Fig. 1

Seven days after exposure to α-syn fibrils, α-syn inclusions localize to axons, increase mEPSC frequency and increase the number of docked presynaptic vesicles. a Untreated primary hippocampal neurons were fixed at DIV 14. Immunofluorescence was performed with antibodies to total α-syn (green) and vGLuT1 (red) to label glutamatergic presynaptic terminals or vGAT1 (red) to label GABAergic terminals. Scale bar = 10 μm. b, c α-syn fibrils (2 μg/mL) were added to primary hippocampal neurons on DIV 7 and neurons were fixed 7 days later (DIV14). Immunofluorescence was performed using antibodies to p-α-syn (green) to label inclusions or tau (red) to label axons. Images were captured with a confocal microscope at an optical thickness of 0.5 μm. Scale bar = 50 μm, top panel, 25 μm, bottom panel. c Primary neurons were exposed to 2 μg/mL monomer, 2 μg/mL fibrils or PBS at DIV 7. Seven days later (DIV14), calcein AM was used to label live cells and ethidium homodimer-1 was used to label dead cells. Each well was scanned and tiled at 10X. Image J was used to quantify live and dead cells. A total of 32,527 PBS treated cells, 40,248 monomer treated cells, and 39,105 fibril treated cells were counted in two independent experiments. Data is expressed as the average live cells/total number of cells (sum of calcein positive and ethidium homodimer positive). p = 0.864 by ANOVA. d, e α-syn fibrils (2 μg/mL) were added to primary hippocampal neurons on DIV 7. Seven days later (DIV14) spontaneous synaptic activity was recorded in the presence of PTX, 100 μM, and TTX, 500 nM to isolate mEPSCs. Representative traces from control neurons and neurons treated with fibrils. f Average (+/− SEM) mEPSC frequencies and cumulative frequency plots of inter-event intervals for control neurons (blue, N-12) and neurons with inclusions (red, N = 9). *** represents p < .001 by independent t-test. **** p < 0.0001 by the Kolmogorov-Smirnov test. Experiments were performed in three independent coverslips. g Average (+/− SEM) mEPSC amplitude and cumulative frequency plot. h Representative images of presynaptic terminal from control hippocampal neurons and from hippocampal neurons 7 days after exposure to fibrils. Scale bar = 100 nm. The average length of the active zone was measured. The number of docked vesicles (≤ 50 nm from the plasma membrane) in excitatory presynaptic terminals was counted and data is expressed as average vesicles normalized to active zone length. Data was collected from two independent experiments. **** p < 0.05 by independent t-test