title: p62, Ref(2)P and ubiquitinated proteins are conserved markers of neuronal aging, aggregate formation and progressive autophagic defects.
abstract: Suppression of macroautophagy, due to mutations or through processes linked to aging, results in the accumulation of cytoplasmic substrates that are normally eliminated by the pathway. This is a significant problem in long-lived cells like neurons, where pathway defects can result in the accumulation of aggregates containing ubiquitinated proteins. The p62/Ref(2)P family of proteins is involved in the autophagic clearance of cytoplasmic protein bodies or sequestosomes. These unique structures are closely associated with protein inclusions containing ubiquitin as well as key components of the autophagy pathway. In this study we show that detergent fractionation followed by western blot analysis of insoluble ubiquitinated proteins (IUP), mammalian p62 and its Drosophila homologue, Ref(2)P can be used to quantitatively assess the activity level of aggregate clearance (aggrephagy) in complex tissues. Using this technique we show that genetic or age-dependent changes that modify the long-term enhancement or suppression of aggrephagy can be identified. Moreover, using the Drosophila model system this method can be used to establish autophagy-dependent protein clearance profiles that are occurring under a wide range of physiological conditions including developmental, fasting and altered metabolic pathways. This technique can also be used to examine proteopathies that are associated with human disorders such as frontotemporal dementia, Huntington and Alzheimer disease. Our findings indicate that measuring IUP profiles together with an assessment of p62/Ref(2)P proteins can be used as a screening or diagnostic tool to characterize genetic and age-dependent factors that alter the long-term function of autophagy and the clearance of protein aggregates occurring within complex tissues and cells.
authors.affiliation: BioScience Center, San Diego State University, CA, USA.
authors.forename: Bryan J
authors.initials: BJ
authors.lastname: Bartlett
authors.forename: Pauline
authors.initials: P
authors.lastname: Isakson
authors.forename: Jan
authors.initials: J
authors.lastname: Lewerenz
authors.forename: Heriberto
authors.initials: H
authors.lastname: Sanchez
authors.forename: Roxanne W
authors.initials: RW
authors.lastname: Kotzebue
authors.forename: Robert C
authors.initials: RC
authors.lastname: Cumming
authors.forename: Greg L
authors.initials: GL
authors.lastname: Harris
authors.forename: Ioannis P
authors.initials: IP
authors.lastname: Nezis
authors.forename: David R
authors.initials: DR
authors.lastname: Schubert
authors.forename: Anne
authors.initials: A
authors.lastname: Simonsen
authors.forename: Kim D
authors.initials: KD
authors.lastname: Finley
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000544:Alzheimer Disease / Q000378:metabolism; D000818:Animals; D030801:Animals, Genetically Modified; D001343:Autophagy; D003593:Cytoplasm / Q000378:metabolism; D004268:DNA-Binding Proteins; D003902:Detergents / Q000494:pharmacology; D029721:Drosophila Proteins / Q000378:metabolism*; D004331:Drosophila melanogaster; D018507:Gene Expression Regulation, Developmental; D006801:Humans; D046529:Microscopy, Electron, Transmission / Q000379:methods; D008954:Models, Biological; D008957:Models, Genetic; D009154:Mutation; D009474:Neurons / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism*; D000071456:Sequestosome-1 Protein; D013997:Time Factors
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D004268:DNA-Binding Proteins; D003902:Detergents; D029721:Drosophila Proteins; D009687:Nuclear Proteins; C099718:SQSTM1 protein, human; D000071456:Sequestosome-1 Protein; C080596:ref(2)P protein, Drosophila