title: Suppression of Aβ toxicity by puromycin-sensitive aminopeptidase is independent of its proteolytic activity.
abstract: The accumulation of β-amyloid (Aβ) peptide in the brain is one of the pathological hallmarks of Alzheimer's disease and is thought to be of primary aetiological significance. In an unbiased genetic screen, we identified puromycin-sensitive aminopeptidase (PSA) as a potent suppressor of Aβ toxicity in a Drosophila model system. We established that coexpression of Drosophila PSA (dPSA) in the flies' brains improved their lifespan, protected against locomotor deficits, and reduced brain Aβ levels by clearing the Aβ plaque-like deposits. However, confocal microscopy and subcellular fractionation of amyloid-expressing 7PA2 cells demonstrated that PSA localizes to the cytoplasm. Therefore, PSA and Aβ are unlikely to be in the same cellular compartment; moreover, when we artificially placed them in the same compartment in flies, we could not detect a direct epistatic interaction. The consequent hypothesis that PSA's suppression of Aβ toxicity is indirect was supported by the finding that Aβ is not a proteolytic substrate for PSA in vitro. Furthermore, we showed that the enzymatic activity of PSA is not required for rescuing Aβ toxicity in neuronal SH-SY5Y cells. We investigated whether the stimulation of autophagy by PSA was responsible for these protective effects. However PSA's promotion of autophagosome fusion with lysosomes required proteolytic activity and so its effect on autophagy is not identical to its protection against Aβ toxicity.
authors.affiliation: Cambridge Institute for Medical Research, Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 0XY, UK; Department of Medicine, University of Cambridge, Box 157, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2QQ, UK; Department of Genetics, University of Cambridge, Downing Street, Cambridge CB2 3EH, UK.
authors.forename: Antonina J
authors.initials: AJ
authors.lastname: Kruppa
authors.forename: Stanislav
authors.initials: S
authors.lastname: Ott
authors.forename: Dhia S
authors.initials: DS
authors.lastname: Chandraratna
authors.forename: James A
authors.initials: JA
authors.lastname: Irving
authors.forename: Richard M
authors.initials: RM
authors.lastname: Page
authors.forename: Elena
authors.initials: E
authors.lastname: Speretta
authors.forename: Tiffany
authors.initials: T
authors.lastname: Seto
authors.forename: Luiz Miguel
authors.initials: LM
authors.lastname: Camargo
authors.forename: Stefan J
authors.initials: SJ
authors.lastname: Marciniak
authors.forename: David A
authors.initials: DA
authors.lastname: Lomas
authors.forename: Damian C
authors.initials: DC
authors.lastname: Crowther
mesh_terms: D000544:Alzheimer Disease / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control*; D000626:Aminopeptidases / Q000494:pharmacology*; D016229:Amyloid beta-Peptides / Q000009:adverse effects*; D000818:Animals; D030801:Animals, Genetically Modified; D001343:Autophagy; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006801:Humans; D007124:Immunoenzyme Techniques; D009447:Neuroblastoma / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control*; D009474:Neurons / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D059748:Proteolysis; D011691:Puromycin / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
chemical_list: D016229:Amyloid beta-Peptides; D012333:RNA, Messenger; D011691:Puromycin; D000626:Aminopeptidases; C025579:enkephalin degrading enzyme
keywords: Alzheimer; Amyloid; Autophagy; Proteolysis; Puromycin-sensitive aminopeptidase
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