Figure 2.

Defective α-syn secretion from both the soma and axons in PARK9 patient DA neurons (Figure 2-1). A, Schematic image of a microfluidic device in which two sets of chambers are connected through 450 μm microgroove groove. Neurons were cultured in the top chambers and extend their axons through grooves into the bottom chamber (Figure 2-1 A–C). B, Representative images of DA neurons cultured with Alexia Fluor-555-labeled α-syn fibrils in microfluidic devices (Figure 2-1 D–E). DA neurons were stained with β-iii-tubulin and visualized as α-syn fibrils conjugated with Alexa Fluor 555. A merged image is shown on the right. C, Fluorescence intensities in the media taken from the top chambers of microfluidic devices. DA neurons were infected with empty lentivirus (left) or lentivirus expressing human PARK9 (right) (Fig. 3-1) (n = 3, *p = 0.0276, **p = 0.0001, ***p = 0.0001). After culturing in media containing α-syn fibrils for 24 h, media was changed to fresh media. After 24 h, the media was collected and fluorescence intensities were analyzed. D, Fluorescence intensities of α-syn fibrils in three control and two PARK9 DA mutant neurons. After culturing in media containing α-syn fibrils for 24 h, the media was replaced with fresh media. The fluorescence intensities were measured at 24, 36, 48, and 60 h (n = 3, *p = 0.0158, **p = 0.0265). E, Representative images of DA neurons (Cont 1 and Mut 1) cultured in media containing Alexa Fluor 555-labeled α-syn fibrils for 24 h and subsequently cultured in fresh media for a week. F, Representative images of PARK9 mutant DA neurons (Mut 1) transfected with empty lentivirus (top) or PARK9 expressing lentivirus (bottom) and subsequently cultured with Alexa Fluor 555-labeled α-syn fibrils. G, Quantification of total α-syn fluorescence intensity in DA neurons (n = 3, *p = 0.0350, **p = 0.0255, Student's t test). H, Representative images of the axons of control (top) and PARK9 mutant (bottom) DA neurons in the bottom chambers of microfluidic devices after adding α-syn fibrils to the top chamber. Arrows show α-syn fibrils. I, Number of α-syn fibrils in each axon of four control and two mutant DA neurons (n = 10–20, *p = 0.0106, **p = 0.0354). J, Fluorescence intensities in the media taken from the bottom chambers of the microfluidic devices. DA neurons were infected with empty lentivirus (left) or lentivirus expressing human PARK9 (right) (n = 3, *p = 0.0016, **p = 0.0354, ***p = 0.0001). After culturing in media containing α-syn fibrils for 24 h, the media was changed to fresh media for another 24 h before the media was collected and fluorescence intensities were analyzed (n = 3, *p < 0.05). Statistical analysis was conducted using one-way ANOVA with Tukey's post hoc test unless otherwise stated. Scale bars: B, 200 μm; D–F, 50 μm; H, 10 μm.