title: A platform to view huntingtin exon 1 aggregation flux in the cell reveals divergent influences from chaperones hsp40 and hsp70.
abstract: Our capacity for tracking how misfolded proteins aggregate inside a cell and how different aggregation states impact cell biology remains enigmatic. To address this, we built a new toolkit that enabled the high throughput tracking of individual cells enriched with polyglutamine-expanded Htt exon 1 (Httex1) monomers, oligomers, and inclusions using biosensors of aggregation state and flow cytometry pulse shape analysis. Supplemented with gel filtration chromatography and fluorescence-adapted sedimentation velocity analysis of cell lysates, we collated a multidimensional view of Httex1 aggregation in cells with respect to time, polyglutamine length, expression levels, cell survival, and overexpression of protein quality control chaperones hsp40 (DNAJB1) and hsp70 (HSPA1A). Cell death rates trended higher for Neuro2a cells containing Httex1 in inclusions than with Httex1 dispersed through the cytosol at time points of expression over 2 days. hsp40 stabilized monomers and suppressed inclusion formation but did not otherwise change Httex1 toxicity. hsp70, however, had no major effect on aggregation of Httex1 but increased the survival rate of cells with inclusions. hsp40 and hsp70 also increased levels of a second bicistronic reporter of Httex1 expression, mKate2, and increased total numbers of cells in culture, suggesting these chaperones partly rectify Httex1-induced deficiencies in quality control and growth rates. Collectively, these data suggest that Httex1 overstretches the protein quality control resources and that the defects can be partly rescued by overexpression of hsp40 and hsp70. Importantly, these effects occurred in a pronounced manner for soluble Httex1, which points to Httex1 aggregation occurring subsequently to more acute impacts on the cell.
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, 30 Flemington Road, University of Melbourne, Melbourne, Victoria 3010, Australia.
authors.forename: Angelique R
authors.initials: AR
authors.lastname: Ormsby
authors.forename: Yasmin M
authors.initials: YM
authors.lastname: Ramdzan
authors.forename: Yee-Foong
authors.initials: YF
authors.lastname: Mok
authors.forename: Kristijan D
authors.initials: KD
authors.lastname: Jovanoski
authors.forename: Danny M
authors.initials: DM
authors.lastname: Hatters
mesh_terms: D000682:Amyloid / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D016923:Cell Death; D002460:Cell Line; D002470:Cell Survival; D005091:Exons; D005786:Gene Expression Regulation; D050956:HSP40 Heat-Shock Proteins / Q000819:agonists / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D000071058:Huntingtin Protein; D051379:Mice; D009419:Nerve Tissue Proteins / Q000819:agonists / Q000378:metabolism*
chemical_list: D000682:Amyloid; C494647:DNAJB1 protein, human; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; C490786:HSPA1A protein, human; C086055:HTT protein, human; D000071058:Huntingtin Protein; D009419:Nerve Tissue Proteins
keywords: Amyloid; Analytical Ultracentrifugation; Chaperone Chaperonin; Flow Cytometry; Oligomers; Polyglutamine; Protein Aggregation; PulSA; huntingtin