Open Access Dissertation
Date of Award
Molecular and Cellular Toxicology
John J. Reiners, Jr.
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.
Kleinman, Miriam Devorah, "Cationic amphiphilic drug-induced autophagosome accumulation is due to autophagosome sequestration within vimentin intermediate filament networks resulting in prolonged autophagosome half-life" (2012). Wayne State University Dissertations. 380.