title: Mutant SOD1 detoxification mechanisms in intact single cells.
abstract: Mutant superoxide dismutase 1 (mtSOD1) causes dominantly inherited amyotrophic lateral sclerosis (ALS). The mechanism for mtSOD1 toxicity remains unknown. Two main hypotheses are the impairment of proteasomal function and chaperone depletion by misfolded mtSOD1. Here, we employed FRET/FLIM and biosensor imaging to quantitatively localize ubiquitination, as well as chaperone binding of mtSOD1, and to assess their effect on proteasomal and protein folding activities. We found large differences in ubiquitination and chaperone interaction levels for wild-type (wt) SOD1 versus mtSOD1 in intact single cells. Moreover, SOD1 ubiquitination levels differ between proteasomal structures and cytoplasmic material. Hsp70 binding and ubiquitination of wt and mtSOD1 species are highly correlated, demonstrating the coupled upregulation of both cellular detoxification mechanisms upon mtSOD1 expression. Biosensor imaging in single cells revealed that mtSOD1 expression alters cellular protein folding activity but not proteasomal function in the neuronal cell line examined. Our results provide the first cell-by-cell-analysis of SOD1 ubiquitination and chaperone interaction. Moreover, our study opens new methodological avenues for cell biological research on ALS.
authors.affiliation: European Neuroscience Institute, Cell Biophysics Group, Waldweg 33, Göttingen 37073, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Ganesan
authors.forename: G
authors.initials: G
authors.lastname: Rohde
authors.forename: K
authors.initials: K
authors.lastname: Eckermann
authors.forename: K
authors.initials: K
authors.lastname: Sroka
authors.forename: M K E
authors.initials: MK
authors.lastname: Schaefer
authors.forename: C P
authors.initials: CP
authors.lastname: Dohm
authors.forename: P
authors.initials: P
authors.lastname: Kermer
authors.forename: G
authors.initials: G
authors.lastname: Haase
authors.forename: F
authors.initials: F
authors.lastname: Wouters
authors.forename: M
authors.initials: M
authors.lastname: Bähr
authors.forename: J H
authors.initials: JH
authors.lastname: Weishaupt
mesh_terms: D000690:Amyotrophic Lateral Sclerosis; D000818:Animals; D002460:Cell Line; D031541:Fluorescence Resonance Energy Transfer; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D018832:Molecular Chaperones / Q000378:metabolism*; D050505:Mutant Proteins / Q000378:metabolism; D009474:Neurons / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D017510:Protein Folding; D051381:Rats; D013482:Superoxide Dismutase / Q000235:genetics / Q000378:metabolism*; D000072105:Superoxide Dismutase-1; D025801:Ubiquitin / Q000378:metabolism; D054875:Ubiquitination
chemical_list: D018840:HSP70 Heat-Shock Proteins; D018832:Molecular Chaperones; D050505:Mutant Proteins; D025801:Ubiquitin; C000606292:Sod1 protein, rat; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1; D046988:Proteasome Endopeptidase Complex