title: Identification of modulators of autophagic flux in an image-based high content siRNA screen.
abstract: Autophagy is the primary process for recycling cellular constituents through lysosomal degradation. In addition to nonselective autophagic engulfment of cytoplasm, autophagosomes can recognize specific cargo by interacting with ubiquitin-binding autophagy receptors such as SQSTM1/p62 (sequestosome 1). This selective form of autophagy is important for degrading aggregation-prone proteins prominent in many neurodegenerative diseases. We carried out a high content image-based siRNA screen (4 to 8 siRNA per gene) for modulators of autophagic flux by monitoring fluorescence of GFP-SQSTM1 as well as colocalization of GFP-SQSTM1 with LAMP2 (lysosomal-associated membrane protein 2)-positive lysosomal vesicles. GFP-SQSTM1 and LAMP2 phenotypes of primary screen hits were confirmed in 2 cell types and profiled with image-based viability and MTOR signaling assays. Common seed analysis guided siRNA selection for these assays to reduce bias toward off-target effects. Confirmed hits were further validated in a live-cell assay to monitor fusion of autophagosomes with lysosomes. Knockdown of 10 targets resulted in phenotypic profiles across multiple assays that were consistent with upregulation of autophagic flux. These hits include modulators of transcription, lysine acetylation, and ubiquitination. Two targets, KAT8 (K[lysine] acetyltransferase 8) and CSNK1A1 (casein kinase 1, α 1), have been implicated in autophagic regulatory feedback loops. We confirmed that CSNK1A1 knockout (KO) cell lines have accelerated turnover of long-lived proteins labeled with (14)C-leucine in a pulse-chase assay as additional validation of our screening assays. Data from this comprehensive autophagy screen point toward novel regulatory pathways that might yield new therapeutic targets for neurodegeneration.
authors.affiliation: a Discovery Technologies, Amgen Inc. , Thousand Oaks , CA , USA.
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Hale
authors.affiliation: b Department of Neuroscience Research , Amgen Inc. , Thousand Oaks , CA , USA.
authors.forename: Qingwen
authors.initials: Q
authors.lastname: Cheng
authors.affiliation: b Department of Neuroscience Research , Amgen Inc. , Thousand Oaks , CA , USA.
authors.forename: Danny
authors.initials: D
authors.lastname: Ortuno
authors.affiliation: b Department of Neuroscience Research , Amgen Inc. , Thousand Oaks , CA , USA.
authors.forename: Ming
authors.initials: M
authors.lastname: Huang
authors.affiliation: a Discovery Technologies, Amgen Inc. , Thousand Oaks , CA , USA.
authors.forename: Dana
authors.initials: D
authors.lastname: Nojima
authors.affiliation: c Genome Analysis Unit, Amgen Inc. , Thousand Oaks , CA , USA.
authors.forename: Paul D
authors.initials: PD
authors.lastname: Kassner
authors.affiliation: c Genome Analysis Unit, Amgen Inc. , Thousand Oaks , CA , USA.
authors.forename: Songli
authors.initials: S
authors.lastname: Wang
authors.affiliation: c Genome Analysis Unit, Amgen Inc. , Thousand Oaks , CA , USA.
authors.forename: Michael M
authors.initials: MM
authors.lastname: Ollmann
authors.affiliation: b Department of Neuroscience Research , Amgen Inc. , Thousand Oaks , CA , USA.
authors.forename: Holly J
authors.initials: HJ
authors.lastname: Carlisle
mesh_terms: D000818:Animals; D001343:Autophagy; D045744:Cell Line, Tumor; D002470:Cell Survival; D055785:Gene Knockdown Techniques; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D021621:Imaging, Three-Dimensional; D034741:RNA, Small Interfering / Q000378:metabolism*; D015203:Reproducibility of Results
chemical_list: D034741:RNA, Small Interfering; D049452:Green Fluorescent Proteins
keywords: Autophagy; CK1α. CSNK1A1; MTOR; RNAi; SQSTM1; p62; pulse-chase; siRNA screen