title: A screen for enhancers of clearance identifies huntingtin as a heat shock protein 90 (Hsp90) client protein.
abstract: Mechanisms to reduce the cellular levels of mutant huntingtin (mHtt) provide promising strategies for treating Huntington disease (HD). To identify compounds enhancing the degradation of mHtt, we performed a high throughput screen using a hippocampal HN10 cell line expressing a 573-amino acid mHtt fragment. Several hit structures were identified as heat shock protein 90 (Hsp90) inhibitors. Cell treatment with these compounds reduced levels of mHtt without overt toxic effects as measured by time-resolved Förster resonance energy transfer assays and Western blots. To characterize the mechanism of mHtt degradation, we used the potent and selective Hsp90 inhibitor NVP-AUY922. In HdhQ150 embryonic stem (ES) cells and in ES cell-derived neurons, NVP-AUY922 treatment substantially reduced soluble full-length mHtt levels. In HN10 cells, Hsp90 inhibition by NVP-AUY922 enhanced mHtt clearance in the absence of any detectable Hsp70 induction. Furthermore, inhibition of protein synthesis with cycloheximide or overexpression of dominant negative heat shock factor 1 (Hsf1) in HdhQ150 ES cells attenuated Hsp70 induction but did not affect NVP-AUY922-mediated mHtt clearance. Together, these data provided evidence that direct inhibition of Hsp90 chaperone function was crucial for mHtt degradation rather than heat shock response induction and Hsp70 up-regulation. Co-immunoprecipitation experiments revealed a physical interaction of mutant and wild-type Htt with the Hsp90 chaperone. Hsp90 inhibition disrupted the interaction and induced clearance of Htt through the ubiquitin-proteasome system. Our data suggest that Htt is an Hsp90 client protein and that Hsp90 inhibition may provide a means to reduce mHtt in HD.
authors: Neuroscience Pathway, Novartis Institutes for BioMedical Research, Novartis Pharma AG, CH-4002 Basel, Switzerland.
authors: Barbara
authors: B
authors: Baldo
authors: Andreas
authors: A
authors: Weiss
authors: Christian N
authors: CN
authors: Parker
authors: Miriam
authors: M
authors: Bibel
authors: Paolo
authors: P
authors: Paganetti
authors: Klemens
authors: K
authors: Kaupmann
mesh_terms: D000818:Animals; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics; D057809:HEK293 Cells; D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000076249:Heat Shock Transcription Factors; D006624:Hippocampus / Q000378:metabolism*; D006801:Humans; D000071058:Huntingtin Protein; D007555:Isoxazoles / Q000494:pharmacology; D051379:Mice; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000235:genetics / Q000378:metabolism; D059748:Proteolysis; D012118:Resorcinols / Q000494:pharmacology; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism
chemical_list: C528044:5-(2,4-dihydroxy-5-isopropylphenyl)-4-(4-morpholin-4-ylmethylphenyl)isoxazole-3-carboxylic acid ethylamide; D004268:DNA-Binding Proteins; C000619706:HSF1 protein, human; D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; C086055:HTT protein, human; D000076249:Heat Shock Transcription Factors; C490017:Hsf1 protein, mouse; C506812:Htt protein, mouse; D000071058:Huntingtin Protein; D007555:Isoxazoles; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D012118:Resorcinols; D014157:Transcription Factors; D025801:Ubiquitin; D046988:Proteasome Endopeptidase Complex