Fig. 3: Addition of exogenous mouse α-syn PFFs to WT and A53T-α-syn-Dendra2 transduced BSCs induces p62 immunoreactivity and Thiazin Red accumulation in the absence of toxicity.
From: Optical pulse labeling studies reveal exogenous seeding slows α-synuclein clearance
BSCs were transduced at 0 DIV with rAAVs to express Dendra2, WT-α-syn-Dendra2 or A53T-α-syn-Dendra2, at 28 DIV mouse α-syn PFFs were added and BSCs were harvested or fixed at 42 DIV. a BSCs were sequentially extracted to prepare soluble and triton-insoluble fractions. Representative western blots of the soluble and triton-insoluble fractions probed for pSer129 α-syn (EP1536Y), total α-syn and β-actin as a loading control are shown. The mobility of molecular mass markers are shown on the left. Black arrow indicates pSer129, asterisks indicate non-specific bands. (n = 3). b BSCs were fixed, immunostained for pSer129 α-syn (EP1536Y) or p62 and confocal imaged. Scale bar = 100 µm. (n = 2). c BSCs were fixed, stained with Thiazin Red and confocal imaged. Scale bar = 100 µm. (n = 2). Enlargement is shown and dystrophic processes are marked with white asterisks. d To assess any acute or chronic toxicity of α-syn overexpression or PFF addition EthD-1 uptake was assessed 24 h and 14 DIV post-PFF application (n = 6, data are mean ± SEM, analyzed by one-way ANOVA).
