A rapid cellular FRET assay of polyglutamine aggregation identifies a novel inhibitor
Open accessAbstract
Many neurodegenerative diseases, including tauopathies, Parkinson's disease, amyotrophic lateral sclerosis, and the polyglutamine diseases, are characterized by intracellular aggregation of pathogenic proteins. It is difficult to study modifiers of this process in intact cells in a high-throughput and quantitative manner, although this could facilitate molecular insights into disease pathogenesis. Here we introduce a high-throughput assay to measure intracellular polyglutamine protein aggregation using fluorescence resonance energy transfer (FRET). We screened over 2800 biologically active small molecules for inhibitory activity and have characterized one lead compound in detail. Y-27632, an inhibitor of the Rho-associated kinase p160ROCK, diminished polyglutamine protein aggregation (EC(50) congruent with 5 microM) and reduced neurodegeneration in a Drosophila model of polyglutamine disease. This establishes a novel high-throughput approach to study protein misfolding and aggregation associated with neurodegenerative diseases and implicates a signaling pathway of previously unrecognized importance in polyglutamine protein processing.
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From an in vitro screen for molecules that reduce polyglutamine toxicity, the ROCK-inhibitor Y27632 was identified and subsequently shown to reduce Htt aggregation in cultured cells and Htt-mediated neurodegeneration in drosophila.
“From an in vitro screen for molecules that reduce polyglutamine toxicity, the ROCK-inhibitor Y27632 was identified and subsequently shown to reduce Htt aggregation in cultured cells and Htt-mediated neurodegeneration in drosophila [197,198].”
From RhoA Signaling in Neurodegenerative Diseases (2022) · cites this paper as [197]
The High-Throughput Screening passage describes identifying Y-27632 in a cellular screen for reduced polyglutamine aggregation, not toxicity. It reports reduced htt exon 1 aggregation in cultured cells, while the Drosophila-model passage reports protection against photoreceptor-neuron loss caused by htt exon 1(Q93). (LLM, conf=0.90)
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- Authors
- Pollitt, S. K., Pallos, J., Shao, J., Desai, U. A., Ma, A. A. K., Thompson, L. M., Marsh, J. L., & Diamond, M. I.
- Affiliations
- Genentech (US)
- University of California, Irvine (US)
- University of California, San Francisco (US)
- Corresponding author
- Diamond, Marc I
- Venue
- Neuron
- Publisher
- Elsevier BV
- Published
- 2003-11
- Volume
- 40
- Issue
- 4
- Pages
- 685-694
- ISSN
- 0896-6273 (ISSN-L)
- OpenAlex
- W2018928754
- Cited by
- 151
- References
- 2
- Citations by year
- 2026: 1 · 2025: 2 · 2024: 3 · 2023: 2 · 2022: 8 · 2021: 3 · 2020: 3 · 2019: 2 · 2018: 2 · 2017: 1 · 2016: 6 · 2015: 7 · 2014: 7 · 2013: 18 · 2012: 11
- FWCI
- 3.63
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Licence
- Locations
- Neuron (publishedVersion)
- PubMed (publishedVersion)
- eScholarship (California Digital Library) (submittedVersion, cc-by)
- Funders
- National Institutes of Health (5r21ns045350-02)
- U.S. Food and Drug Administration (BM3355)
- National Institute of Neurological Disorders and Stroke (1R21NS045350, NS045283, R01NS045283)
- Topics
- Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Genetic Neurodegenerative Diseases
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genetics and Neurodevelopmental Disorders
- Health Sciences › Medicine › Neurology › Amyotrophic Lateral Sclerosis Research
- Keywords
- polyglutamine aggregation, protein misfolding, neurodegenerative diseases, polyglutamine diseases, high-throughput screening, FRET assay, Y-27632, Rho kinase, small molecule inhibitors, Drosophila model, neurodegeneration
- MeSH terms
- Huntingtin Protein, Amides, Animals, Biological Assay, Inclusion Bodies, Disease Models, Animal, Drosophila melanogaster, Drug Evaluation, Preclinical, Enzyme Inhibitors, Humans, Nerve Tissue Proteins, Nuclear Proteins, Peptides, Pyridines, Signal Transduction, Down-Regulation, Cell Death, Protein Serine-Threonine Kinases, Protein Folding, Photoreceptor Cells, Invertebrate, COS Cells, Neurodegenerative Diseases, Trinucleotide Repeat Expansion, Animals, Genetically Modified, Fluorescence Resonance Energy Transfer, Intracellular Signaling Peptides and Proteins, rho-Associated Kinases
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