Figure 7. Defective autophagosome clearance in PD-iPSC-derived DA neurons.

A-C. DAn (TH positive, white) cultured on astrocytes for 75 days stained for LC3 (green) and LAMP1 (red) after 6 h of treatment with leupeptin from CONTROL (cell line SP11.1, A), ID-PD (cell line SP10.2, B) and LRRK2-PD (cell line SP05.1, C) iPSC. Z-stack views are shown on the merged images (right panels). Nuclei are counterstained with DAPI, shown in blue. Scale bars, 5 µm.

D. Quantitative analyses of the colocalization of LAMP1 and LC3. Data for each group is the average of 15, 15 and 14 DAn representing three different Ctrl-, ID-PD and LRKK2-PD iPSC lines, respectively. Asterisks denote statistically significant differences between CONTROL and ID-PD (p < 0.001) or LRRK2-PD (p < 0.001) [F(2,41) = 41.628; p < 0.001].

E. Quantitative analyses of the total neurite length per neuron with or without treatment with rapamycin and leupeptin. Under basal conditions (without treatment), data for CONTROL is the average of 97 DAn from 4 iPSC lines, for ID-PD is from 127 DAn from 7 iPSC lines and for LRRK2-PD is from 80 DAn from 4 iPSC lines. After treatment with rapamycin and leupeptin (RAPA + LEUP), data for CONTROL is the average of 60 DAn from 4 iPSC lines, for ID-PD is from 112 DAn from 7 iPSC lines and for LRRK2-PD is from 74 DAn from 4 iPSC lines. Asterisks above data bars denote statistically significant differences under basal conditions [F(2,301) = 5.886; p = 0.003] between CONTROL and ID-PD (p = 0.003), and between CONTROL and LRRK2-PD (p = 0.022), and after treatment with rapamycin and leupeptin [F(2,243) = 48.812; p < 0.001] between CONTROL and ID-PD (p < 0.001), and between CONTROL and LRRK2-PD (p < 0.001). Asterisks comparing data sets denote statistically significant differences in total neurite length after treatment with rapamycin and leupeptin in DAn from ID-PD (p = 0.001) and LRRK2-PD (p < 0.001), but not from CONTROL (p = 0.090). In (D–E) bars represent average with SEM as error bars.