Late-stage results from the probe development effort to identify transcriptional activators of heat shock protein 70 (Hsp70): fluorescence-based cell-based microscopic assay to assess aggregation of polyQ35 in C. elegans larvae
Name: Late-stage results from the probe development effort to identify transcriptional activators of heat shock protein 70 (Hsp70): fluorescence-based cell-based microscopic assay to assess aggregation of polyQ35 in C. elegans larvae. ..more
BioActive Compound: 1
Source (MLSCN Center Name): The Scripps Research Institute Molecular Screening Center
Center Affiliation: The Scripps Research Institute (TSRI)
Assay Provider: Richard Morimoto, Northwestern University
Network: Molecular Library Screening Center Network (MLSCN)
Grant Proposal Number: 5 R21 NS056337-02
Grant Proposal PI: Richard Morimoto
External Assay ID: HSP70_AG_FLUO_POLYQ35_AGGREGATION
Name: Late-stage results from the probe development effort to identify transcriptional activators of heat shock protein 70 (Hsp70): fluorescence-based cell-based microscopic assay to assess aggregation of polyQ35 in C. elegans larvae.
The human heat shock protein 70 (Hsp70) family is evolutionarily conserved among all organisms from archaebacteria to humans, suggesting an essential role in cell survival (1, 2). Under circumstances of transient cell stress, the heat shock response and activities of molecular chaperones can restore protein homeostasis. In human disease, however, misfolded proteins can accumulate when polyglutamine-expansion proteins are chronically expressed over the life of the cell. Elevated expression of molecular chaperones suppresses protein misfolding/aggregation and toxicity phenotypes in various model systems of Huntington's disease, Alzheimer's disease, Parkinson's disease, and Amyotrophic Lateral Sclerosis (ALS). Mutations in the respective proteins huntingtin, tau, alpha-synuclein, and superoxide dismutase (SOD1), associated with these diseases, result in the appearance of misfolded species that adopt alternate conformations. These observations led to the proposal that a common feature of diverse diseases of protein conformation is the appearance of alternate folded states that self-associate and form toxic species and protein aggregates.
A role for Hsp70 family proteins in controlling these events has been widely studied. Studies with mammalian tissue culture cells, transgenic mice, Drosophila, and C. elegans have established that the heat shock response can be activated in cells expressing aggregation-prone proteins, suggesting a role for molecular chaperones as an adaptive survival response (3, 4). Moreover, a direct relationship with polyglutamine diseases is suggested by the co-localization of several heat shock proteins, including Hdj-1, Hdj-2, Hsp70 and ubiquitin with polyglutamine aggregates in the tissues of affected individuals, transgenic mice and tissue culture cells (5). Finally, overexpression of Hsp70 can suppress the toxicity associated with the accumulation of misfolded proteins (6-8). High throughput screening initiatives aimed at the identification of compounds that enhance the heat shock response, in particular Hsp70, will provide insights into this conserved cellular process and may lead to novel therapeutics for these devastating disorders.
1.Gupta, R.S., and Singh, B. 1994. Phylogenetic analysis of 70 kD heat shock protein sequences suggests a chimeric origin for the eukaryotic cell nucleus. Curr Biol 4:1104-1114.
2.Lindquist, S., and Craig, E.A. 1988. The heat-shock proteins. Annu Rev Genet 22:631-677.
3.Satyal, S.H., Schmidt, E., Kitagawa, K., Sondheimer, N., Lindquist, S., Kramer, J.M., and Morimoto, R.I. 2000. Polyglutamine aggregates alter protein folding homeostasis in Caenorhabditis elegans. Proc Natl Acad Sci U S A 97:5750-5755.
4.Wyttenbach, A., Carmichael, J., Swartz, J., Furlong, R.A., Narain, Y., Rankin, J., and Rubinsztein, D.C. 2000. Effects of heat shock, heat shock protein 40 (HDJ-2), and proteasome inhibition on protein aggregation in cellular models of Huntington's disease. Proc Natl Acad Sci U S A 97:2898-2903.
5.Cummings, C.J., Mancini, M.A., Antalffy, B., DeFranco, D.B., Orr, H.T., and Zoghbi, H.Y. 1998. Chaperone suppression of aggregation and altered subcellular proteasome localization imply protein misfolding in SCA1. Nat Genet 19:148-154.
6.Krobitsch, S., and Lindquist, S. 2000. Aggregation of huntingtin in yeast varies with the length of the polyglutamine expansion and the expression of chaperone proteins. Proc Natl Acad Sci U S A 97:1589-1594.
7.Kazemi-Esfarjani, P., and Benzer, S. 2000. Genetic suppression of polyglutamine toxicity in Drosophila. Science 287:1837-1840.
8.Warrick, J.M., Chan, H.Y., Gray-Board, G.L., Chai, Y., Paulson, H.L., and Bonini, N.M. 1999. Suppression of polyglutamine-mediated neurodegeneration in Drosophila by the molecular chaperone HSP70. Nat Genet 23:425-428.
9. Brenner S. 1974. The genetics of Caenorhabditis elegans. Genetics. 1974 May;77(1):71-94.
10. Morley, J.F., Brignull, H.R., Weyers, J.J. & Morimoto, R.I. The threshold for polyglutamine-expansion protein aggregation and cellular toxicity is dynamic and influenced by aging in Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA 99, 10417-10422 (2002).
11. Hirsh D, Oppenheim D, Klass M. 1976. Development of the reproductive system of Caenorhabditis elegans. Dev Biol. 1976 Mar;49(1):200-219.
12. Nollen EA, Garcia SM, van Haaften G, Kim S, Chavez A, Morimoto RI, Plasterk RH. Genome-wide RNA interference screen identifies previously undescribed regulators of polyglutamine aggregation. Proc Natl Acad Sci U S A. 2004 Apr 27;101(17):6403-6408.
13. Garcia SM, Casanueva MO, Silva MC, Amaral MD, Morimoto RI. Neuronal signaling modulates protein homeostasis in Caenorhabditis elegans post-synaptic muscle cells. Genes Dev. 2007 Nov 15;21(22):3006-3016.
14. Waza M, Adachi H, Katsuno M, Minamiyama M, Sang C, Tanaka F, Inukai A, Doyu M, Sobue G. 17-AAG, an Hsp90 inhibitor, ameliorates polyglutamine-mediated motor neuron degeneration. Nat Med. 2005 Oct;11(10):1088-1095.
15. Fujikake N, Nagai Y, Popiel HA, Okamoto Y, Yamaguchi M, Toda T. Heat shock transcription factor 1-activating compounds suppress polyglutamine-induced neurodegeneration through induction of multiple molecular chaperones. J Biol Chem. 2008 Sep 19;283(38):26188-26197.
Hsp70, HSPA1A, HSF1, heat shock transcription factor 1, chaperone, agonist, activator, C. elegans, C. elegans larvae, polyglutamine, polyglutamine disease, aggregation, foci, microscopic, counterscreen, fluorescence, late stage, late stage AID, assay provider, powders, Scripps, Scripps Research Institute Molecular Screening Center, Molecular Library Screening Center Network, MLSCN.
The purpose of this assay is to test powder sample of a compound identified as a transcriptional activator of heat shock protein 70 (Hsp70) in a C. elegans model that has many of the characteristics of polyglutamine diseases such as Huntington's disease, and has been a valuable tool in the identification of genetic and chemical modifiers of aggregation and toxicity (12, 13). This C. elegans model for the expression of expanded polyglutamines (35 glutamines fused to YFP, referred to as polyQ35-YFP) in body wall muscles, shows age-dependent aggregation in the muscle cells (10). Age-synchronized animals expressing polyQ35 are treated with test compound and scored on aggregation when the animals are 6 days old. 17-AAG is used as a positive control inducer of the heat shock response that induces chaperone expression and reduces polyglutamine aggregation (14, 15). As designed, compounds that are active in regulating proteostasis will reduce the observed number of foci of aggregation.
C. elegans strains: The polyglutamine strain expressing 35 CAG-repeats fused with YFP (Q35::YFP) (10) was maintained according to standard methods, at 20 C on nematode growth media (NGM) with OP50 E. coli (9).
C. elegans assays for aggregation: C. elegans expressing YFP-tagged Q35 protein were treated with either DMSO or test compound at different concentrations (1, 5, 10 and 15 uM) for 4 days at 20 C. Test compound was dissolved and diluted in 100% DMSO, and the animals were incubated at a maximum concentration of 1.5% DMSO to avoid solvent-specific developmental defects and toxicity. OP50-only and DMSO-only controls were used. 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) was used as positive control for induction of the heat shock response at concentrations of 0.5, 1, 5 and 50 uM. Treatment with chemicals was performed in liquid culture in a 96-well plate format. Each well contained a final volume of 60 uL, comprising 20 to 25 L2 (larval 2 stage) age-synchronized animals, compound at the appropriate concentration, and OP50 bacteria to a final OD595nm of 0.8 in the microtiter plate. Animals and bacteria were resuspended in S-medium (11) supplemented with streptomycin, penicillin, and nystatin. To obtain the age synchronized population of L2 larvae, gravid adults were bleached with a NaOCl solution (250 mM NaOH and 1:4 (v/v) dilution of commercial bleach) and the eggs were allowed to hatch in M9 buffer overnight at 20 C. The first larval stage (L1) animals were transferred to OP50 plates to allow them to develop into L2 stage at 20 C. The animals were then washed with M9 buffer, resuspended in S-medium to the appropriate concentration, and transferred into the 96-well plates. The animals were scored for changes in aggregation (number of fluorescent foci) at day 6 using the stereomicroscope Leica MZ16FA equipped for epifluorescence. The total number of foci were counted in each animal treated with either DMSO or test compound.
The percent of foci number in compound-treated animals relative to the DMSO control (set as 100%) was calculated as follows:
Relative percent foci in compound-treated worm = ( [number of foci in compound-treated worm] / [number of foci in DMSO-treated worm] ) x 100
PubChem Activity Outcome and Score:
Compounds that resulted in less than or equal to 50% relative foci compared to the DMSO control (set as 100%) were active in this assay.
The PubChem Activity Score is assigned a value of 100 for active compounds, and 0 for inactive compounds.
The PubChem Activity Score range for active compounds is 100-100. There are no inactive compounds.
List of reagents:
Q35::YFP C. elegans strain (AM140) (C. elegans Genetic Center, CGC)
M9 Minimal Salts media, 5x (Sigma, part #M6030)
This assay was performed by the assay provider with powder samples of purchased test compound.
** Test Concentration.
Data Table (Concise)