Figure 2. AIM-100 induces endogenous HA-DAT endocytosis and oligomerization in mouse dopaminergic neurons.

(A–C) Cultured post-natal mesencephalic neuronal cultures were pre-incubated with HA11 antibodies for 30 min at 37°C, and then further incubated with vehicle (DMSO) or 10 µM AIM-100 for 2 hr at 37°C. After fixation, cultures were stained with secondary anti-mouse antibodies conjugated with Cy5 (surface HA-DAT), permeabilized with Triton X-100 and incubated with rat-anti-DAT antibody, and then stained with secondary anti-mouse conjugated with Cy3 (internalized HA-DAT) and secondary anti-rat antibody conjugated with Alexa488 (total HA-DAT immunoreactivity). 3D images were acquired through 488 (Alexa488, green), 561 (Cy3, red) and 640 nm (Cy5, blue) channels. Individual confocal sections are presented. (A) Representative images. Arrows point on examples of Cy3 enriched puncta (endosomes) that localize in neuronal processes. (B) Representative images of endosomal HA-DAT (Cy3 fluorescence) in axonal varicosities in AIM-100-treated cells. Scale bars, 10 µm. (C) Quantification of Cy3/Cy5 ratio values from 3D images exemplified in (A). Results are presented as mean values (±S.E.M.; n = 13-15). P value is calculated for ‘AIM-100’ compared to ‘vehicle’. (D) Striatal synaptosomes prepared from adult HA-DAT mice were incubated with vehicle or 10–20 µM AIM-100 for 2 hr at 37°C. Lysates were resolved by electrophoresis and probed by western blotting with DAT antibody. M, monomers, O, oligomers; hO, high-order oligomers. O/M ratios are presented as a mean of the ratio values obtained from two mice.