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.

Metabolic changes may precede proteostatic dysfunction in a Drosophila model of amyloid beta peptide toxicity

“…This stock has been characterized previously ( Luheshi et al., 2007 ). Unless otherwise stated, all other experiments were carried out with single transgenic Aβ 40 , Aβ 42 , and Arctic Aβ 42 flies which have been described previously ( Kruppa et al., 2013 ). The Aβ transgene was inserted into the 51D location on the second chromosome using the φC31 integrase system ( Bischof et al., 2007 ).…”

Iron is a specific cofactor for distinct oxidation- and aggregation-dependent Aβ toxicity mechanisms

“…Drosophila FerHCH and FerLCH stocks have been described previously ( Rival et al, 2009 ). The UAS-Arctic Aβ 42 , UAS-Aβ 42 and UAS-Aβ 40 transgenic flies that were used for all survival assays were created by site-directed insertion at the 51D location on the second chromosome using the φC31 integrase system as previously described ( Bischof et al, 2007 ; Kruppa et al, 2013 ). The ‘pre-dimerised’ tandem Aβ 42 isoform (with a 12 amino acid linker peptide) has been described previously ( Speretta et al, 2012 ).…”

“…Successful mutagenesis was confirmed by DNA sequencing (Source Bioscience) of the insert. The Aβ 42 His>Ala constructs were subcloned into a pUAST-AttB vector (GenBank EF362409) and transgenic Drosophila lines were created by site-directed genomic insertion using the φC31 system at the 51D location on the second chromosome as described previously ( Bischof et al, 2007 ; Kruppa et al, 2013 ). Plasmids carrying multiple site-directed mutations were made by sequential rounds of mutagenesis.…”

“…There is accumulating evidence that Aβ, along with these amyloid-forming polypeptides, can exert a generic toxicity on cells ( Baglioni et al, 2006 ). The toxicity is generic in two regards: first, oligomeric aggregates of peptides damage a wide variety of mammalian cell types ( Barucker et al, 2014 ; Jang et al, 2014 ; Kinghorn et al, 2006 ; Kruppa et al, 2013 ); second, oligomers comprising a wide range of polypeptides can exhibit cytotoxicity ( Bucciantini et al, 2002 ; Cascella et al, 2013 ; Demuro et al, 2005 ; Newington et al, 2011 ; Zhao et al, 2009 ). Studies into the toxicity of amyloid aggregates have been undertaken in a wide range of model systems, not only in mammalian tissues and cells but also using vertebrate and invertebrate in vivo model systems.…”

Modeling the complex pathology of Alzheimer's disease in Drosophila

“…Co-expression with Aβ42 rescued the toxicity of neuroserpin in flies, supporting the biological relevance of this interaction. Puromycin-sensitive aminopeptidase (PSA) was initially identified as a suppressor of tau pathogenesis (Karsten et al, 2006) and was later isolated as a suppressor of Aβ42 toxicity in a genetic screen, but the mechanisms mediating its protective activity remain to be elucidated (Kruppa et al, 2013). Despite the robust protective activity of molecular chaperones against several intracellular amyloids, particularly polyglutamines, the in vivo role of chaperones against extracellular amyloids such as Aβ42 have received less attention.…”