Access Type
Open Access Dissertation
Date of Award
January 2012
Degree Type
Dissertation
Degree Name
Ph.D.
Department
Molecular and Cellular Toxicology
First Advisor
John J. Reiners, Jr.
Abstract
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.
Recommended Citation
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.
https://digitalcommons.wayne.edu/oa_dissertations/380