Figure 1. Exogenous αS pre-formed fibril stably accumulates as a truncated variant in primary cortical neurons.

Primary cortical neurons (PCN) cultured from newborn C57BL6/J mouse brains at 7 days in vitro (DIV) were used. PCN were pre-incubated with 4 μg/ml (unless otherwise indicated) (A, B) monomeric αS or (C-F) αS PFF for 2h and washed to removed extracellular αS or αS PFF that was not internalized. Cells were provided with fresh media and then incubated for the indicated time before harvesting. A) Following αS monomer treatment, cell lysates were immunoblotted for total (Tot) αS or Hu-αS. Equal loading of proteins was verified using total protein stain (Ponceau S, PonS). B) Quantitative analysis of TotαS immunoblot shows that internalized αS monomers are rapidly metabolized within 3 hours of internalization. Mean±SEM; n=3. b, p<0.001 vs 0-hour, One-way ANOVA. C) Immunoblot analyses for TotαS following PFF treatment show that internalized full-length (FL) αS is truncated (Δ) and stably accumulate as the truncated species. Shown are two different exposure levels to show details. D) Quantitative analysis of TotαS immunoblot in (C). Bar and line graph show the kinetics of degradation and accumulation of αS species over time. Mean±SEM; n=3. a, p<0.01; b, p<0.001 vs. total αS (FL+Δ) at 0-hour, One-way ANOVA. E) To determine if the amount of αS PFFs internalized impacts the truncation and/or stability of internalized αS PFFs, PCN was treated with 0.5-, 2-, and 4-μg/ml of αS PFF and the status of TotαS was analyzed at indicated times. Even at very low amounts of PFFs (0.5 μg/ml), internalized PFFs are rapidly truncated and stably accumulate. F) To determine if endogenous αS impacts the metabolism of internalized αS PFFs PCN established from αS KO mice were treated with αS PFFs. The results show that the lack of endogenous αS does not impact the metabolism of internalized αS PFFs. Shown are two different exposures for TotalαS to show details. Equal loading of proteins was verified by immunoblotting for α-tubulin (αTub).