Fig. 7.

TLR2 activation with α-syn PFF addition inhibits autophagy in astrocytes. Differentiated midbrain cells derived from PD patient iPSCs were treated with 1 μg/ml Pam3CSK4 and/or 1 μg/ml PFFs, or medium only for the untreated control. a Cells were fixed at day 2 and 7 after treatment and stained for autophagy marker P62 (green) in S100β positive astrocytes (red). Confocal images were taken at 60 × magnification with 8 images captured per condition, scale bar, 10 μm. b Graph shows the fold change in P62 intensity in astrocytes as normalised to the control as mean ± SEM (n = 6–9). c Cells were stained with Lysotracker (red) for 1 h at 37 °C prior to fixation at day 2 and 7 post treatment, and then co-stained for S100β (green). Confocal images were taken at 60 × magnification with 8 images captured per condition and used for the analysis. Scale bar, 10 μm. d Graph shows the average number of lysosomes in astrocytes ± SEM (n = 6). e Graph shows the percentage of astrocytes with enlarged lysosomes (> 2 μm²), displayed by mean ± SEM (n = 6). f To assess autophagy flux cells were treated with Bafilomycin A1 for 4 h, fixed and stained for P62 (green) and S100β (red). Confocal images were taken at 60 × magnification with 8 images captured per condition. Scale bar, 10 μm. g Graph shows autophagy flux measured as P62 expression in Bafilomycin A1 treated/Control treated S100β positive astrocytes, displayed as mean ± SEM (n = 6). h Graph shows the proportion of LAMP2A puncta in perinuclear positioning as percentage of total LAMP2A puncta measured in astrocytes, displayed mean ± SEM (n = 3). i Cells were fixed at day 14 after treatment and stained for LAMP2A (red) in GFAP positive astrocytes (green). Confocal images were taken at 60 × magnification with 8 images captured per condition, scale bar, 10 μm. For all graphs **P < 0.01, ***P < 0.001, ****P < 0.0001, ns = not significant