Biochemical characterization of ER-derived cytoplasmic inclusions in cells expressing the amyotrophic lateral sclerosis-associated mutant form of VAP-B

VAP-B (Vesicle-Associated Membrane Protein-Associated Protein B) is an ER resident tail-anchored protein that functions as adaptor for lipid-exchange proteins. A dominantly inherited mutant, P56S-VAPB, causes a familial form of amyotrophic lateral sclerosis (ALS) and forms intracellular inclusions, which have been recently shown to derive from the ER membrane and classified as a novel form of organized smooth ER (OSER) (Fasana et al., in press). In order to elucidate the molecular mechanisms of P56S-VAPB pathogenicity, we analyzed both the formation and turnover of P56S-VAPB inclusions in cellular models expressing this exogenous mutant and the endogenous wild type VAPB at comparable levels, similarly to what is found in heterozigote ALS patients. To this aim, we generated stable Hela Tet-Off cell lines expressing the mutant protein in a doxycycline-dependent fashion. Both immunofluorescence and western blot analysis show that a progressive accumulation of P56S-VAP-B can be achieved by progressively decreasing exposure doses of doxycycline. In particular, at the concentration of antibiotic that allows mutant and endogenous VAPB comparable expression levels, small, though still detectable, P56S-VAPB inclusions can be observed. In these conditions, mutant VAP-B accumulation does not correlate with a slower turnover rate, as assessed by a 24 hour protein synthesis inhibition. Long time-spanning pulse-chase analysis is being performed in order to confirm these preliminary data. To investigate the preferential cellular pathway of protein degradation followed by P56S-VAPB we are evaluating its state of ubiquitination, before and after proteasome inhibition. Moreover, the involvement of autophagy by means of LC3 clusterization and LC3-I to LC3-II maturation is being clarified by immunofluorescence and western blot analyses, respectively. Preliminary results have shown a non-prominent co-localization of P56S VAP-B inclusions with ubiquitin-positive structures. Finally, to compare the effects of P56S-VAPB on the functional viability of non neuronal and neuronal cells and obtain important information on the mechanisms that could underlie ALS pathogenesis, we are generating stable Tet-off cell lines of NSC34 cells, a cell culture model for motoneuronal cells.