ER-derived Cytoplasmic Inclusions generated by Mutant, Amyotrophic Lateral Sclerosis-associated VAPB are cleared by the proteasome

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 clarified the ultrastructure of those inclusions and their precise relationship with the ER, demonstrating that they are formed by a profound remodeling of ER membranes with pairs of ER cisternae separated by a thin layer of electron-dense cytosol. Here, we used stable HeLa-TetOff cell lines inducibly expressing wild type (wt) and P56S-VAPB to investigate the dynamics of inclusion generation and degradation. Shortly after synthesis, the mutant protein forms small, polyubiquitinated clusters, which then concentrate in large juxtanuclear structures independently from the integrity of the microtubule cytoskeleton. The rate of degradation of the aggregated mutant is higher than that of the wt protein and 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. Moreover, we investigated a possible involvement of the p97/VCP AAA-ATPase in the extraction of poly-ubiquitinated P56S-VAPB from the ER membrane. Transfection of a dominant-negative p97 mutant stabilizes P56S-VAPB, indicating that it is degraded by the ER-Associated Degradation (ERAD) pathway. Our results suggest that the slow onset of P56S-linked familial ALS is not a consequence of the progressive accumulation of the mutant protein over time and may be related to a reduction of the intracellular levels of wt VAPB. Future work on the effects of the inhibition of wt VAPB expression in neuronal cells will provide a deeper insight into the role of mutant VAPB in the pathogenesis of ALS.