Figure 4

The inhibition of dynamin GTPases decreases the internalization of α -synuclein by neuronal and oligodendroglial cells. A, The expression profile of various dynamin isoforms across neuronal and oligodendroglial cells. Dynamin 2 was widely expressed in both types of cells, whereas dynamin 1 was strongly expressed in dopaminergic neuronal cells. However, the expression of dynamin 1 was weak (MO3.13) or absent (KG1C) in cells of the oligodendroglial lineage. Equal loading was confirmed using α-tubulin as control. B, The pharmacological inhibition of dynamin GTPase activity via sertraline treatment (0–10 μM for 30 min) dose-dependently prevented both monomeric (arrowhead) and oligomeric αSYN accumulation in SH-SY5Y (a) and KG1C (b) cells. Both cells were treated with 5 μM αSYN for 30 min with different concentrations of sertraline, as indicated. The control cells were treated with solvent (0.1% DMSO) alone. Fifty micrograms of each lysate was 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, Consistent with the results shown in Figure 4B, the inhibition of dynamin 1 through the use of either the K44A DN mutant (a) or siRNA (b) resulted in a marked reduction of internalized αSYN in SH-SY5Y cells. Cells were treated with 5 μM αSYN (for 30 min) 48 hours after either the DN-dynamin1 transfection or the siRNA silencing of dynamin 1. Note that the αSYN monomer and the SDS-stable HMW oligomers were increased in cells overexpressing wild-type dynamin 1. Representative immunoblots from three independent experiments are shown.