Cationic amphiphilic drug-induced autophagosome accumulation is due to autophagosome sequestration within vimentin intermediate filament networks resulting in prolonged autophagosome half-life

Accumulations of autophagosomes and non-esterified cholesterol are observed in several cell lines derived from lysosomal storage diseases, including Niemann Pick Type C (NPC). The relationship between autophagosome accumulation and lysosomal non-esterified cholesterol is unclear. Exposure of murine hepatoma 1c1c7 cultures to the cationic amphiphilic drugs (CADs) U18666A, imipramine and clozapine caused lysosomal non-esterified cholesterol and autophagosome accumulation. Measurement of LC3-II conversion in the presence of lysosomal inhibitors bafilomycin A1 and NH4Cl, degradation of long-lived proteins, and colocalization of GFP-LC3 and LAMP1 indicated an increase in autophagosome synthesis without compensatory increase in clearance. Autophagosome synthesis was blocked using 3-MA to monitor pre-existing autophagosome degradation. Autophagosomes generated by leucine starvation or treatment with rapamycin, U18666A or clozapine had an estimated half-life of ~0.7, 2.5, 29 and 26 h, respectively. Shifting U18666A-treated cultures to leucine-starvation media enhanced autophagosome clearance (half-life ~2.6 h), without effecting non-esterified cholesterol content suggesting lysosomal non-esterified cholesterol content did not inhibit autophagosome-lysosome fusion. Therefore, U18666A-mediated effects on trafficking were investigated.;Fluorescent microscopy analysis revealed U18666A treatment affected F-actin and vimentin, but not microtubules. Rhodamine-phalloidin staining indicated U18666A induced F-actin depolymerization. Actin repolymerized when cultures were shifted to leucine-starvation medium. However, this effect did not mediate enhanced autophagosome clearance. Immunofluorescence staining patterns of vimentin filaments increased in number and complexity in U18666A-treated normal human fibroblasts similar to NPC fibroblasts. Inhibition of PKC by bisindolylmaleimide 1 (Bis-1) treatment of 1c1c7 cultures favored the formation of filamentous vimentin, accumulation of non-esterified cholesterol and autophagosomes, and decreased GFP-LC3 and LAMP1 colocalization. Confocal microscopy revealed that autophagosomes associated with vimentin filaments following U18666A and Bis-1 treatment, but not with leucine starvation. The addition of PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA) to U18666AA-treated cultures resulted in vimentin filament dissociation and decreases in 1c1c7 and NPC fibroblast culture cholesterol content. Analyses of GFP-LC3 indicated enhanced autophagosome clearance (half-life reduced to ~3.6 h) in 1c1c7 cultures. Western blot analysis showed that LC3-II decreased in NPC fibroblasts within 24 h of TPA-treatment. The cumulative data suggest that autophagosome accumulation in NPC fibroblasts or in response to CAD-treatment is due to increased autophagosome synthesis paired with inefficient degradation due to sequestration of autophagosomes within vimentin filament networks.