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• W1978577114 abstract "Several neurodegenerative diseases, including Huntington disease (HD), are associated with aberrant folding and aggregation of polyglutamine (polyQ) expansion proteins. Here we established the zebrafish, Danio rerio, as a vertebrate HD model permitting the screening for chemical suppressors of polyQ aggregation and toxicity. Upon expression in zebrafish embryos, polyQ-expanded fragments of huntingtin (htt) accumulated in large SDS-insoluble inclusions, reproducing a key feature of HD pathology. Real time monitoring of inclusion formation in the living zebrafish indicated that inclusions grow by rapid incorporation of soluble htt species. Expression of mutant htt increased the frequency of embryos with abnormal morphology and the occurrence of apoptosis. Strikingly, apoptotic cells were largely devoid of visible aggregates, suggesting that soluble oligomeric precursors may instead be responsible for toxicity. As in nonvertebrate polyQ disease models, the molecular chaperones, Hsp40 and Hsp70, suppressed both polyQ aggregation and toxicity. Using the newly established zebrafish model, two compounds of the N′-benzylidene-benzohydrazide class directed against mammalian prion proved to be potent inhibitors of polyQ aggregation, consistent with a common structural mechanism of aggregation for prion and polyQ disease proteins. Several neurodegenerative diseases, including Huntington disease (HD), are associated with aberrant folding and aggregation of polyglutamine (polyQ) expansion proteins. Here we established the zebrafish, Danio rerio, as a vertebrate HD model permitting the screening for chemical suppressors of polyQ aggregation and toxicity. Upon expression in zebrafish embryos, polyQ-expanded fragments of huntingtin (htt) accumulated in large SDS-insoluble inclusions, reproducing a key feature of HD pathology. Real time monitoring of inclusion formation in the living zebrafish indicated that inclusions grow by rapid incorporation of soluble htt species. Expression of mutant htt increased the frequency of embryos with abnormal morphology and the occurrence of apoptosis. Strikingly, apoptotic cells were largely devoid of visible aggregates, suggesting that soluble oligomeric precursors may instead be responsible for toxicity. As in nonvertebrate polyQ disease models, the molecular chaperones, Hsp40 and Hsp70, suppressed both polyQ aggregation and toxicity. Using the newly established zebrafish model, two compounds of the N′-benzylidene-benzohydrazide class directed against mammalian prion proved to be potent inhibitors of polyQ aggregation, consistent with a common structural mechanism of aggregation for prion and polyQ disease proteins. A common feature of neurodegenerative diseases, such as Parkinson disease, Alzheimer disease, and polyglutamine (polyQ) 3The abbreviations used are: polyQ, polyglutamine; HD, Huntington disease; NBB, N′-benzylidene-benzohydrazide; htt, huntingtin; GFP, green fluorescent protein; YFP, yellow fluorescent protein; HA, hemagglutinin. diseases, including Huntington disease (HD), is the accumulation and deposition of characteristic amyloid or amyloid-like fibrils of the respective disease proteins in the brain (1Ross C.A. Poirier M.A. Nat. Rev. Mol. Cell. Biol. 2005; 6: 891-898Crossref PubMed Scopus (515) Google Scholar, 2Aguzzi A. Haass C. Science. 2003; 302: 814-818Crossref PubMed Scopus (203) Google Scholar). In HD, it is the intracellular deposition of inclusion bodies that is associated with the degeneration of neurons, primarily in the striatum region of the brain. These inclusion bodies contain fibrillar aggregates of the disease protein, huntingtin (htt), and likely represent the final manifestation of a multi-step aggregation pathway involving several intermediate species (3Poirier M.A. Li H. Macosko J. Cai S. Amzel M. Ross C.A. J. Biol. Chem. 2002; 277: 41032-41037Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar). The pathogenic length of the polyQ stretch within the N-terminal segment of htt is ∼38Q or greater, and correlates with its ability to form detergent-insoluble fibrillar aggregates (4Davies S.W. Turmaine M. Cozens B.A. DiFiglia M. Sharp A.H. Ross C.A. Scherzinger E. Wanker E.E. Mangiarini L. Bates G.P. Cell. 1997; 90: 537-548Abstract Full Text Full Text PDF PubMed Scopus (1918) Google Scholar, 5DiFiglia M. Sapp E. Chase K.O. Davies S.W. Bates G.P. Vonsattel J.P. Aronin N. Science. 1997; 277: 1990-1993Crossref PubMed Scopus (2331) Google Scholar, 6Scherzinger E. Lurz R. Turmaine M. Mangiarini L. Hollenbach B. Hasenbank R. Bates G.P. Davies S.W. Lehrach H. Wanker E.E. Cell. 1997; 90: 549-558Abstract Full Text Full Text PDF PubMed Scopus (1090) Google Scholar). N-terminal fragments containing the pathogenic polyQ stretch have been detected in inclusions in HD brain tissue, and their generation is considered to be a critical step in both fibril formation and toxicity (4Davies S.W. Turmaine M. Cozens B.A. DiFiglia M. Sharp A.H. Ross C.A. Scherzinger E. Wanker E.E. Mangiarini L. Bates G.P. Cell. 1997; 90: 537-548Abstract Full Text Full Text PDF PubMed Scopus (1918) Google Scholar, 7Lunkes A. Lindenberg K.S. Ben-Haiem L. Weber C. Devys D. Land-wehrmeyer G.B. Mandel J.L. Trottier Y. Mol. Cell. 2002; 10: 259-269Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar). Molecular dynamics simulations of polyQ fragments suggest that the core structure of a htt fibril is composed of peptides arranged in a triangular β-helical structure (8Stork M. Giese A. Kretzschmar H.A. Tavan P. Biophys. J. 2005; 88: 2442-2451Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Such fibrillar structures would grow by the attachment of further coils at the top and bottom ends of the β-helix. Although inclusion bodies are closely associated with neurodegenerative disease, there has been considerable debate about the role of aggregates in pathogenesis. Increasing evidence suggests that inclusion bodies, and the fibrillar aggregates therein, may not represent the primary neurotoxic agent (1Ross C.A. Poirier M.A. Nat. Rev. Mol. Cell. Biol. 2005; 6: 891-898Crossref PubMed Scopus (515) Google Scholar, 9Walsh D.M. Klyubin I. Fadeeva J.V. Cullen W.K. Anwyl R. Wolfe M.S. Rowan M.J. Selkoe D.J. Nature. 2002; 416: 535-539Crossref PubMed Scopus (3721) Google Scholar, 10Cleary J.P. Walsh D.M. Hofmeister J.J. Shankar G.M. Kuskowski M.A. Selkoe D.J. Ashe K.H. Nat. Neurosci. 2005; 8: 79-84Crossref PubMed Scopus (1500) Google Scholar, 11Arrasate M. Mitra S. Schweitzer E.S. Segal M.R. Finkbeiner S. Nature. 2004; 431: 805-810Crossref PubMed Scopus (1627) Google Scholar). Instead, soluble intermediates in the aggregation pathway are likely to be the key toxic species, giving rise to neurodegeneration through various mechanisms including inactivation of essential transcription factors and inhibition of the ubiquitin proteasome system (12Schaffar G. Breuer P. Boteva R. Behrends C. Tzvetkov N. Strippel N. Sakahira H. Siegers K. Hayer-Hartl M. Hartl F.U. Mol. Cell. 2004; 15: 95-105Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar, 13Bennett E.J. Bence N.F. Jayakumar R. Kopito R.R. Mol. Cell. 2005; 17: 351-365Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar). Much attention has therefore been focused on identifying means to disrupt or alter the polyQ aggregation pathway (14Di Prospero N.A. Fischbeck K.H. Nat. Rev. Genet. 2005; 6: 756-765Crossref PubMed Scopus (125) Google Scholar), and several compounds with anti-aggregation activity have been identified in vitro and in cell-based assays (15Heiser V. Engemann S. Brocker W. Dunkel I. Boeddrich A. Waelter S. Nordhoff E. Lurz R. Schugardt N. Rautenberg S. Herhaus C. Barnickel G. Bottcher H. Lehrach H. Wanker E.E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16400-16406Crossref PubMed Scopus (196) Google Scholar, 16Hockly E. Tse J. Barker A.L. Moolman D.L. Beunard J.L. Revington A.P. Holt K. Sunshine S. Moffitt H. Sathasivam K. Woodman B. Wanker E.E. Lowden P.A. Bates G.P. Neurobiol. Dis. 2006; 21: 228-236Crossref PubMed Scopus (74) Google Scholar). We set out to develop a zebrafish HD model system suitable for whole organism validation of candidate compounds identified as aggregation inhibitors in vitro. The zebrafish has several key advantages over other in vivo models of HD. It is a vertebrate organism and contains homologues to many human genes, including the htt gene (17Karlovich C.A. John R.M. Ramirez L. Stainier D.Y. Myers R.M. Gene (Amst.). 1998; 217: 117-125Crossref PubMed Scopus (71) Google Scholar), making it a valuable tool to model human diseases such as HD. Additional important advantages include: the ease and cost efficiency with which in vivo drug tests can be performed; the transparency of zebrafish embryos allowing for visualization of morphological and physiological features in the live embryo; and the aqueous environment of the zebrafish, which facilitates drug administration (18Zon L.I. Peterson R.T. Nat. Rev. Drug Discov. 2005; 4: 35-44Crossref PubMed Scopus (1118) Google Scholar). Importantly, the zebrafish model system allows for the evaluation of drug effectiveness in a whole organism, taking into account the stability and cellular targeting of the tested compound, as well as the assessment of potential side effects of the drug at active concentrations. Here, we describe the characterization of a newly established zebrafish model of HD. Mutant htt expressed in zebrafish embryos accumulated in large SDS-insoluble inclusions, recapitulating a cardinal feature of HD pathology. Mutant htt also increased the frequency of embryos with abnormal morphology and dead embryos, as well as the occurrence of apoptosis. As in nonvertebrate polyQ disease models, the molecular chaperones, Hsp40 and Hsp70, suppressed both polyQ aggregation and toxicity. Using this zebrafish model, two anti-prion compounds of the N′-benzylidene-benzohydrazide class were identified as novel inhibitors of polyQ aggregation, suggesting that polyQ aggregates and prions may share common structural epitopes. We also address, for the first time in a vertebrate model system, the controversial question of whether inclusion bodies of polyQ-expanded htt directly contribute to toxicity. Our observation that the majority of apoptotic cells were devoid of visible inclusions suggests that polyQ toxicity is not primarily caused by insoluble aggregates but is more likely caused by soluble aggregation intermediates. Animal Husbandry—All of the experiments were performed in compliance with the guidelines of the German Council on Animal Care. The zebrafish were maintained, mated, and raised as described (19Mullins M.C. Hammerschmidt M. Haffter P. Nusslein-Volhard C. Curr. Biol. 1994; 4: 189-202Abstract Full Text Full Text PDF PubMed Scopus (631) Google Scholar). The embryos were kept at 28 °C and staged as described (20Kimmel C.B. Ballard W.W. Kimmel S.R. Ullmann B. Schilling T.F. Dev. Dyn. 1995; 203: 253-310Crossref PubMed Scopus (8862) Google Scholar). The wild type line AB was used for all experiments. Constructs and Embryo Injection—pCS2+Q25GFP and pCS2+Q102GFP were generated by subcloning the inserts from pYES2-htt(25 or 102Q)-GFP (21Meriin A.B. Zhang X. He X. Newnam G.P. Chernoff Y.O. Sherman M.Y. J. Cell Biol. 2002; 157: 997-1004Crossref PubMed Scopus (303) Google Scholar) into the AflII and XbaI sites of the pCS2+-AflII vector. PCR mutagenesis of pCS2+Q25GFP was used to generate a Q4GFP fragment, which was cloned into NdeI and BstAPI cut pCS2+Q25GFP backbone. To generate pCS2+Hsp70-YFP, an Hsp70-YFP fragment derived from pEYFP-N1-Hsp70 (22Kim S. Nollen E.A. Kitagawa K. Bindokas V.P. Morimoto R.I. Nat. Cell Biol. 2002; 4: 826-831Crossref PubMed Scopus (252) Google Scholar) was cloned into EcoRI, XbaI cut pCS2+. The construct used for transcription of human Hdj1 (Hsp40) was a kind gift from A. Haacke; the β-galactoside construct was purchased from Invitrogen. In vitro transcription of all of these constructs was performed with the MessageMachine Kit (Ambion, TX), and the resulting mRNA was injected at a concentration of 1 μg/μl into the yolk of embryos at the first or second cell stage. Co-injections were performed with 1 μg/μl polyQ-GFP mRNA and either 1 μg/μl β-Gal mRNA as a control or 0.5 μg/μl Hdj1 and 0.5 μg/μl Hsp70-YFP mRNA. The embryos were cultured at 28.5 °C in E3 (5 mm NaCl, 0.17 mm KCl, 0.33 mm CaCl2 0.33 mm MgSO4, and 0.1% methylene blue), the unfertilized eggs were removed, and the embryos were scored 24 h post-fertilization according to their appearance. Microscopy—Injected zebrafish embryos were dechorionated, anesthetized with Tricaine (0.016% w/v), and orientated in 3% methyl cellulose on coverslips. Fluorescence was visualized with an LSM510 META inverted confocal microscope (Zeiss, Oberkochen, Germany). The images were captured digitally with a Zeiss MRM AxioCam camera and assembled in Photoshop 8.0 (Adobe Systems, Mountain View, CA). For nuclear staining, the embryos were immersed in 1:1000 dilution of TOPRO-3 stain (monomeric cyanine nucleic acid stain; Molecular Probes) for 30 min at room temperature and washed three times with E3. To assess apoptotic cell death, the embryos were additionally immersed in 3 μg/ml acridine orange (acridinium chloride hemi[zinc chloride]; Molecular Probes) for 15 min and washed three times with E3. For three-dimensional imaging, a stack of 10 frames was collected at intervals of 10-24 μm between adjacent slices, and the images were assembled to obtain a three-dimensional projection. To dissociate the GFP/acridine orange or GFP/YFP fluorescence signals, analysis was performed by emission fingerprinting in confocal lambda mode. A stack of two or three frames of the embryonic tail region 24 h post-fertilization was collected at intervals of 20 μm, and the number of acridine orange-stained cells was counted to estimate the amount of apoptotic cells. For the time lapse analysis of the living cells, zebrafish embryos 24 h post-fertilization were orientated in 1.2% low melting agarose (1.2% in E3 with Tricaine 0.09% w/v) and covered with E3. Images taken 24, 28, and 32 h post-fertilization were corrected for photobleaching effects, which reduced the signal ∼22.4% over the course of the experiment. Cell Fractionation, Western Blot Analysis, and the Filter Trap Assay—Injected embryos were resuspended in cold lysis buffer (0.5% Triton X-100, 0.5% sodium deoxycholate, and protease inhibitor mixture (Roche Applied Science) in phosphate-buffered saline) and lysed by passing through a 0.6-mm needle. After freezing and thawing the extracts, benzonase was added (75 units), and the extracts were incubated at 4 °C for 1 h with gentle agitation. The extracts were centrifuged at 2,000 × g for 3 min at 4 °C in the presence of glass beads to facilitate separation of the chorion debris. The supernatant was collected, and protein concentration was determined with a protein assay kit (Bio-Rad). For the detection of Hsp70-YFP (and Q102-GFP in Fig. 4b), the embryos were first dechorionated, and yolk was dissected, as previously described (23Evans T.G. Yamamoto Y. Jeffery W.R. Krone P.H. Cell Stress Chaperones. 2005; 10: 66-78Crossref PubMed Scopus (55) Google Scholar). For subcellular fractionation, the extracts were centrifuged at 100,000 × g for 30 min to obtain the supernatant and pellet fractions. The pellet was washed once with lysis buffer, centrifuged at 100,000 × g for 15 min, and either dissolved in SDS sample buffer or 100% formic acid. The latter was incubated for 3 h at 37°C with agitation, vacuum-dried, and resuspended in SDS sample buffer, as previously reported (24Hazeki N. Tukamoto T. Goto J. Kanazawa I. Biochem. Biophys. Res. Commun. 2000; 277: 386-393Crossref PubMed Scopus (80) Google Scholar). The fractions were heated to 95 °C for 5 min and analyzed using SDS-PAGE and Western blotting (anti-GFP, anti-Hsp40, or anti-β-galactosidase), according to the standard procedure. For the filter trap assay, an equal volume of 4% SDS, 100 mm dithiothreitol was added to embryo extracts, and the mixture was heated for 5 min at 95 °C. Several dilutions of between 25 and 1000 μg were filtered through a cellulose acetate membrane (pore size, 0.2 μm), according to protocol (6Scherzinger E. Lurz R. Turmaine M. Mangiarini L. Hollenbach B. Hasenbank R. Bates G.P. Davies S.W. Lehrach H. Wanker E.E. Cell. 1997; 90: 549-558Abstract Full Text Full Text PDF PubMed Scopus (1090) Google Scholar). Aggregates retained on the filter were detected with an anti-GFP antibody. In all cases, the extracts were analyzed in parallel via SDS-PAGE and Western analysis to ensure equal expression levels among all conditions. For quantitating the results of Western blotting or the filter trap assay, an LAS-3000 imager (Fujifilm Image Reader) was used. Chemical Treatments—Chemicals from the DIVERSet library (313B02, 293G02, and 306H03) were obtained from ChemBridge Corporation (San Diego, CA). Isoproterenol hydrochloride was dissolved in H2O, and the other compounds were dissolved in Me2SO, at the indicated concentrations. After the injection, the embryos were randomly split into two groups; one group was transferred into E3 supplemented with 1% Me2SO and the indicated concentration of compound, and the other group was transferred into E3 supplemented with 1% Me2SO as a control. The chorion was carefully disrupted using fine forceps to facilitate penetration of the compound through the chorion, and unfertilized eggs were removed several hours post-fertilization. The embryos were cultured at 28.5 °C for 24 h post-fertilization, and extracts were prepared and analyzed as described above. The filter trap assay signal associated with extracts derived from Me2SO- or H2O-treated embryos was set to 100%. In Vitro Aggregation Reactions—In vitro aggregation reactions were performed essentially as described (12Schaffar G. Breuer P. Boteva R. Behrends C. Tzvetkov N. Strippel N. Sakahira H. Siegers K. Hayer-Hartl M. Hartl F.U. Mol. Cell. 2004; 15: 95-105Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar). Briefly, purified glutathione S-transferase-htt exon 1 fusions (3 μm) were mixed with 3 or 30 μm Congo red, 313B02 (3Poirier M.A. Li H. Macosko J. Cai S. Amzel M. Ross C.A. J. Biol. Chem. 2002; 277: 41032-41037Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar), 293G02 (4Davies S.W. Turmaine M. Cozens B.A. DiFiglia M. Sharp A.H. Ross C.A. Scherzinger E. Wanker E.E. Mangiarini L. Bates G.P. Cell. 1997; 90: 537-548Abstract Full Text Full Text PDF PubMed Scopus (1918) Google Scholar), 306H03 (5DiFiglia M. Sapp E. Chase K.O. Davies S.W. Bates G.P. Vonsattel J.P. Aronin N. Science. 1997; 277: 1990-1993Crossref PubMed Scopus (2331) Google Scholar), or Me2SO. To initiate the aggregation reaction, 2.5 units of PreScission Protease in buffer A (50 mm Tris-HCl, pH 7.4; 150 mm KCl) was added. The reactions were incubated at 30 °C for 8 h and then stopped by adding an equal volume of 4% SDS, 100 mm dithiothreitol. The amount of SDS-insoluble aggregates per reaction was analyzed using the filter trap assay (anti-HA), as described above. Statistical Analyses—The data were compared using Bonferroni's post hoc test and one-way analysis of variance (α-level was set to 0.05), as indicated. Docking of Compounds to a Triangular PolyQ Dimer—Using the program AutoDock Version 3.0 (25Morris G.M. Goodsell D.S. Halliday R.S. Huey R. Hart W.E. Belew R.K. Olson A.J. J. Comp. Chem. 1998; 19: 1639-1662Crossref Scopus (9301) Google Scholar), we simulated the docking of models for the examined compounds (293G02, 306H03, 313B02, isoproterenol, mycophenolic acid, PGL-135, and Congo red) to a specific β-helical model for a polyQ proto-fibril. The structural model of a polyQ protofibril was adopted from the work of Stork et al. (8Stork M. Giese A. Kretzschmar H.A. Tavan P. Biophys. J. 2005; 88: 2442-2451Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar), who had suggested a left-handed triangular β-helix as the secondary structure motif for polyQ amyloid fibers. More specifically, a dimer of two β-helical polyQ peptides, each covering 36 residues, was selected. Five structural snapshots of the dimer solvated in water were taken at a temporal distance of 50 ps from the molecular dynamics trajectory, which served for equilibration of the modeled dimer structure with its aqueous environment at room temperature and ambient pressure in the quoted paper. The polyQ dimer snapshots were prepared for the AutoDock simulations using respective AutoDockTools, which assigned partial charges and solvation parameters to the atoms of the receptor peptides. Structural models for the ligand molecules were generated, and partial charges were assigned using the quantum chemistry program MNDO (26Dewar M.J.S. Thiel W. J. Am. Chem. Soc. 1977; 99: 4899-4907Crossref Scopus (6024) Google Scholar). Rotatable bonds within the ligands were defined through AutoDockTools. AutoDock applies an inter-polation scheme to evaluate the energy of a ligand in the vicinity of a receptor using grid maps that are precalculated over the receptor for each atom type in the ligand. We calculated the grid maps with 127 × 127 × 127 points and a grid spacing of 0.375 Å centered at the polyQ dimers using the AutoGrid tool within AutoDockTools. For each of the five polyQ dimer snapshots, we simulated the docking of the seven compounds using the Lamarckian Genetic Algorithm available in AutoDock. For each receptor-ligand pair, 100 separate dockings were performed and sorted by ascending free energy. The 20 docking structures of lowest free energy were visualized for each ligand using the software VMD (27Humphrey W. Dalke A. Schulten K. J. Mol. Graph. 1996; 14: 33-38Crossref PubMed Scopus (39882) Google Scholar). PolyQ-expanded htt Is Deposited into Inclusions of SDS-insoluble Aggregates in Danio rerio—We first evaluated whether we could reproduce key features of HD in zebrafish. Embryos at the first or second cell stage were injected with mRNA encoding an N-terminal fragment of htt containing 4, 25, or 102 Q fused to green fluorescent protein (Q4-GFP, Q25-GFP, and Q102-GFP, respectively). Q4-GFP and Q25-GFP fusion proteins were diffusely distributed, whereas the mutant polyQ-expanded htt fusion protein, Q102-GFP, accumulated in inclusion body-like aggregates throughout most embryos after 24 h (Fig. 1A). Higher magnification images of the hatching gland indicated that Q102-GFP, but not the shorter polyQ variants, accumulated in large, cytoplasmic inclusions adjacent to the nuclei (Fig. 1, B and C). Analysis of embryos at later time points using time lapse microscopy showed that Q102-GFP inclusions grew in size by efficient incorporation of soluble Q102-GFP, resulting in its depletion from the cytoplasm (Fig. 1D and supplemental Video S1). When total cellular extracts of injected embryos 24 h post-fertilization were subjected to high speed centrifugation, both Q4- and Q25-GFP remained exclusively in the soluble supernatant fraction, whereas a significant portion of Q102-GFP was found in the SDS-insoluble pellet fraction and could only be dissolved by formic acid treatment (Fig. 2A) (24Hazeki N. Tukamoto T. Goto J. Kanazawa I. Biochem. Biophys. Res. Commun. 2000; 277: 386-393Crossref PubMed Scopus (80) Google Scholar). Analysis of total embryo extracts by an established filter trap assay (6Scherzinger E. Lurz R. Turmaine M. Mangiarini L. Hollenbach B. Hasenbank R. Bates G.P. Davies S.W. Lehrach H. Wanker E.E. Cell. 1997; 90: 549-558Abstract Full Text Full Text PDF PubMed Scopus (1090) Google Scholar) confirmed that Q102-GFP, but not Q4-GFP or Q25-GFP, formed large SDS-insoluble aggregates that were retained by the 200-nm poresized membrane (Fig. 2B).FIGURE 2Q102-GFP forms SDS-insoluble aggregates in D. rerio. A, analysis of the solubility of the polyQ-GFP fusions. Extracts from embryos (24 h post-fertilization) expressing Q4-GFP, Q25-GFP, or Q102-GFP were subjected to high speed centrifugation, and the resulting fractions were analyzed by SDS-PAGE and Western blotting with anti-GFP antibody. T, total cell lysate; S, supernatant; P, pellet fraction dissolved in SDS; FA, pellet fraction dissolved in formic acid. Asterisk, free GFP cleaved from the fusion protein. B, filter trap analysis of polyQ-GFP fusions. Increasing amounts (25, 50, and 100 μg) of total cell extracts from A were analyzed by filter trap assay in the presence (bottom row) and absence (top row) of SDS/dithiothreitol at 95 °C.View Large Image Figure ViewerDownload Hi-res image Download (PPT) PolyQ Length-dependent Toxicity Is Independent of Detectable Inclusions—To determine whether the polyQ-GFP fusion proteins caused toxicity, we evaluated the phenotypes of embryos expressing Q4-, Q25-, or Q102-GFP, 24 h post-fertilization. The embryos were scored as visibly normal, abnormal, or dead. Q102-GFP expression was associated with increased numbers of dead or morphologically abnormal embryos when compared with Q4- or Q25-GFP expression (Fig. 3A). Acridine orange staining of embryos indicated that Q102-GFP expression caused increased apoptosis (Fig. 3B and supplemental Fig. S1). Low levels of apoptosis and abnormal phenotypes in embryos expressing Q4-GFP and Q25-GFP were also observed, which was likely due to background toxicity caused by these fusion proteins (Fig. 3, A and B). Interestingly, further analyses showed that nearly all (∼95%) of the apoptotic cells were devoid of visible inclusions, which must be more than ∼0.5 μmin diameter for detection in our experimental setup. On the other hand, almost all cells containing detectable inclusions were nonapoptotic (∼99%), suggesting that prefibrillar Q102-GFP may instead be the toxic agent (Fig. 3C, top panels). Indeed, apoptotic cells containing diffusely distributed Q102-GFP were detected (Fig. 3C, bottom panels). Thus, our results indicate that expression of polyQ-expanded htt is toxic to the zebrafish embryo, but toxicity is not associated with the presence of visible inclusions. Hsp70 and Hsp40 Suppress Both PolyQ Aggregation and Toxicity—Molecular chaperones are known to modulate both polyQ aggregation and toxicity (28Barral J.M. Broadley S.A. Schaffar G. Hartl F.U. Semin. Cell Dev. Biol. 2004; 15: 17-29Crossref PubMed Scopus (251) Google Scholar). We next evaluated whether we could reproduce this important feature by testing the effect of the human chaperones Hsp70 and Hsp40 on Q102-GFP aggregation and toxicity in the zebrafish embryo. Co-expression of Hsp40 and Hsp70 fused to YFP (Hsp70-YFP) reduced the amount of SDS-insoluble aggregates of Q102-GFP, as judged by the filter trap assay, whereas total protein levels of Q102-GFP were not detectably altered (Fig. 4, A and B). Fluorescence microscopy revealed that Hsp70-YFP associated with inclusions of Q102-GFP (Fig. 4C), suggesting that the chaper-one machinery recognized Q102-GFP as an aberrantly folded species. Expression of Hsp40 and Hsp70-YFP reduced the number of detectable inclusions (∼41.8% of control; data not shown), apoptotic cells (∼80% of control; data not shown), and dead embryos (Fig. 4D) while increasing the number of phenotypically normal embryos expressing Q102-GFP. Thus, aggregation and toxicity associated with Q102-GFP expression in zebrafish is partially suppressed by Hsp40 and Hsp70. Identification of Compounds That Inhibit PolyQ Aggregation—We next tested the effect of several classes of compounds on the aggregation of Q102-GFP in the zebrafish embryo (Fig. 5). First, we evaluated the capacity of known inhibitors of the polyQ aggregation process to suppress Q102-GFP aggregation in this vertebrate system. PGL-135 and Congo red (16Hockly E. Tse J. Barker A.L. Moolman D.L. Beunard J.L. Revington A.P. Holt K. Sunshine S. Moffitt H. Sathasivam K. Woodman B. Wanker E.E. Lowden P.A. Bates G.P. Neurobiol. Dis. 2006; 21: 228-236Crossref PubMed Scopus (74) Google Scholar, 29Sanchez I. Mahlke C. Yuan J. Nature. 2003; 421: 373-379Crossref PubMed Scopus (454) Google Scholar) reduced Q102-GFP aggregation in zebrafish, as judged by the filter trap assay (Fig. 6A). In contrast, isoproterenol HCl did not alter Q102-GFP aggregation in the zebrafish embryo, consistent with a recent study in a cell culture model (30Wang W. Duan W.Z. Igarashi S. Morita H. Nakamura M. Ross C.A. Neurobiol. Dis. 2005; 20: 500-508Crossref PubMed Scopus (39) Google Scholar). Surprisingly, mycophenolic acid, a compound known to suppress htt inclusion formation in cell culture (30Wang W. Duan W.Z. Igarashi S. Morita H. Nakamura M. Ross C.A. Neurobiol. Dis. 2005; 20: 500-508Crossref PubMed Scopus (39) Google Scholar), had no effect on Q102-GFP aggregation in zebrafish at the maximum tolerated dose. Thus, in the case of PGL-135, Congo red, and isoproterenol HCl, our results substantiate observations in other HD models. However, in contrast to data obtained in cell culture (30Wang W. Duan W.Z. Igarashi S. Morita H. Nakamura M. Ross C.A. Neurobiol. Dis. 2005; 20: 500-508Crossref PubMed Scopus (39) Google Scholar), our findings suggest that mycophenolic acid is not an effective inhibitor of polyQ aggregation in the vertebrate system.FIGURE 6N′-benzylidene-benzohydrazides inhibit Q102-GFP aggregation in vitro and in D. rerio. A, effect of the indicated chemical compounds on HA-httEx1Q53 aggregation in vitro, as assessed by the filter trap assay (anti-HA) with quantitation. The signal intensity from the control sample containing solvent alone was set to 100%. The data are the mean values of three experiments ± S.D. B, filter trap analysis of extracts derived from embryos expressing Q102-GFP in the presence and absence of the indicated compounds. Embryos expressing Q102-GFP" @default.
• W1978577114 created "2016-06-24" @default.
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• W1978577114 date "2007-03-01" @default.
• W1978577114 modified "2023-10-18" @default.
• W1978577114 title "Identification of Anti-prion Compounds as Efficient Inhibitors of Polyglutamine Protein Aggregation in a Zebrafish Model" @default.
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