Figure 3

Lysosomal inhibition facilitates α-synuclein oligomer formation and impairs autophagic flux. A, A portion of the αSYN-positive aggregates found in the αSYN-treated SH-SY5Y cells (5 μM recombinant αSYN for 24 hours) showed partial co-localization with markers of early endosomes (Rab5A) and lysosomes (Lamp-1). The crossed lines indicate the positions of the xz- and yz-planes. Immunostaining was performed three times and exhibited consistent results. Size bar: 10.0 μm. B, Pretreatment of the cells with bafilomycin A1 (0–5 nM) increased the intracellular accumulation of the αSYN oligomers (X2 and higher) in a dose-dependent manner. Furthermore, this increase was mainly observed in the hydrophilic fractions of the αSYN-exposed cells, whereas the level of the αSYN monomer (X1) was unchanged. Fifty micrograms of lysates were analyzed by immunoblotting and the blot was probed with an anti-αSYN Ab. Hsp90 and Na⁺/K⁺ ATPase α were used as markers for the cytosol and the plasma membrane, respectively. The asterisk indicates the non-specific band. C, In the αSYN-exposed SH-SY5Y cells (5 μM recombinant αSYN for 24 hours), bafilomycin (5 nM) treatment augmented the induction of the LC3-II protein, whereas no cleaved fragments of caspase-3 were detected. Representative western blots from three independent experiments are presented.