Access Type

Open Access Dissertation

Date of Award

January 2012

Degree Type


Degree Name



Molecular and Cellular Toxicology

First Advisor

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.