title: Niclosamide prevents the formation of large ubiquitin-containing aggregates caused by proteasome inhibition.
abstract: BACKGROUND
  Protein aggregation is a hallmark of many neurodegenerative diseases and has been linked to the failure to degrade misfolded and damaged proteins. In the cell, aberrant proteins are degraded by the ubiquitin proteasome system that mainly targets short-lived proteins, or by the lysosomes that mostly clear long-lived and poorly soluble proteins. Both systems are interconnected and, in some instances, autophagy can redirect proteasome substrates to the lysosomes.
  
  
  RESULTS
  To better understand the interplay between these two systems, we established a neuroblastoma cell population stably expressing the GFP-ubiquitin fusion protein. We show that inhibition of the proteasome leads to the formation of large ubiquitin-containing inclusions accompanied by lower solubility of the ubiquitin conjugates. Strikingly, the formation of the ubiquitin-containing aggregates does not require ectopic expression of disease-specific proteins. Moreover, formation of these focused inclusions caused by proteasome inhibition requires the lysine 63 (K63) of ubiquitin. We then assessed selected compounds that stimulate autophagy and found that the antihelmintic chemical niclosamide prevents large aggregate formation induced by proteasome inhibition, while the prototypical mTORC1 inhibitor rapamycin had no apparent effect. Niclosamide also precludes the accumulation of poly-ubiquitinated proteins and of p62 upon proteasome inhibition. Moreover, niclosamide induces a change in lysosome distribution in the cell that, in the absence of proteasome activity, may favor the uptake into lysosomes of ubiquitinated proteins before they form large aggregates.
  
  
  CONCLUSIONS
  Our results indicate that proteasome inhibition provokes the formation of large ubiquitin containing aggregates in tissue culture cells, even in the absence of disease specific proteins. Furthermore our study suggests that the autophagy-inducing compound niclosamide may promote the selective clearance of ubiquitinated proteins in the absence of proteasome activity.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada.
authors.forename: Esther
authors.initials: E
authors.lastname: Gies
authors.forename: Inga
authors.initials: I
authors.lastname: Wilde
authors.forename: Jason M
authors.initials: JM
authors.lastname: Winget
authors.forename: Maria
authors.initials: M
authors.lastname: Brack
authors.forename: Barak
authors.initials: B
authors.lastname: Rotblat
authors.forename: Carolina Arias
authors.initials: CA
authors.lastname: Novoa
authors.forename: Aruna D
authors.initials: AD
authors.lastname: Balgi
authors.forename: Poul H
authors.initials: PH
authors.lastname: Sorensen
authors.forename: Michel
authors.initials: M
authors.lastname: Roberge
authors.forename: Thibault
authors.initials: T
authors.lastname: Mayor
mesh_terms: D000969:Antinematodal Agents / Q000494:pharmacology; D001343:Autophagy; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D008247:Lysosomes / Q000378:metabolism; D000076222:Mechanistic Target of Rapamycin Complex 1; D008870:Microtubules / Q000378:metabolism; D046912:Multiprotein Complexes; D019636:Neurodegenerative Diseases / Q000378:metabolism; D009534:Niclosamide / Q000494:pharmacology*; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D020123:Sirolimus / Q000494:pharmacology; D012995:Solubility; D058570:TOR Serine-Threonine Kinases; D025801:Ubiquitin / Q000737:chemistry*
chemical_list: D000969:Antinematodal Agents; D046912:Multiprotein Complexes; D011506:Proteins; D025801:Ubiquitin; D049452:Green Fluorescent Proteins; D009534:Niclosamide; D000076222:Mechanistic Target of Rapamycin Complex 1; D058570:TOR Serine-Threonine Kinases; D046988:Proteasome Endopeptidase Complex; D020123:Sirolimus