Structure, biogenesis, and clearance of ER-derived inclusions generated by the ALS-linked mutant of VAPB

Background: VAP-B (Vesicle-Associated Membrane Protein-Associated Protein B) is a ubiquitously expressed, ER-resident tail-anchored adaptor protein implicated in interorganellar lipid exchange, generation of membrane contact sites and membrane traffic. Its mutant form, P56S-VAPB, has been 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 showed (1) that newly synthesized mutant VAPB is normally targeted to the ER where it rapidly aggregates, generating a restructured ER domain consisting of ribbons of stacked cisternae apparently held together by the mutated cytosolic domain of VAPB. The P56S-VAPB structures are formed also when the mutant protein is expressed at physiological levels, and are continuous with the surrounding normally organized ER, which is, however, devoid of the mutant protein. Objectives: To investigate the dynamics of inclusion generation and degradation. Methods: We used stable HeLa-TetOff cell lines inducibly expressing a Myc-tagged version of wild type VAPB or P56S-VAPB to establish a 'pulse-chase' protocol in which cells were first allowed to accumulate the mutant protein in the absence of Doxycyclin; to stop synthesis of VAPB, the antibiotic was reintroduced into the medium and the rate of decay of the two proteins was studied by analysing the levels of Myc-tagged proteins during this chase period. Results: At variance with most pathological inclusion bodies (IBs), P56S-VAPB inclusions undergo turnover, and the rate of degradation of the aggregated polyubiquitinated mutant is faster than that of the wild type protein. Degradation of the mutant protein involves extraction of P56S-VAPB from the ER membrane by the p97 ATPase, as suggested by the observation that transfection of a dominant negative p97 stabilizes mutant VAPB. Clearance of the inclusions occurs via the proteasome degradation pathway with no apparent participation of macro-autophagy (2). Involvement of the proteasome in P56S-VAPB elimination does not impair the proteasome's ability to clear a classical ERAD substrate. Autophagocytosis does not appear to be slowed in cells expressing mutant VAPB. Discussion and Conclusions: P56S-VAPB inclusions differ from other IBs, both in the mechanisms of their genesis and of their clearance from the cell. Our results reveal surprisingly efficient extraction from the ER and proteasomal degradation of this severely aggregated mutant protein. Furthermore, they 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 that the dominant inheritance of the mutant allele may rather be related to haploinsufficiency. References: Fasana E, Fossati M, Ruggiano A et al FASEB J 2010; 24:1419-1430 Papiani G, Ruggiano A, Fossati M et al J Cell Sci 2012; 125: 3601-3611 Acknowledgements: This work was supported by Cariplo Foundation, Regione Lombardia (TERDISMENTAL) and PNR-CNR Aging Program. Keywords: Endoplasmic Reticulum, Inclusion bodies, Protein degradation