title: Restructured endoplasmic reticulum generated by mutant amyotrophic lateral sclerosis-linked VAPB is cleared by the proteasome.
abstract: VAPB (vesicle-associated membrane protein-associated protein B) is a ubiquitously expressed, ER-resident tail-anchored protein that functions as adaptor for lipid-exchange proteins. Its mutant form, P56S-VAPB, is linked to a dominantly inherited form of amyotrophic lateral sclerosis (ALS8). P56S-VAPB forms intracellular inclusions, whose role in ALS pathogenesis has not yet been elucidated. We recently demonstrated that these inclusions are formed by profoundly remodelled stacked ER cisternae. Here, we used stable HeLa-TetOff cell lines inducibly expressing wild-type VAPB and P56S-VAPB, as well as microinjection protocols in non-transfected cells, to investigate the dynamics of inclusion generation and degradation. Shortly after synthesis, the mutant protein forms small, polyubiquitinated clusters, which then congregate in the juxtanuclear region independently of the integrity of the microtubule cytoskeleton. The rate of degradation of the aggregated mutant is higher than that of the wild-type protein, so that the inclusions are cleared only a few hours after cessation of P56S-VAPB synthesis. At variance with other inclusion bodies linked to neurodegenerative diseases, clearance of P56S-VAPB inclusions involves the proteasome, with no apparent participation of macro-autophagy. Transfection of a dominant-negative form of the AAA ATPase p97/VCP stabilizes mutant VAPB, suggesting a role for this ATPase in extracting the aggregated protein from the inclusions. Our results demonstrate that the structures induced by P56S-VAPB stand apart from other inclusion bodies, both in the mechanism of their genesis and of their clearance from the cell, with possible implications for the pathogenic mechanism of the mutant protein.
authors.affiliation: Consiglio Nazionale delle Ricerche Institute of Neuroscience and Department of Pharmacology, University of Milan, Milano,  Italy.
authors.forename: Giulia
authors.initials: G
authors.lastname: Papiani
authors.forename: Annamaria
authors.initials: A
authors.lastname: Ruggiano
authors.forename: Matteo
authors.initials: M
authors.lastname: Fossati
authors.forename: Andrea
authors.initials: A
authors.lastname: Raimondi
authors.forename: Giovanni
authors.initials: G
authors.lastname: Bertoni
authors.forename: Maura
authors.initials: M
authors.lastname: Francolini
authors.forename: Roberta
authors.initials: R
authors.lastname: Benfante
authors.forename: Francesca
authors.initials: F
authors.lastname: Navone
authors.forename: Nica
authors.initials: N
authors.lastname: Borgese
mesh_terms: D000690:Amyotrophic Lateral Sclerosis / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D002479:Inclusion Bodies / Q000235:genetics / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport; D014162:Transfection; D054875:Ubiquitination; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: C496748:VAPB protein, human; D033921:Vesicular Transport Proteins; D046988:Proteasome Endopeptidase Complex