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.