Fig. 1

α-synuclein autoantibodies cause a dose-dependent loss of α-synuclein overexpressing neurons. (A) Experimental outline showing generation of neuron-astrocyte co-cultures. Cells were transduced with adeno-associated viral (AAV) vectors encoding Bcl-XL and the calcium sensor GCaMP6f under control of the strictly neuron-specific synapsin1 promoter. Additionally, cells were transduced with bi-cistronic AAV vectors encoding α-synuclein (α-syn) and the fluorescent label nuclear-targeted mCherry (NmC) from independent, neuron-specific transcription units, or expressing only NmC as control. At DIV 14, cells were treated with human serum containing α-syn autoantibodies (α-syn AAb). (B) Concentration and range of α-syn AAbs in human serum (left panel) and in culture medium (right panel). A total of 8 serum samples were used. Depending on the concentration of the α-syn AAbs, serum samples were categorized as “low” or “high”, each with 4 samples obtained from 2 healthy subjects and 2 PD patients. (C) Live imaging of NmC - positive cells in α-syn + NmC or NmC only transduced cultures after 1 d of exposure with serum-containing low (upper panel) or high levels (lower panel) of α-syn AAbs. (D) Neuronal survival represented by the quantification of NmC-positive cells after 1 d of exposure to serum-containing low or high levels of α-syn-AAbs. (E) Correlation analysis between serum concentration and surviving NmC-expressing cells upon serum treatment. A significant, negative correlation was observed in α-syn + NmC overexpressing cells treated with serum. N = 8–10 biological and 32–40 technical replicates for (D, E). Statistics by one-way ANOVA with Tukey’s multiple comparisons test; statistical power (1-ß error probability) > 0.9 for all conditions. ** = p < 0.01; *** = p < 0.001