Figure 4. GGA modifies secreted α-syn oligomer species and exacerbates extracellular α-syn toxicity. GGA1, 2, 3 or myc control were expressed alone or with S1 and S2 in N2A cells. Then, 48h after transfection, conditioned media were collected and analyzed for increased cytotoxicity using the ToxiLight assay (Lonza). Neither GGA1 nor GGA2 co-expression with S1 and S2 showed enhanced toxicity compared to myc-control whereas a slight increase was observed upon GGA3 expression (A, left panel). The mean fold change over control ±SEM of n=6 independent experiments is shown. To test for the GGA-dependent increase in cytotoxicity, GGA1, 2, 3 or myc control were expressed without S1/ S2 in N2A cells. GGA3 expression slightly increased toxicity compared to control, GGA1 and GGA2 (A, right panel). The mean fold change over control ±SEM of n=3 independent experiments is shown. Statistical analysis was performed using Kruskal-Wallis one-way ANOVA on ranks followed by multiple comparisons versus control group (Dunn's Method) with *=p<0.05. CM from cells expressing GGA1, 2, 3 or myc-control alone or with S1 and S2 was transferred to naïve N2A cells. After 72h, cells were analyzed for altered Caspase 3/7 activity. We found that CM from N2A cells co-expressing S1/S2 together with GGA1, 2 and 3 caused a 2-fold increase in Caspase 3/7 activity on naïve N2A cells compared to CM from N2A cells that were co-transfected with S1/S2 together myc-control plasmids (B, left panel). In contrast, CM from N2A overexpressing GGA1, 2, 3 or myc control alone had no influence on Caspase 3/7 activity (B, right panel). These findings indicate that GGA not only alters the amount of secreted α-synuclein oligomers but also the quality of oligomeric species in the conditioned media. The mean fold change over control ±SEM of n=3 independent experiments is shown. Statistical analysis was performed using Kruskal-Wallis one-way ANOVA on ranks followed by multiple comparisons versus control group (Dunn's Method) with *=p<0.05. CM from HEK293 cells co-expressing S1/S2 and GGA3 or myc-control was analyzed by size exclusion chromatography (SEC). Indicated in grey are the calculated sizes of different α-syn oligomers and their estimated elution volumes. Fractions of 0.5 ml were collected and further analyzed for α-syn by luciferase assay and Dot Blots. We detected a heterogeneous population of oligomeric α-syn in CM of S1/S2 transfected cells secreted from cells ranging from multimers to dimers (C). GGA co-expression increased extracellular α-syn dimers but also multimers. These results support the idea that co-expression of GGAs change α-syn oligomer formation, species and secretion.