Research Paper Volume 9, Issue 7 pp 1677—1697

Figure 1. GGAs alter α-synuclein oligomer secretion in N2A cells. N2A cells were co-transfected with α-syn fused to non-bioluminescent N- or C-terminal fragments of Gaussia princeps luciferase (α-syn-hGLuc1 (S1) and α-syn-hGLuc2 (S2)) and either empty control or one member of the GGA protein family. Then, 48 h after transfection, luciferase activity, indicative for α-syn oligomers, was determined in conditioned media as well as cells. GGA 1 and 3 co-transfected cells showed significantly higher luciferase activity in the conditioned media compared to controls (A), while intracellular levels of α-syn oligomers were not changed (GGA 2, 3) or even reduced (GGA1) (B). The ratio of secreted α-syn oligomers in the conditioned medium to intracellular α-syn oligomers was built showing increased secretion of α-syn oligomers for GGA1 and GGA3 (C). Experiments were carried out in triplicate; the results of n=8 independent experiments are shown. Densitometric analysis of corresponding Western blots revealed no significant difference in total α-syn levels upon expression of GGA2, 3 and the control . The mean fold change over control ±SEM of n=4 independent experiments is shown. Statistical analysis was performed using Kruskal-Wallis one-way analysis of variance (ANOVA) on ranks followed by multiple comparisons versus the control group (Dunn's Method) with *=p<0.05. The siRNA mediated knockdown of single endogenous Gga’s abolished but did not reverse the effect of enhanced α-syn oligomer secretion in N2A cells (D). Experiments were carried out in triplicate; the results of n=7 independent experiments are shown. Statistical analysis was performed using Kruskal-Wallis one-way analysis of variance (ANOVA) on ranks (*=p<0.05). Simultaneous knockdown of all 3 Gga’s significantly reduces α-syn oligomer secretion. Experiments were carried out in triplicate; the results of n=5 independent experiments are shown. Statistical analysis was performed using Mann-Whitney Rank Sum Test (**=p<0.008).