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.
","abstract_html":"<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>","abstract_has_math":false,"creators":["Kleinman, Miriam Devorah"],"institution":null,"degree_name":"Ph.D.","degree_level":"Open Access Dissertation","degree_discipline":"Molecular and Cellular Toxicology","degree_department":null,"school":null,"contributors":["John J. Reiners, Jr."],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2012,"date_issued":"2012-01-01T08:00:00Z","date_published":"2012-01-01T08:00:00Z","updated_at":"2026-07-24T05:58:57Z","subjects":["Autophagosome, Autophagy, Cationic amphiphilic drugs, Cytoskeleton, Niemann Pick Type C, Trafficking","Biology","Pharmacology","Toxicology"],"languages":[],"rights":[],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"https://digitalcommons.wayne.edu/oa_dissertations/380","outbound_label":"Repository record","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["John J. Reiners, Jr."]},{"key":"dc:creator","label":"Author","values":["Kleinman, Miriam Devorah"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.available","label":"Dc Date Available","values":["2011-01-01T08:00:00Z"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Molecular and Cellular Toxicology"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Open Access Dissertation"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Ph.D."]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Autophagosome, Autophagy, Cationic amphiphilic drugs, Cytoskeleton, Niemann Pick Type C, Trafficking","Biology","Pharmacology","Toxicology"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["https://digitalcommons.wayne.edu/oa_dissertations/380"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["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.
"]},{"key":"dc:title","label":"Title","values":["Cationic amphiphilic drug-induced autophagosome accumulation is due to autophagosome sequestration within vimentin intermediate filament networks resulting in prolonged autophagosome half-life"]}]}],"canonical_facts":{"dc:contributor":["John J. Reiners, Jr."],"dc:creator":["Kleinman, Miriam Devorah"],"dc:date.available":["2011-01-01T08:00:00Z"],"dc:description.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.
"],"dc:identifier":["https://digitalcommons.wayne.edu/oa_dissertations/380"],"dc:subject":["Autophagosome, Autophagy, Cationic amphiphilic drugs, Cytoskeleton, Niemann Pick Type C, Trafficking","Biology","Pharmacology","Toxicology"],"dc:title":["Cationic amphiphilic drug-induced autophagosome accumulation is due to autophagosome sequestration within vimentin intermediate filament networks resulting in prolonged autophagosome half-life"],"thesis:degree_discipline":["Molecular and Cellular Toxicology"],"thesis:degree_level":["Open Access Dissertation"],"thesis:degree_name":["Ph.D."]},"updated_at":"2026-07-24T05:58:57Z"}