Blood Plasma of Patients with Parkinson’s Disease Increases Alpha-Synuclein Aggregation and Neurotoxicity

Figure 4

α-syn oligomers incubated with PD plasma accumulated in primary neurons. (a) and (b) Freshly fixed cells were double-immunostained for thioflavin S (green) and α-syn (red). Neurons treated with PD plasma stained positive with thioflavin S. Note the colocalization in an α-syn-positive and thioflavin S-positive plaque within a cytoplasmic inclusion (arrow). (a) Scale bar = 10 µM. (b) Scale bar = 5 µM. (c) Quantification of thioflavin S-positive neurons. Digitized images of cells were taken at 20x magnification under confocal microscopy. Images of 5 fields per dish were taken with an average of 10–20 cells per field. The number of thioflavin S-positive neurons with at least one plaque was counted for each group. Values are indicated as the number of cells per field of view. All values represent mean ± SEM, . (d) Primary neurons were fixed after treatment with PD plasma and α-syn for 14 d and then double-stained for thioflavin S and ubiquitin (red). Colocalization of thioflavin S-positive and ubiquitin-positive staining was found within a cytoplasmic inclusion (arrow) that remained following detergent extraction. Scale bar = 5 µM. (e) Percentage of ubiquitin-positive neurons containing thioflavin S-positive inclusions for α-syn plus PD plasma group. 71.19% of neurons were found to have thioflavin S-positive plaque after being treated with α-syn plus PD plasma and a total of 62.71% of neurons were found colocalized with thioflavin S and ubiquitin. Mean ± SEM, .

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