A Rapid Cellular FRET Assay of Polyglutamine Aggregation Identifies a Novel Inhibitor

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.

[1]  A. Hall,et al.  Rho GTPases in cell biology , 2002, Nature.

[2]  M. Sherman,et al.  Intracellular Aggregation of Polypeptides with Expanded Polyglutamine Domain Is Stimulated by Stress-Activated Kinase Mekk1 , 2001, The Journal of cell biology.

[3]  P. Peters,et al.  Arfaptin 2 regulates the aggregation of mutant huntingtin protein , 2002, Nature Cell Biology.

[4]  Richard I. Morimoto,et al.  Polyglutamine protein aggregates are dynamic , 2002, Nature Cell Biology.

[5]  Claire-Anne Gutekunst,et al.  A YAC Mouse Model for Huntington’s Disease with Full-Length Mutant Huntingtin, Cytoplasmic Toxicity, and Selective Striatal Neurodegeneration , 1999, Neuron.

[6]  B. Stockwell,et al.  Biological mechanism profiling using an annotated compound library. , 2003, Chemistry & biology.

[7]  J. Walcott,et al.  Expression of expanded repeat androgen receptor produces neurologic disease in transgenic mice. , 2001, Human molecular genetics.

[8]  S. Narumiya,et al.  Use and properties of ROCK-specific inhibitor Y-27632. , 2000, Methods in enzymology.

[9]  W. Kennedy,et al.  Progressive proximal spinal and bulbar muscular atrophy of late onset , 1998, Neurology.

[10]  W. Welch,et al.  Glucocorticoid modulation of androgen receptor nuclear aggregation and cellular toxicity is associated with distinct forms of soluble expanded polyglutamine protein. , 2001, Human molecular genetics.

[11]  H. Zoghbi,et al.  Interaction of Akt-Phosphorylated Ataxin-1 with 14-3-3 Mediates Neurodegeneration in Spinocerebellar Ataxia Type 1 , 2003, Cell.

[12]  M Alter,et al.  Progressive proximal spinal and bulbar muscular atrophy of late onset. A sex-linked recessive trait. , 1968, Neurology.

[13]  S. Narumiya,et al.  Molecular Dissection of the Rho-associated Protein Kinase (p160ROCK)-regulated Neurite Remodeling in Neuroblastoma N1E-115 Cells , 1998, The Journal of cell biology.

[14]  J. Chant,et al.  Rac and Cdc42 Induce Actin Polymerization and G1 Cell Cycle Progression Independently of p65PAK and the JNK/SAPK MAP Kinase Cascade , 1996, Cell.

[15]  K. Yamamoto,et al.  Regulation of expanded polyglutamine protein aggregation and nuclear localization by the glucocorticoid receptor. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[16]  Fabrice P Cordelières,et al.  The IGF-1/Akt pathway is neuroprotective in Huntington's disease and involves Huntingtin phosphorylation by Akt. , 2002, Developmental cell.

[17]  A. Hackam,et al.  Kennedy's Disease , 1999, Journal of neurochemistry.

[18]  Ammasi Periasamy,et al.  Fluorescence resonance energy transfer (FRET) microscopy imaging of live cell protein localizations , 2003, The Journal of cell biology.

[19]  H. Lehrach,et al.  Membrane filter assay for detection of amyloid-like polyglutamine-containing protein aggregates. , 1999, Methods in enzymology.

[20]  K. Fujisawa,et al.  p160ROCK, a Rho‐associated coiled‐coil forming protein kinase, works downstream of Rho and induces focal adhesions , 1997, FEBS letters.

[21]  S. Narumiya,et al.  Pharmacological properties of Y-27632, a specific inhibitor of rho-associated kinases. , 2000, Molecular pharmacology.

[22]  E. Sztul,et al.  Characterization and Dynamics of Aggresome Formation by a Cytosolic Gfp-Chimera✪ , 1999, The Journal of cell biology.

[23]  R. Wetzel,et al.  A microtiter plate assay for polyglutamine aggregate extension. , 2001, Analytical biochemistry.

[24]  K. Fischbeck,et al.  Intranuclear Inclusions of Expanded Polyglutamine Protein in Spinocerebellar Ataxia Type 3 , 1997, Neuron.

[25]  S. W. Davies,et al.  Aggregation of huntingtin in neuronal intranuclear inclusions and dystrophic neurites in brain. , 1997, Science.

[26]  R. Kopito,et al.  Aggresomes: A Cellular Response to Misfolded Proteins , 1998, The Journal of cell biology.

[27]  Manish S. Shah,et al.  A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes , 1993, Cell.

[28]  R. Day,et al.  Conformation of CCAAT/Enhancer-binding Protein α Dimers Varies with Intranuclear Location in Living Cells* , 2003, The Journal of Biological Chemistry.

[29]  D. Housman,et al.  Histone deacetylase inhibitors arrest polyglutamine-dependent neurodegeneration in Drosophila , 2001, Nature.

[30]  Fumiaki Tanaka,et al.  Aggresomes protect cells by enhancing the degradation of toxic polyglutamine-containing protein. , 2003, Human molecular genetics.

[31]  C. Cepko,et al.  Multipotent neural cell lines can engraft and participate in development of mouse cerebellum , 1992, Cell.

[32]  K. Fischbeck,et al.  Androgen receptor gene mutations in X-linked spinal and bulbar muscular atrophy , 1991, Nature.

[33]  D. Rubinsztein,et al.  Effects of heat shock, heat shock protein 40 (HDJ-2), and proteasome inhibition on protein aggregation in cellular models of Huntington's disease. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[34]  J. Baxter,et al.  Ligand-selective interactions of ER detected in living cells by fluorescence resonance energy transfer. , 2002, Molecular endocrinology.

[35]  Fumiaki Tanaka,et al.  Nuclear inclusions of the androgen receptor protein in spinal and bulbar muscular atrophy , 1998, Annals of neurology.

[36]  W. Welch,et al.  Polyglutamine protein aggregation and toxicity are linked to the cellular stress response. , 2003, Human molecular genetics.

[37]  J H Zhang,et al.  Confirmation of primary active substances from high throughput screening of chemical and biological populations: a statistical approach and practical considerations. , 2000, Journal of combinatorial chemistry.

[38]  H. Lehrach,et al.  Huntingtin aggregation monitored by dynamic light scattering. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[39]  Effat S. Emamian,et al.  Serine 776 of Ataxin-1 Is Critical for Polyglutamine-Induced Disease in SCA1 Transgenic Mice , 2003, Neuron.

[40]  S. Fields,et al.  Requirement of an intact microtubule cytoskeleton for aggregation and inclusion body formation by a mutant huntingtin fragment , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[41]  K. Fischbeck,et al.  Cleavage, aggregation and toxicity of the expanded androgen receptor in spinal and bulbar muscular atrophy. , 1998, Human molecular genetics.