title: Amyotrophic lateral sclerosis-linked mutant VAPB inclusions do not interfere with protein degradation pathways or intracellular transport in a cultured cell model.
abstract: VAPB is a ubiquitously expressed, ER-resident adaptor protein involved in interorganellar lipid exchange, membrane contact site formation, and membrane trafficking. Its mutant form, P56S-VAPB, which has been linked to a dominantly inherited form of Amyotrophic Lateral Sclerosis (ALS8), generates intracellular inclusions consisting in restructured ER domains whose role in ALS pathogenesis has not been elucidated. P56S-VAPB is less stable than the wild-type protein and, at variance with most pathological aggregates, its inclusions are cleared by the proteasome. Based on studies with cultured cells overexpressing the mutant protein, it has been suggested that VAPB inclusions may exert a pathogenic effect either by sequestering the wild-type protein and other interactors (loss-of-function by a dominant negative effect) or by a more general proteotoxic action (gain-of-function). To investigate P56S-VAPB degradation and the effect of the inclusions on proteostasis and on ER-to-plasma membrane protein transport in a more physiological setting, we used stable HeLa and NSC34 Tet-Off cell lines inducibly expressing moderate levels of P56S-VAPB. Under basal conditions, P56S-VAPB degradation was mediated exclusively by the proteasome in both cell lines, however, it could be targeted also by starvation-stimulated autophagy. To assess possible proteasome impairment, the HeLa cell line was transiently transfected with the ERAD (ER Associated Degradation) substrate CD3δ, while autophagic flow was investigated in cells either starved or treated with an autophagy-stimulating drug. Secretory pathway functionality was evaluated by analyzing the transport of transfected Vesicular Stomatitis Virus Glycoprotein (VSVG). P56S-VAPB expression had no effect either on the degradation of CD3δ or on the levels of autophagic markers, or on the rate of transport of VSVG to the cell surface. We conclude that P56S-VAPB inclusions expressed at moderate levels do not interfere with protein degradation pathways or protein transport, suggesting that the dominant inheritance of the mutant gene may be due mainly to haploinsufficiency.
authors.affiliation: Institute of Neuroscience, Consiglio Nazionale delle Ricerche, and Department of Medical Biotechnology and Translational Medicine (BIOMETRA), Università degli Studi di Milano, Milano, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Genevini
authors.affiliation: Institute of Neuroscience, Consiglio Nazionale delle Ricerche, and Department of Medical Biotechnology and Translational Medicine (BIOMETRA), Università degli Studi di Milano, Milano, Italy.
authors.forename: Giulia
authors.initials: G
authors.lastname: Papiani
authors.affiliation: Institute of Neuroscience, Consiglio Nazionale delle Ricerche, and Department of Medical Biotechnology and Translational Medicine (BIOMETRA), Università degli Studi di Milano, Milano, Italy.
authors.forename: Annamaria
authors.initials: A
authors.lastname: Ruggiano
authors.affiliation: Department of Molecular Biochemistry and Pharmacology, Istituto di Ricerche Farmacologiche "Mario Negri", Milan, Italy.
authors.forename: Lavinia
authors.initials: L
authors.lastname: Cantoni
authors.affiliation: Institute of Neuroscience, Consiglio Nazionale delle Ricerche, and Department of Medical Biotechnology and Translational Medicine (BIOMETRA), Università degli Studi di Milano, Milano, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Navone
authors.affiliation: Institute of Neuroscience, Consiglio Nazionale delle Ricerche, and Department of Medical Biotechnology and Translational Medicine (BIOMETRA), Università degli Studi di Milano, Milano, Italy; Department of Health Science, Magna Graecia University of Catanzaro, Catanzaro, Italy.
authors.forename: Nica
authors.initials: N
authors.lastname: Borgese
mesh_terms: D000690:Amyotrophic Lateral Sclerosis / Q000378:metabolism / Q000473:pathology*; D001343:Autophagy / Q000187:drug effects; D017252:CD3 Complex / Q000378:metabolism; D002460:Cell Line; D004317:Doxorubicin / Q000633:toxicity; D006056:Golgi Apparatus / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism; D007976:Leupeptins / Q000494:pharmacology; D018613:Microscopy, Confocal; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D059748:Proteolysis / Q000187:drug effects; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D017252:CD3 Complex; C472562:CD3delta antigen; D007976:Leupeptins; C496748:VAPB protein, human; D033921:Vesicular Transport Proteins; D004317:Doxorubicin; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde