TY - JOUR
T1 - A rapid cellular FRET assay of polyglutamine aggregation identifies a novel inhibitor
AU - Pollitt, Sonia K.
AU - Pallos, Judit
AU - Shao, Jieya
AU - Desai, Urvee A.
AU - Ma, Aye Aye K.
AU - Thompson, Leslie Michels
AU - Marsh, J. Lawrence
AU - Diamond, Marc I.
N1 - Funding Information:
Fred Schaufele provided invaluable assistance performing FRET measurements in C17-2 cells. Brent Stockwell kindly provided the Annotated Compound Library. The authors also wish to thank the following individuals for critical review of the manuscript: Henry Bourne, Ivan Diamond, William Welch, Huda Zoghbi, Diane Merry, J. Paul Taylor, and George Jackson. Fei Wang kindly provided chemicals used in the limited chemical screen. This work was supported by a Cure HD Initiative of the Hereditary Disease Foundation (L.M.T. and J.L.M.), Coalition for the Cure of the Huntington's Disease Society of America (L.M.T.), NINDS award NS045283 (J.L.M.), NINDS award 1R21NS045350-01 (M.I.D.), the Muscular Dystrophy Association (M.I.D.), the Sandler Family Supporting Foundation (M.I.D.), and NIH Grant BM3355 (J.P.). This work was also made possible through access to the National Drosophila Stock Center in Bloomington, IN.
PY - 2003/11/13
Y1 - 2003/11/13
N2 - 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 (EC50 ≅ 5 μM) 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.
AB - 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 (EC50 ≅ 5 μM) 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.
UR - http://www.scopus.com/inward/record.url?scp=0242657586&partnerID=8YFLogxK
U2 - 10.1016/S0896-6273(03)00697-4
DO - 10.1016/S0896-6273(03)00697-4
M3 - Article
C2 - 14622574
AN - SCOPUS:0242657586
SN - 0896-6273
VL - 40
SP - 685
EP - 694
JO - Neuron
JF - Neuron
IS - 4
ER -