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

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

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Open Access Dissertation

Discipline thesis:degree_discipline

Molecular and Cellular Toxicology

Year dc:date.available

2012

Author dc:creator

• Kleinman, Miriam Devorah

Contributors dc:contributor

• John J. Reiners, Jr.

• Autophagosome, Autophagy, Cationic amphiphilic drugs, Cytoskeleton, Niemann Pick Type C, Trafficking
• Biology
• Pharmacology
• Toxicology

Repository record dc:identifier

https://digitalcommons.wayne.edu/oa_dissertations/380

OAI identifier oai:identifier

oai:digitalcommons.wayne.edu:oa_dissertations-1379