post-transduction. b αSyn-mediated toxicity. Cellular toxicity was monitored by lactate dehydrogenase (LDH) activity in the CM at the indicated readout times. Cells were either left untreated (Ctrl), challenged with a control AV expressing green fluorescent protein (GFP) or an AV expressing wild type αSyn. At DIV8, αSyn overexpression induced a considerably high toxicity, whilst GFP overexpression induced a mild toxicity, compared to untreated cells. LDH release values were related to a cell lysis positive control representing 100%. Data are presented as mean + standard error of the mean (SEM) from at least 3 biological repeats. ns: not significant, *p < 0.05, **p < 0.005, ***p < 0.001; two-way ANOVA with Tukey’s post hoc test. c αSyn-overexpression in cell homogenates. Cell lysates were analysed by Western blot at the indicated readout times. αSyn overexpression levels were stable between DIV4 and DIV8. Aggregation of αSyn is visible in overexpressing cells, but not in untreated control or GFP overexpression conditions. αSyn bands smaller than 15 kDa were not detected, indicating absence of fragmented αSyn. f: fragments; m: monomer; o: oligomer; *: unspecific band. Actin was used as loading control. d αSyn in CM. Western blot analysis of αSyn in CM at the indicated times reveals the presence of several αSyn species at DIV6 and DIV8. An oligomer band appears at 37 kDa (o), a monomer band at 15 kDa (m) and several fragmented αSyn bands ranging from 13 to 6 kDa (f). CM of untreated and GFP overexpressing cells did not contain detectable levels of αSyn. Unconditioned medium (Med.) was used as control for unspecific bands (*). GAPDH was used as control for cytoplasmic content in the CM.