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• W2058182560 abstract "Huntington disease (HD) is caused by an expansion of more than 35–40 polyglutamine (polyQ) repeats in the huntingtin (htt) protein, resulting in accumulation of inclusion bodies containing fibrillar deposits of mutant htt fragments. Intriguingly, polyQ length is directly proportional to the propensity for htt to form fibrils and the severity of HD and is inversely correlated with age of onset. Although the structural basis for htt toxicity is unclear, the formation, abundance, and/or persistence of toxic conformers mediating neuronal dysfunction and degeneration in HD must also depend on polyQ length. Here we used atomic force microscopy to demonstrate mutant htt fragments and synthetic polyQ peptides form oligomers in a polyQ length-dependent manner. By time-lapse atomic force microscopy, oligomers form before fibrils, are transient in nature, and are occasionally direct precursors to fibrils. However, the vast majority of fibrils appear to form by monomer addition coinciding with the disappearance of oligomers. Thus, oligomers must undergo a major structural transition preceding fibril formation. In an immortalized striatal cell line and in brain homogenates from a mouse model of HD, a mutant htt fragment formed oligomers in a polyQ length-dependent manner that were similar in size to those formed in vitro, although these structures accumulated over time in vivo. Finally, using immunoelectron microscopy, we detected oligomeric-like structures in human HD brains. These results demonstrate that oligomer formation by a mutant htt fragment is strongly polyQ length-dependent in vitro and in vivo, consistent with a causative role for these structures, or subsets of these structures, in HD pathogenesis. Huntington disease (HD) is caused by an expansion of more than 35–40 polyglutamine (polyQ) repeats in the huntingtin (htt) protein, resulting in accumulation of inclusion bodies containing fibrillar deposits of mutant htt fragments. Intriguingly, polyQ length is directly proportional to the propensity for htt to form fibrils and the severity of HD and is inversely correlated with age of onset. Although the structural basis for htt toxicity is unclear, the formation, abundance, and/or persistence of toxic conformers mediating neuronal dysfunction and degeneration in HD must also depend on polyQ length. Here we used atomic force microscopy to demonstrate mutant htt fragments and synthetic polyQ peptides form oligomers in a polyQ length-dependent manner. By time-lapse atomic force microscopy, oligomers form before fibrils, are transient in nature, and are occasionally direct precursors to fibrils. However, the vast majority of fibrils appear to form by monomer addition coinciding with the disappearance of oligomers. Thus, oligomers must undergo a major structural transition preceding fibril formation. In an immortalized striatal cell line and in brain homogenates from a mouse model of HD, a mutant htt fragment formed oligomers in a polyQ length-dependent manner that were similar in size to those formed in vitro, although these structures accumulated over time in vivo. Finally, using immunoelectron microscopy, we detected oligomeric-like structures in human HD brains. These results demonstrate that oligomer formation by a mutant htt fragment is strongly polyQ length-dependent in vitro and in vivo, consistent with a causative role for these structures, or subsets of these structures, in HD pathogenesis. Huntington disease (HD), 3The abbreviations used are: HDHuntington diseasepolyQpolyglutamineAFMatomic force microscopyDTTdithiothreitolHD20Qmutant htt fragment with 20 glutamine repeatsHD35Qmutant htt fragment with 35 glutamine repeatsHD46Qmutant htt fragment with 46 glutamine repeatsHD53Qmutant htt fragment with 53 glutamine repeatsGSTglutathione S-transferaseAGEagarose gel electrophoresisEMimmunoelectron microscopyGFPgreen fluorescent protein. a fatal neurodegenerative disorder, is caused by an expansion of a polyglutamine (polyQ) repeat in the protein huntingtin (htt) (1The Huntington's Disease Collaborative Research Group Cell. 1993; 72: 971-983Abstract Full Text PDF PubMed Scopus (7118) Google Scholar). This expanded polyQ domain mediates the deposition of cytoplasmic and intranuclear inclusion bodies that contain fibrillar material as determined by electron microscopy of HD brain tissues (2DiFiglia 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). PolyQ expansions are also responsible for a growing number of less common neurodegenerative disorders, such as the spinocerebellar ataxias (3Zoghbi H.Y. Orr H.T. Annu Rev. Neurosci. 2000; 23: 217-247Crossref PubMed Scopus (1109) Google Scholar). One of the most fascinating aspects of polyQ diseases is that the age of onset and severity of disease are tightly correlated with the length of the polyQ expansion. For example, HD repeat lengths of fewer than 35 do not result in disease, 35–39 repeats may or may not cause disease, 40–60 repeats elicits adult onset HD, and more than 60 repeats results in juvenile forms of HD (4Penney Jr., J.B. Vonsattel J.P. MacDonald M.E. Gusella J.F. Myers R.H. Ann. Neurol. 1997; 41: 689-692Crossref PubMed Scopus (535) Google Scholar, 5Snell R.G. MacMillan J.C. Cheadle J.P. Fenton I. Lazarou L.P. Davies P. MacDonald M.E. Gusella J.F. Harper P.S. Shaw D.J. Nat. Genet. 1993; 4: 393-397Crossref PubMed Scopus (607) Google Scholar, 6Tobin A.J. Signer E.R. Trends Cell Biol. 2000; 10: 531-536Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). Huntington disease polyglutamine atomic force microscopy dithiothreitol mutant htt fragment with 20 glutamine repeats mutant htt fragment with 35 glutamine repeats mutant htt fragment with 46 glutamine repeats mutant htt fragment with 53 glutamine repeats glutathione S-transferase agarose gel electrophoresis immunoelectron microscopy green fluorescent protein. Biochemical analyses of purified mutant htt fragments with polyQ repeats longer than 39Q demonstrated the polyQ length-dependent formation of detergent-insoluble protein aggregates characteristic of amyloid fibrils (7Scherzinger 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, 8Scherzinger E. Sittler A. Schweiger K. Heiser V. Lurz R. Hasenbank R. Bates G.P. Lehrach H. Wanker E.E. Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 4604-4609Crossref PubMed Scopus (581) Google Scholar). Synthetic polyQ peptides with expanded polyQ repeats also form amyloid-like fibrils, but surprisingly, in contrast to studies with htt exon 1, synthetic peptides with non-disease-causing polyQ lengths readily aggregated into fibrils (9Chen S. Berthelier V. Hamilton J.B. O'Nuallain B. Wetzel R. Biochemistry. 2002; 41: 7391-7399Crossref PubMed Scopus (282) Google Scholar). In cell culture, mutant htt fragments form intranuclear and cytoplasmic inclusion bodies in a polyQ length-dependent manner (10Lunkes A. Mandel J.L. Hum. Mol. Genet. 1998; 7: 1355-1361Crossref PubMed Scopus (174) Google Scholar, 11Saudou F. Finkbeiner S. Devys D. Greenberg M.E. Cell. 1998; 95: 55-66Abstract Full Text Full Text PDF PubMed Scopus (1371) Google Scholar, 12Hackam A.S. Singaraja R. Wellington C.L. Metzler M. McCutcheon K. Zhang T. Kalchman M. Hayden M.R. J. Cell Biol. 1998; 141: 1097-1105Crossref PubMed Scopus (288) Google Scholar). Thus, polyQ length directly correlates with the kinetics of aggregation into fibrils and formation of inclusion bodies. Mutant htt fragments and other expanded polyQ proteins, as demonstrated with transmission electron microscopy and atomic force microscopy (AFM), also assemble into spherical and annular oligomeric structures similar in size and morphology to those formed by Aβ, α-synuclein, and other proteins that have been implicated in neurodegeneration (13Poirier 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, 14Tanaka M. Morishima I. Akagi T. Hashikawa T. Nukina N. J. Biol. Chem. 2001; 276: 45470-45475Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 15Wacker J.L. Zareie M.H. Fong H. Sarikaya M. Muchowski P.J. Nat. Struct. Mol. Biol. 2004; 11: 1215-1222Crossref PubMed Scopus (249) Google Scholar). However, whether or not such structures form in a polyQ length-dependent manner has not been rigorously established. Although inclusion bodies are a major neuropathological hallmark of HD (3Zoghbi H.Y. Orr H.T. Annu Rev. Neurosci. 2000; 23: 217-247Crossref PubMed Scopus (1109) Google Scholar), whether or not intraneuronal aggregates mediate HD pathogenesis is still not resolved. Considering the direct correlation between mutant htt aggregation and severity of pathogenesis with polyQ length (7Scherzinger 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), aggregation was thought to be causative in neurodegeneration. However, how the misfolded/aggregated form(s) mediates pathogenesis is unclear. Although, the onset of symptoms trails inclusion body formation in a transgenic mouse model of HD (16Davies 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), it might also be a conserved cellular mechanism to protect against diffuse, soluble, toxic forms of mutant htt (11Saudou F. Finkbeiner S. Devys D. Greenberg M.E. Cell. 1998; 95: 55-66Abstract Full Text Full Text PDF PubMed Scopus (1371) Google Scholar, 17Muchowski P.J. Ning K. D'Souza-Schorey C. Fields S. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 727-732Crossref PubMed Scopus (117) Google Scholar, 18Arrasate M. Mitra S. Schweitzer E.S. Segal M.R. Finkbeiner S. Nature. 2004; 431: 805-810Crossref PubMed Scopus (1627) Google Scholar). Indeed, such soluble aggregates that precede symptoms may exist (19Weiss A. Klein C. Woodman B. Sathasivam K. Bibel M. Régulier E. Bates G.P. Paganetti P. J. Neurochem. 2008; 104: 846-858PubMed Google Scholar). In agreement with this hypothesis, a stable, toxic, monomeric β-sheet conformation of a thioredoxin-polyQ fusion protein was recently reported (20Nagai Y. Inui T. Popiel H.A. Fujikake N. Hasegawa K. Urade Y. Goto Y. Naiki H. Toda T. Nat. Struct. Mol. Biol. 2007; 14: 332-340Crossref PubMed Scopus (267) Google Scholar), suggesting that polyQ adopts multiple, distinct conformations that may have toxic consequences even in the absence of higher order aggregation. However, a mutant htt fragment may also misfold into distinct amyloid conformations and, depending on whether the polyQ domain was exposed or buried in a β-sheet, the amyloids can be either toxic or nontoxic, respectively (21Nekooki-Machida Y. Kurosawa M. Nukina N. Ito K. Oda T. Tanaka M. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 9679-9684Crossref PubMed Scopus (175) Google Scholar). Our recent studies used AFM and other techniques to show that an expanded polyQ domain in a mutant htt fragment adopts a stable multitude of monomeric (22Legleiter J. Lotz G.P. Miller J. Ko J. Ng C. Williams G.L. Finkbeiner S. Patterson P.H. Muchowski P.J. J. Biol. Chem. 2009; 284: 21647-21658Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar) and oligomeric 4E. J. Mitchell, G. Lotz, B. Apostol, A. Weiss, G. P. Bates, P. Paganetti, P. J. Muchowski, C. Glabe, and L. M. Thompson, manuscript in preparation. conformations that can readily be discriminated by monoclonal antibodies in vitro and in situ (22Legleiter J. Lotz G.P. Miller J. Ko J. Ng C. Williams G.L. Finkbeiner S. Patterson P.H. Muchowski P.J. J. Biol. Chem. 2009; 284: 21647-21658Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar).4 Because polyQ length only above a critical threshold (35–40Q) causes HD and is correlated to the onset and severity of the disease, the formation, abundance, and/or persistence of toxic conformers that mediate neuronal dysfunction and degeneration in HD must also be polyQ length-dependent. Here, we sought to systematically quantify the effects of polyQ length on the kinetics, morphologies, and abundance of aggregates formed by a mutant htt fragment in comparison to those formed by synthetic peptides. Because traditional light and electron microscopic approaches have important limitations for studying heterogeneous mixtures of htt aggregates, we used AFM to characterize the morphology of the assembly states. AFM is uniquely well suited to study amyloidogenic proteins. It yields three-dimensional surface maps with nanometer resolution in solution in the absence of artifacts from sample processing. Our aim was to determine the precise relationship between aggregate morphology, kinetics of formation, and polyQ length. Glutathione S-transferase (GST)-HD fusion proteins were purified as described (24Muchowski P.J. Schaffar G. Sittler A. Wanker E.E. Hayer-Hartl M.K. Hartl F.U. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 7841-7846Crossref PubMed Scopus (547) Google Scholar). Cleavage of the GST moiety by a PreScission Protease (GE Healthcare) initiates aggregation. Fresh, unfrozen GST-HD fusion protein was used for each experiment. Solutions with all fusion proteins were centrifuged at 20,000 × g for 30 min at 4 °C to remove any preexisting aggregates before the addition of the PreScission protease. All peptides were custom synthesized and obtained from 21st Century Biochemicals (Marlboro, MA). The flanking lysines were included to ensure solubility. Disaggregation and solubilization of polyQ peptides were performed as described (25Chen S. Berthelier V. Yang W. Wetzel R. J. Mol. Biol. 2001; 311: 173-182Crossref PubMed Scopus (282) Google Scholar). Briefly, peptides were dissolved in a 50:50 mixture of trifluoroacetic acid and hexafluoroisopropanol. After vortexing, the solution was incubated at room temperature for 30 min with agitation. The solvent was then evaporated off. The remaining peptide residue was resuspended in water adjusted to pH 3 with trifluoroacetic acid and to a concentration of 0.5 mg/ml. This was in turn diluted into Buffer A (50 mm Tris-HCl, pH 7, 150 mm NaCl). For experiments on monomeric preparations of mutant htt proteins, GST-HD fusion proteins were incubated at 30 and 2 μm with 1 mm dithiothreitol (DTT) or at 60 μm without DTT in buffer A (50 mm Tris-HCl, pH 7, 150 mm NaCl). PreScission protease (4 units of 100 μg of fusion protein) was added at time 0 to initiate GST cleavage and aggregation. Samples were incubated at 37 °C and 1400 rpm for the duration of the experiment. At 1, 3, 5, 8, and 24 h after cleavage of the GST, a sample (5 μl) of each incubation solution was deposited on freshly cleaved mica (Ted Pella Inc., Redding, CA) and allowed to sit for 30 s. Then the substrate was washed with 200 μl of ultrapure water and dried under a gentle steam of air. For experiments on preformed fibrils, 40 μm solutions of HD53Q were incubated without DTT for 5–6 h after the removal of the GST tag to allow the formation of fibrils. Each sample was imaged ex situ using a MFP3D scanning probe microscope (Asylum Research, Santa Barbara, CA). Images were taken with silicon cantilever with a nominal spring constant of 40 newtons/m and resonance frequency of ∼ 300 kHz. Typical imaging parameters were drive amplitude, 150–500 kHz with set points of 0.7–0.8 V, scan frequencies of 2–4 Hz, image resolution 512 by 512 points, and scan size of 5 μm. All experiments were performed in triplicate. For in situ AFM experiments tracking aggregation of monomeric preparations of HD53Q or synthetic peptide, bare substrate was imaged in buffer A, and the sample was injected directly into the fluid cell to reach the final concentration for the experiment. For in situ AFM experiments of preformed HD53Q fibrils, solutions containing preformed fibrils of HD53Q were allowed to rest on mica until several fibrils were present on the surface. Then the substrate was washed with buffer A to remove proteins remaining in solution, and then fresh monomeric preparations of HD53Q that had been incubated with protease on ice for 1 h was injected directly into the fluid cell. All in situ AFM images were taken with V-shaped oxide-sharpened silicon nitride cantilever with a nominal spring constant of 0.5 newtons/m. Scan rates were set at 1–2 Hz with cantilever drive frequencies ranging from ∼8 to 12 kHz. Images were taken with an image resolution of 1024 by 1024 points with scan sizes ranging from 10 to 20 μm. Image analysis of all AFM images was performed with Matlab with the image processing toolbox (Mathworks, Natick, MA) (for more details, see the supplemental material). Volume measurements were partially corrected for error associated with the finite size of the AFM probe based on geometric models (26Legleiter J. DeMattos R.B. Holtzman D.M. Kowalewski T. J. Colloid Interface Sci. 2004; 278: 96-106Crossref PubMed Scopus (31) Google Scholar). The analysis of mutant htt fragments by SDS-AGE with Western analysis was performed as described (19Weiss A. Klein C. Woodman B. Sathasivam K. Bibel M. Régulier E. Bates G.P. Paganetti P. J. Neurochem. 2008; 104: 846-858PubMed Google Scholar). Mutant htt oligomers were resolved by immunoreactions with the EM48 antibody (Chemicon, MAB5374). HD20Q and HD53Q proteins were purified as described (15Wacker J.L. Zareie M.H. Fong H. Sarikaya M. Muchowski P.J. Nat. Struct. Mol. Biol. 2004; 11: 1215-1222Crossref PubMed Scopus (249) Google Scholar, 24Muchowski P.J. Schaffar G. Sittler A. Wanker E.E. Hayer-Hartl M.K. Hartl F.U. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 7841-7846Crossref PubMed Scopus (547) Google Scholar). Fresh, unfrozen HD53Q and HD20Q proteins were used for all experiments. Before each experiment, HD20Q or HD53Q was centrifuged (20,000 × g) for 30 min at 4 °C to remove pre-existing aggregates. HD20Q or HD53Q were incubated at 12 μm in 20 mm Tris-HCl, pH 8, 150 mm NaCl, 1 mm DTT at 37 °C with shaking at 800 rpm. Solutions of PreScission Protease (4 units/100 μg of fusion protein, Amersham Biosciences) were added at time 0 to initiate the aggregation of HD53Q. At the indicated time points after GST cleavage, equal amounts of protein (15 μg) were removed from the 12 μm aggregation reactions, diluted 1:1 into non-reducing Laemmli sample buffer (150 mm Tris-HCl, pH 6.8, 33% glycerol, 1.2% SDS, and bromphenol blue), and analyzed by SDS-AGE and Western analysis with EM48 (1:1000). Molecular mass standards used in SDS-AGE were ferritin (400 kDa), thyroglobulin (660 kDa), and IgM (1000 kDa). Complete Httex1p plasmids encoding either a normal range (25Q), intermediate (46Q), or expanded (97Q) polyQ tract with alternating CAG/CAA amino acid repeats were fused in-frame to GFP at the C terminus and subcloned into pcDNA3.1 (Invitrogen) as described (27Steffan J.S. Agrawal N. Pallos J. Rockabrand E. Trotman L.C. Slepko N. Illes K. Lukacsovich T. Zhu Y.Z. Cattaneo E. Pandolfi P.P. Thompson L.M. Marsh J.L. Science. 2004; 304: 100-104Crossref PubMed Scopus (561) Google Scholar, 28Kazantsev A. Preisinger E. Dranovsky A. Goldgaber D. Housman D. Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 11404-11409Crossref PubMed Scopus (394) Google Scholar). All constructs were verified by sequencing and transient transfection into ST14A cells to observe expression using light microscopy (Zeiss AxioObserver.Z1). Immortalized rat striatal neurons (ST14A cells) were grown in 6-well plates and transiently transfected with Httex1p plasmids (1 μg) with Lipofectamine 2000 (Invitrogen). ST14A cells were grown in Dulbecco's modified Eagle's medium and 10% fetal bovine serum at 33 °C (all reagents from Invitrogen). Each plasmid was transfected in duplicate per experiment, and three independent experiments were performed. At 24 and 48 h post-transfection, cells were lysed in radioimmune precipitation assay buffer (10 mm Tris, pH 7.5, 150 mm NaCl, 1 mm EDTA (from a concentrated stock at pH 8.0), 1% Nonidet P-40, 0.5% SDS) containing Complete Protease Inhibitor (Roche Diagnostics). A DC protein assay (Bio-Rad) was performed to determine protein concentration. Lysate (30 μg) was added in a 1:1 ratio to loading buffer (150 mm Tris, pH 6.8, 33% glycerol, 1.2% SDS) and loaded onto a 1% agarose gel containing 0.1% SDS and run until the dye front had migrated at least 12 cm to allow for maximum resolution of aggregates from the dye front. The proteins were then semidry-blotted (Owl HEP-1) onto a polyvinylidene difluoride membrane in transfer buffer (192 mm glycine, 2 5 mm Tris-base, 0.1% SDS, 15% MeOH). This blot was blocked for 1 h in 5% bovine serum albumin in Tris-buffered saline containing 0.1% Tween 20 at room temperature. The blot was then probed with either EM48 (Millipore, 1:1000) or anti-GFP antibody (Clontech, 1:1000). Peroxidase-conjugated AffiniPure goat anti-mouse secondary (Jackson ImmunoResearch Laboratories) was used at 1:50,000 for 1 h at room temperature. Blots were detected using PICO detection reagent (Pierce). The same lysates used in the SDS-AGE assays were analyzed by cellulose acetate filter-retardation assays. Lysate (30 μg) was diluted to 200 μl with 2% SDS and filtered through cellulose acetate membrane (Schleicher & Schuell, 0.2-μm pore size) with a Bio-Rad dot blot filtration unit. The assay was performed as described (7Scherzinger 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). The blot was then probed with primary and secondary antibodies and developed as described above. At 4, 7, and 14 weeks of age R6/2 and wild type cortex were homogenized with 10 volumes of ice-cold sample buffer (100 mm Tris-HCl, pH 7.4, 150 mm NaCl, and a protease inhibitor mixture) by a ball-bearing homogenizer and sonification with 10 pulses/min and 15% power. Samples were either analyzed without centrifugation (total homogenates) or were centrifuged at 20,000 × g for 45 min at 4 °C and were then diluted 1:1 into non-reducing Laemmli sample buffer (150 mm Tris-HCl, pH 6.8, 33% glycerol, 1.2% SDS, and bromphenol blue). Bradford assays were used to determine protein concentration in the homogenates before the addition of Laemmli sample buffer. Total protein (50 μg) was loaded per SDS-AGE well. Mutant htt oligomers were imaged after immunoreaction with EM48 antibody as described above. Immunoelectron microscopy (EM) analysis of human HD cortex and MCF-7 cells transiently transfected with FH969–100Q were performed as described (2DiFiglia 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, 29Qin Z.H. Wang Y. Sapp E. Cuiffo B. Wanker E. Hayden M.R. Kegel K.B. Aronin N. DiFiglia M. J. Neurosci. 2004; 24: 269-281Crossref PubMed Scopus (150) Google Scholar). Specifically, the cortex was dissected from a HD postmortem brain and fixed in 4% paraformaldehyde in phosphate-buffered saline overnight, immersed in 20–30% sucrose for 1 to 2 days, frozen on dry ice, and stored at −70 °C. The number of CAG repeats identified in the HD allele was 69. Immunoperoxidase labeling was performed by cutting frozen sections (40 μm thick), incubated in 5% normal goat serum (NGS), 1% bovine serum albumin, 0.2% Triton X-100, and 1% H2O2 in phosphate-buffered saline, and then incubated with a primary antibody specific to htt1–17 at a concentration of 1 μg/ml in 5% NGS and 1% bovine serum albumin for 40 h at 4 °C. Control experiments included omission of the primary antibody and preadsorption with 50 μg of N-terminal peptide. Both of these controls resulted in the absence of staining. For analysis of ultrastructure, some immunoperoxidase-labeled sections were embedded in Epon, and thin sections were cut on an ultramicrotome and mounted on Formvar-coated slot grids. MCF-7 cells were cultured onto poly-l-lysine-coated microslips and transfected with FH969–100. Cells were fixed in 4% paraformaldehyde in phosphate-buffered saline 3 days after transfection. Cells were incubated with antibody M5 and horseradish peroxidase-conjugated anti-mouse IgG antibody. The htt bodies were visualized with diaminobenzidine. Cells were postfixed in 2.5% glutaraldehyde, incubated in 1% osmium tetroxide and 1% uranyl acetate, dehydrated in increasing grades of alcohol, and embedded in an ethanol-soluble resin (LX112, LADD). Embedded cells were sectioned (Ultracut E; Reichert-Jung). Both cortex and MCF-7 cells were examined with a JEOL 100CX electron microscope. In these experiments we used mutant htt fragments with various polyQ repeat lengths (HD20Q, HD35Q, HD46Q, and HD53Q) that were purified from Escherichia coli as a fusion to GST (Fig. 1A) (24Muchowski P.J. Schaffar G. Sittler A. Wanker E.E. Hayer-Hartl M.K. Hartl F.U. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 7841-7846Crossref PubMed Scopus (547) Google Scholar). After purification and centrifugation to remove small aggregates that might serve as seeds, GST-HD fusion proteins appeared to be non-aggregated by AFM (data not shown). Cleavage of the GST moiety with a site-specific protease (PreScission protease) released the intact mutant htt fragments, initiating aggregation in a time-dependent manner as reported (8Scherzinger E. Sittler A. Schweiger K. Heiser V. Lurz R. Hasenbank R. Bates G.P. Lehrach H. Wanker E.E. Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 4604-4609Crossref PubMed Scopus (581) Google Scholar, 15Wacker J.L. Zareie M.H. Fong H. Sarikaya M. Muchowski P.J. Nat. Struct. Mol. Biol. 2004; 11: 1215-1222Crossref PubMed Scopus (249) Google Scholar). To determine the effects of polyQ length on the kinetics and morphologies of aggregates formed by a mutant htt fragment, preparations of soluble HD20Q, HD35Q, HD46Q, or HD53Q were incubated at concentrations of 2 and 30 μm and sampled for ex situ AFM analysis at 0, 1, 3, 5, 8, and 24 h after the addition of protease (Fig. 2). To quantify the relationship between oligomers, fibrils, and amorphous aggregates, the number of each aggregate type per μm2 was measured as a function of time at both concentrations (Fig. 3). Oligomers were defined as 2–10 nm in height with an aspect ratio (longest distance across to shortest distance across) less than 2.5, indicating a globular structure. Fibrils were defined as aggregates greater than 4 nm in height that had an aspect ratio greater than 2.5. Amorphous aggregates were defined as any aggregate greater than 10 nm in height that did not have any obvious fibrillar morphology. These criteria were based on measured characteristics of representative examples of each respective aggregate type (Fig. 1, C–E). The number of fibrils in a bundle was estimated by counting the number of fibrils visible in the bundle.FIGURE 3Aggregate species of mutant htt fragments change temporally in a polyQ length- and concentration-dependent manner. Quantification of aggregate types observed in AFM images for incubations HD20Q, HD35Q, HD46Q, and HD53Q at 2 μm and 30 μm. The numbers of oligomers (A), fibrils (B), and amorphous (C) aggregates per μm2 were calculated for all images in Fig. 2. The appearance of fibrils occurred earlier for longer polyQ repeat lengths. Although HD20Q formed oligomers at both concentrations, fibrils were not observed. For HD35Q, a peak population of oligomers preceded the formation of amorphous aggregates (2 μm) or fibrils (30 μm). A peak population of oligomers also preceded the appearance of fibrils of HD46Q at both concentrations. As fibrils of HD53Q appeared early in the experiment, a peak oligomer population preceding the appearance of fibrils was only observed if the kinetics of aggregation reactions were decreased (see supplemental Fig. 2).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Oligomeric aggregates were observed for all htt fragments studied, including HD20Q. As reported (7Scherzinger 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, 8Scherzinger E. Sittler A. Schweiger K. Heiser V. Lurz R. Hasenbank R. Bates G.P. Lehrach H. Wanker E.E. Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 4604-4609Crossref PubMed Scopus (581) Google Scholar), the appearance of fibrils was polyQ length and concentration-dependent, with fibrils appearing earlier, growing longer, and forming bundles with longer polyQ lengths and higher concentrations. Quantification of distinct aggregate types per unit area (Fig. 3) demonstrated that aggregation results in a complex mixture of aggregate types at any given time for mutant htt fragments. Oligomers were detected at both concentrations of HD20Q but gradually dissipated (2 μm) or remained relatively stable (30 μm) in number, depending on concentration. Importantly, fibrils were not observed for HD20Q at any time, unlike those observed with longer polyQ lengths (see below). For both concentrations of HD20Q, low amounts (< 0.3 aggregates/μm2) of amorphous aggregates were observed relative to other aggregate types. For HD35Q, a significant number of large amorphous aggregates (>1.0 aggregates/μm2) and very few (<0.1 fibrils/μm2) short putative fibrils formed at 2 μm after 8–24 h of incubation (FIGURE 2, FIGURE 3). Short fibrillar species became the predominant aggregate type for HD35Q incubated at 30 μm after 8 h. The appea" @default.
• W2058182560 created "2016-06-24" @default.
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• W2058182560 date "2010-05-01" @default.
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• W2058182560 title "Mutant Huntingtin Fragments Form Oligomers in a Polyglutamine Length-dependent Manner in Vitro and in Vivo" @default.
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