Structure of Inclusions of Huntington's Disease Brain Revealed by

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Structure of Inclusions of Huntington’s Disease Brain Revealed by Synchrotron Infrared Microspectroscopy: Polymorphism and Relevance to Cytotoxicity William André,† Christophe Sandt,‡ Paul Dumas,‡ Philippe Djian,*,†,§ and Guylaine Hoffner*,†,§ †

CNRS, Génétique Moléculaire et Défense Antivirale, Centre Universitaire des Saints-Pères, Université Paris Descartes, 75006 Paris, France ‡ Synchrotron SOLEIL, 91192 Saint-Aubin, France S Supporting Information *

ABSTRACT: Huntington’s disease is caused by a polyglutamine expansion in huntingtin. Affected brain regions contain characteristic aggregates of the misfolded expanded protein. Studies in cells and animals show that aggregates are polymorphic and that the secondary structure of the aggregates is likely to condition their cytotoxicity. Therefore knowing the structure of aggregates is important as neurotoxic secondary structures may be specifically targeted during the search for prophylactic or therapeutic drugs. The structure of aggregates in the brain of patients is still unknown. Using synchrotron based infrared microspectroscopy we demonstrate that the brains of patients with Huntington disease contain putative oligomers and various kinds of microscopic aggregates (inclusions) that can be distinguished by their differential absorbance at 1627 cm−1 (amyloid β sheets) and 1639 cm−1 (β sheets/unordered). We also describe the parallel/antiparallel organization of the β strands. As the inclusions enriched in both β sheets and β sheets/unordered structures are characteristic of severely affected brain regions, we conclude that this kind of amyloid inclusions is likely to be particularly toxic to neurons.

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structure of the aggregates found in the brain of HD patients remains unknown and there is no direct evidence that the aggregates contain β-sheet structures. Synchrotron-based Fourier transform-infrared (sFT-IR) microspectroscopy is the method of choice to study the structure of the inclusions of HD brain, because of its unique ability to achieve the high-sensitivity and high-resolution needed for such small objects. Using sFT-IR, we demonstrate that the proteins aggregated in the inclusions of HD brain adopt polymorphic secondary structures, which vary according to the length of the polyQ, the subcellular location of the inclusions, and the affected brain region. We discuss the neurotoxicity of these different kinds of secondary structures.

untington’s disease (HD) is caused by expansion of the glutamine codon CAG in the first exon of the huntingtin (htt) gene.1 The disease occurs when the polyQ expands beyond 35−40 repeats: a polyQ in excess of 60 glutamines produces juvenile disease (JHD), whereas 40−60 glutamines produce the much more frequent adult form (AHD). HD is characterized by the progressive destruction of neurons in the striatum and to a lesser extent the cortex2 and by the formation in the affected brain regions of inclusions, predominantly nuclear in JHD and cytoplasmic in AHD. The inclusions contain N-terminal fragments of htt bearing the expanded polyQ,3,4 coaggregated with a variety of other proteins.5 Misfolding and aggregation of the expanded polyQ are thought to be key pathogenic events in polyQ diseases, although how they cause neuronal toxicity has not yet been established.6−8 In 1994, Max Perutz postulated that, beyond a critical length, polyQ misfolds and aggregates by forming antiparallel β-sheets stabilized by hydrogen bonds between their main-chain and sidechain amides.9 Subsequent studies showed that expanded polyQ undergoes a conformational transition leading to the formation of fibrils with high β-sheet content.10−13 A variety of polyQ aggregates have been observed in vitro.7,14−16 A mutant htt fragment has been shown to misfold in vitro into distinct amyloid conformations, whose cytotoxicity varies. There is circumstantial evidence suggesting that htt amyloid fibrils that form in different brain regions of R6/2 mice have distinct conformations.17 The © 2013 American Chemical Society

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EXPERIMENTAL SECTION

Preparation of Samples. Frozen brain samples were obtained from the Harvard Brain Tissue Resource Center (Belmont) and the Hôpital de la Pitié-Salpêtrière (Laboratoire de Neuropathologie R. Escourolle, France). Patients with juvenile and adult HD as well as normal persons (controls) Received: January 19, 2013 Accepted: March 4, 2013 Published: March 4, 2013 3765

dx.doi.org/10.1021/ac400038b | Anal. Chem. 2013, 85, 3765−3773

Analytical Chemistry

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Figure 1. AHD Cis: (A) frozen striatal section of case 3701 stained with an antibody directed against the N-terminus of huntingtin (green) and counterstained with Hoechst 33258 for DNA (blue). The arrow shows inclusion. Scale bar: 20 μm. (B) Comparison of Cis spectra with those of cytoplasm. Left panels show average spectra of Cis (red) and cytoplasm (black), spectra of Cis most divergent from cytoplasm (green) and corresponding second derivative spectra (with same colors). Arrowheads mark enrichments in peaks assigned to the β-sheet, at 1627 cm−1, 1681 cm−1, and 1693 cm−1. Peak at 1627 cm−1 demonstrates the amyloid nature of inclusions. Arrows in cases 3701 and 3491 show the additional contribution of a peak assigned to a β-sheet/unordered structure at 1639 cm−1. Middle and right panels show the score plots and loading plots of PCA, respectively. The score plots show PC1 vs PC2, with spectra of Cis and cytoplasm in red and black, respectively. PC1, but not PC2, separates Cis from cytoplasm. The loading plots show that the component at 1627 cm−1 mostly influences PC1. Co, cortex; St, striatum.

BaF2 or CaF2 slides (Crystal GmbH). The final thickness of the sections was approximately 8 μm because of dehydration. Staining of formalin-fixed inclusions with a polyclonal anti Nterminal huntingtin antibody coupled to an Alexa 488-bearing

were examined. The characteristics of the cases analyzed are given in Table S1 in the Supporting Information. Indirect Immunofluorescence. Frozen sections (12-μm thick) of either cortex or striatum were deposited on 1-mm thick 3766



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Table 1. Analysis of Amide I Banda

a

All values determined by univariate data analysis. Statistical significance assessed using the Kruskal−Wallis test after Bonferonni correction. Cis and Nis that differed significantly from surrounding cytoplasm and nuclei, respectively, are in red type. Nuclei that differed significantly from cytoplasm are in green type (p ≤ 0.001). β/α, β-sheet/α-helix; βU/α, [β-sheet/unordered]/α-helix; OS, analysis on either original (OS) or second derivative spectra (SD).

secondary antibody was carried out as previously described18 except that the last wash consisted of bidistillated water and preceded air-drying of the sections, which were then stored at −80° until FT-IR analysis. sFT-IR Microspectroscopy and Data Acquisition. sFTIR microspectroscopy was performed at the SMIS beamline of the SOLEIL synchrotron (Saint-Aubin, France), using a ThermoNicolet Continuμm XL microscope equipped with a 32× magnification/0.6 numerical aperture Schwarzschild objective. The microscope was coupled to a Nicolet 5700 spectrometer equipped with a Michelson interferometer and a KBr beamsplitter (Thermo). An Olympus fluorescence accessory was coupled to the IR microscope and a Basler Scout camera. The microscope was operated in dual aperture mode. Under these conditions, the synchrotron source was about 100fold brighter than conventional sources.

All spectra were collected in transmission mode using an aperture size of 6 × 6 μm2. High quality spectra were collected in the 4000−800 cm−1 mid-IR range at a resolution of 6 cm−1 with 250 to 1500 coadded scans. Background spectra were collected from a clean area of each BaF2 or CaF2 slide. Spectra with visible Mie scattering or edge effect were eliminated. Typical examples of uncorrected spectra of a cytoplasmic inclusion and nearby cytoplasm are given in Supporting Information, Figure S1. Neither Mie nor resonant Mie scattering is present. IR Spectral Analysis. Analyses of IR spectra and chemical images were performed using the OMNIC software (Thermo Scientific). For all spectra, a linear baseline correction was applied from 1450 cm−1 to 1735 cm−1. Derivation of the spectra to the second order was used to increase the number of discriminative features and was achieved by using a Savitsky− 3767



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Figure 2. Chemical maps of β-sheet content in and around Cis. Immunofluorescence labeling and chemical maps obtained from cortex of case 955 (A− C) and striatum of case 3701 (D−F): (A and D) fluorescent labeling of Cis (green) and nuclei (blue); (B and E) β-sheet/α-helix ratio displayed by a color scale going from red (highest) to blue (lowest or nil). Correspondence between inclusion and highest β-sheet content is obvious. (C and F) Analysis of representative IR spectra acquired on maps. Left panels show spectra acquired on points scattered on the surface of inclusions (red) and on surrounding tissue (black); they also display either the corresponding second derivative spectra with identical colors (C) or the variance between both categories of spectra in green (F). Arrowhead in part C points to the shoulder around 1627 cm−1 in amide I bands in spectra acquired on inclusions. The variance between both groups of spectra shows the contribution of the peak indicative of β-sheet in striatal Cis (F, arrowhead). Middle and right panels show PCA. Spectra of Cis (red) and their surrounding tissue (black) form well separated clusters along the PC1 axis, with spectra of Cis being enriched in the 1627 cm−1 component representative of the β-sheet. The PC2 axis does not separate the two categories of spectra.

Golay algorithm with a seven-points filter and a polynomial order of three.19 Maps were collected on predefined areas (≈100 μm2) encompassing fluorescently labeled cytoplasmic inclusions with

an aperture of 6 × 6 μm2 and a step size of 1 μm. They were processed to represent the relative protein secondary structure by calculating the area ratio of a range attributed to a β-sheet (1625−1631 cm−1) to one due to an α-helix (1654−1660 cm−1), 3768



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Figure 3. JHD Cis: analysis of spectra of Cis and cytoplasm. Spectra of Cis (red) and cytoplasm (black) are not visually distinguishable and are not separated by PCA. Co, cortex; St, striatum.

with a linear baseline of 1300−1800 cm−1. The statistical analysis is described in the Supporting Information.

observed that their amide I bands differed from those of the cytoplasm by the presence of a shoulder at the right side. This shoulder was more or less pronounced but clearly visible on average spectra in all five cases examined (Figure 1B, left panels). Second derivatives revealed that the shoulder resulted from an increased contribution of a peak at 1627 cm−1. In addition, elevation of a peak at 1639 cm−1 was obvious in the average second derivative spectra of striatal Cis in case 3491 (and present in all spectra) and in some spectra of striatal Cis in case 3701. Because of its proximity to affiliations to unordered structures, the assignment of the 1639 cm−1 wavenumber is equivocal. It could either correspond to β-sheet (maybe a more soluble βsheet than the 1627 cm−1 mode which is considered amyloid) or to unordered structures. Therefore we assigned the 1639 cm−1 wavenumber to “β-sheet/unordered” structures. Smaller increases in contributions of peaks at around 1681 cm−1 and 1693 cm−1 were also observed in some second derivative spectra of Cis and in average spectra. The increases at 1627 cm−1, 1681 cm−1, and 1693 cm−1 showed that the inclusions present in the cortex and the striatum of AHD brain were enriched in β sheets, while the increase at 1627 cm−1 indicated their amyloid nature. We then performed principal component analyses (PCA). In all five cases, spectra of Cis and cytoplasm were separated along the principal component 1 (PC1) axis, which alone contributed 63−89% of total variability (Figure 1B, middle panels).

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RESULTS Nuclear and cytoplasmic inclusions (Nis and Cis) were localized by immunofluorescence staining of huntingtin, and IR spectra of the inclusions and the surrounding tissue were obtained by sFTIR. The patients analyzed and the number of collected spectra are listed in the Supporting Information, Tables S1 and S2, respectively. We examined the amide I protein band to determine the protein secondary structure. The assignment of spectroscopic IR bands to protein secondary structures is well established, with marginal variations depending on the authors:20−24 IR bands located between 1648 cm−1 and 1660 cm−1 are assigned to the α-helix, 1640−1648 to the unordered structure, 1623−1641 and 1670−1695 to the β-sheet. An increase in the 1620−1630 cm−1 component has been considered a signature of the amyloid fiber.25 AHD Cis Have Different Amyloid Structures in Cortex and Striatum. We acquired IR spectra from five AHD cases: three cortices and two striata (Figure 1). We first acquired the spectra of inclusion-free cytoplasm and we found that, as in normal persons, cytoplasmic proteins predominantly existed in α-helical conformation, with an amide I maximum at 1656.6 ± 0.8 cm−1. We then acquired the spectra of Cis (Figure 1A) and 3769



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Figure 4. JHD Nis: (A) double staining of inclusions (green) and DNA (blue) in cortex of case 3815. Scale bar: 20 μm. (B) Analysis of spectra of Nis (red) and nuclei (black) in three JHD cases. Amide I bands of average spectra of Nis show a right shoulder, conspicuous for a spectrum of Nis differing most from nuclei (green). Second derivative spectra reveal an increase in peaks indicative of β sheets at 1627 cm−1, 1681 cm−1, and 1693 cm−1 (arrowheads) and of β-sheet/unordered structures at 1639/1640 cm−1 (arrow). Spectra of Nis and nuclei are well separated along the PC1 axis in cases 3815 and 3482 but not in case 3123. As the PC1 axis is influenced by variations in the 1627 cm−1 range, spectra of Nis are enriched in the β-sheet compared to nuclei in cases 3815 and 3482. A few spectra of Nis are further enriched in the β-sheet in case 3123 (circled in red). JHD Nis are amyloid. Co, cortex; St, striatum.

As the shoulder at 1627 cm−1 in Cis was more or less marked, even within a given patient, we wondered whether this variability reflected differences in the inclusion size. Because the area examined is constant, the cytoplasm surrounding the inclusions contributes to a larger extent to the spectra when inclusions are small. However, we found no correlation between length of the long axis and peak depth at 1627 cm−1 on second derivative spectra of nine inclusions of case 955 (regression coefficient was 0.14). We conclude that β-sheet enrichment truly reflected intrinsic secondary structure variability. Finally we generated chemical maps representing the β-sheet/ α-helix ratio. For this we photographed areas encompassing a fluorescently labeled Cis in cortical sections of case 955 and striatal sections of case 3701. We then immediately collected the corresponding IR spectra at points spaced by 1 μm and processed them to generate the maps. For both inclusions, the immunolabeled area (Figure 2A,D) and the area with the highest β-sheet/α-helix ratio (Figure 2B,E) were clearly superimposable. Representative spectra of Cis were virtually superimposable and

Corresponding loading plots confirmed that the greatest contributor to the variance of PC1 was the 1627 cm−1 wavenumber (Figure 1B, right panels). We also developed a univariate data analysis method to evaluate if spectral differences in the shapes of amide I bands, β-sheet enrichment, and β-sheet/ unordered enrichment were statistically significant (see the Supporting Information). Original spectra were used to evaluate the amide I bandwidth and gravity center as well as β sheet enrichment. Although enrichment in β sheet/unordered structures at 1639 cm−1 was visible in the original spectra as a change in slope, it could not be estimated on the original spectra because of the overlap with the larger β sheet peak at 1627 cm−1 and we therefore used second derivative spectra. The enrichment in the β sheet was statistically significant in all five AHD cases examined (p ≤ 0.001) and the difference in the amide I band shape (width and gravity center) in four of five cases. The increase in the [β-sheet/unordered]/α-helix ratio was only significant in the inclusions of case 3491 (Table 1). 3770



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Table 2. Analysis of Amide I Band of Inclusions According to Subcellular Localization and Type of HD (AHD or JHD)a

a β/α, β-sheet/α-helix; βU/α, [β-sheet/unordered]/α-helix; AP/α, antiparallel β sheet/α helix; AP/β, antiparallel β sheet/β sheet. Values of Cis and Nis differing from non-HD controls are in red type. Values of JHD Nis differing from AHD Cis (discriminating amyloid inclusions) are circled. OS, analysis on original spectra; SD, analysis on second derivative spectra.

and possibly unordered structures. PCA revealed that spectra of Nis and nuclei in cases 3815 and 3482 formed two clusters and confirmed that the range 1627−1639 cm−1 was responsible for most of the variance observed (Figure 4B, middle and right panels). Enrichment in β-sheet and β-sheet/unordered structures was statistically significant in these two patients (Table 1). Case 3123 differed from cases 3815 and 3482 because its Nis resembled nuclei in the amide I bandwidth, β-sheet/α-helix ratio, and [β-sheet/unordered]/α-helix ratio. However, 3123 nuclei stand out from those of the other juvenile cases by their elevated absorption at 1627 and 1639 cm−1 (Table 1). Therefore the Nis of case 3123 should be considered as enriched in β-sheet and βsheet/unordered structures. A subpopulation of case 3123 Nis (5/39) possessed a higher β-sheet and β-sheet/unordered content than the surrounding nucleoplasm (Figure 4B, middle panel). In conclusion, we demonstrate enrichment in β-sheet and βsheet/unordered structures in cortical and striatal Nis. Because one of the main contributions was at 1627 cm−1, we conclude that JHD Nis are amyloid. We also observed that JHD Nis shared with AHD Cis of striatum but not with AHD Cis of cortex, [βsheet/unordered]/α-helix enrichment in the component at 1639 cm−1. HD Inclusions Possess Polymorphic Protein Secondary Structures. We undertook a global comparison of the inclusions across all HD cases. The average spectra of both AHD Cis and JHD Nis differed strikingly from those of controls by the presence of a right shoulder in the amide I band, with elevation of the component at 1627 cm−1 (Supporting Information Figure S2A, upper panel). PCA showed that AHD Cis and JHD Nis had higher β-sheet content than controls, based on the intensity at 1627 cm−1 (Figure S2A in the Supporting Information, PC1 in lower panels and Table 2). Although average cortical spectra of JHD Nis and AHD Cis were remarkably similar, they differed at wavelength 1639 cm−1 (Supporting Information Figure S2A, upper panel and PC3 in lower panels). The difference is particularly visible in the Supporting Information, Figure S2B, which illustrates two spectra with similar β-sheet enrichment. Univariate data analysis confirmed that JHD Nis possessed a higher content in β-sheet/ unordered structures than AHD Cis and that AHD Cis tended to

so were cytoplasmic spectra, but the two kinds of spectra diverged from each other in the 1627 cm−1 region. As a consequence, the spectra of inclusions and those of cytoplasm formed two clustered groups by PCA (Figure 2C,F). Differences in the β-sheet/α-helix ratio as well as the amide I band shape were statistically significant (Table 1). Our results suggest that the structure of proteins inside a Cis is quite homogeneous. In summary, we demonstrate that in AHD (1) both cortical and striatal Cis are enriched in the β-sheet and can be considered amyloid, (2) the amyloid structure of cortical Cis is quite homogeneous, as it always originates predominantly from an increase in the component at 1627 cm−1, and (3) striatal Cis differ from cortical Cis by the presence of an additional component at 1639 cm−1. JHD Cis Do Not Differ Structurally from the Surrounding Cytoplasm. We were surprised to discover that JHD Cis did not differ appreciably from the surrounding cytoplasm (Figure 3, left panels). PCA analyses showed that the spectra of Cis could not be separated from those of cytoplasm in any of the four cases examined (Figure 3, middle and right panels). This was confirmed by univariate statistical analyses (Table 1). Because on average Cis are smaller in JHD brain than in AHD brain,4 it was conceivable that our inability to detect particular arrangements in JHD Cis was due to insufficient resolution of our technique. However even the largest JHD Cis, which reach the size of AHD Cis did not appreciably differ from cytoplasm. An alternative explanation may have been that the β-sheet enrichment was too low to be detected. In any case, we conclude that the Cis present in the JHD brain, whether cortex or striatum, lack structural rearrangement and do not possess a detectable amyloid structure. JHD Nis Possess an Amyloid Structure Resembling That of Striatal AHD Cis. We collected spectra centered on Nis and nuclei in the cortex of JHD cases 3815 and 3123 and the striatum of case 3482 (Figure 4A). In cases 3815 and 3482, amide I bands of Nis had a right shoulder. The shoulder was conspicuous on average spectra and was particularly striking in spectra of Nis differing most from nuclei (Figure 4B, left panels). Second derivatives of average spectra showed major increases at 1627 cm−1 and 1639 cm−1 and smaller increases at 1681 cm−1 and 1693 cm−1. These components are ascribed to the β-sheet 3771



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Figure 5. Summary of secondary structure of inclusions in JHD and AHD brains.

cytoplasmic spectra were significantly different from their nuclear counterparts in case 955 (Table 1). Among juvenile cases, 3123 alone showed nuclear/cytoplasmic differences with increased contributions at 1627 cm−1, 1639 cm−1, and 1693 cm−1 in the cortical nuclei (Supporting Information Figure S3); the β-sheet/α-helix ratio was about one-third higher in nuclei than in cytoplasm (Table 1). We ascribe these differences to the presence of large amounts of submicroscopic aggregates (oligomers), which are nuclear in JHD case 3123 and cytoplasmic in AHD case 955, in keeping with the location of the microscopic aggregates in JHD and AHD. The putative oligomers contain antiparallel β-sheets (1627 cm−1 and 1693 cm−1) and β-sheet/unordered structures (1639 cm−1).

possess a greater abundance of such structures in the striatum than in the cortex (Table 2). In contrast to AHD Cis and JHD Nis, JHD Cis were similar to controls (Supporting Information Figure S2 and Table 2). Overall, comparison of inclusions and controls supported previous conclusions that there exists three kinds of inclusions: AHD Cis enriched in β-sheet, JHD Nis enriched in β-sheet and β-sheet/unordered structures, and JHD Cis lacking structural rearrangement. Parallel and antiparallel β-sheets can be distinguished by their amide I region.22,26−28 Antiparallel β-sheets possess both an increased peak at 1623−1641 cm−1 and a weaker peak at 1670− 1695 cm−1, whereas parallel β-sheets possess only an increased peak at 1623−1641 cm−1. It can be seen in the Supporting Information, Figure S2 that the average amide I bands of AHD Cis and JHD Nis possessed a small left shoulder, because of higher absorptions than controls at 1682 cm−1 and 1693 cm−1. The weak contribution of the component at 1693 cm−1 together with the strong contribution at 1627 cm−1 is also visible on the loading plot of PC1 in the same figure, suggesting an antiparallel β-sheet structure. Both AHD Cis and JHD Nis showed an elevation in the antiparallel β-sheet/α-helix ratio, which suggested that these inclusions had high amounts of antiparallel β-sheets (Table 2). AHD Cis and JHD Nis also showed a reduced antiparallel β-sheet/β-sheet (AP/β) ratio, which indirectly suggested enrichment in parallel β-sheets. As the AP/β ratio was lower in AHD Cis than in JHD Nis, parallel β-sheets may be more abundant in AHD Cis than in JHD Nis. We conclude that the amyloid structures of AHD Cis and JHD Nis contain antiparallel and also probably parallel β-sheets (Table 2). Structural Changes Outside of the Inclusions in One Juvenile and One Adult HD Case. We thought it was of interest to determine whether patients showed alteration of protein secondary structure outside of the inclusions. Because of the aforementioned interindividual variability, we compared nuclei and cytoplasm in the same patient. Such a comparison should be informative since the aggregation process is predominantly nuclear in JHD and cytoplasmic in AHD. As a prerequisite, we demonstrated that the nuclear and cytoplasmic amide I bands were undistinguishable in the four normal persons examined (see case 1402 in Supporting Information Figure S3 and Table 1). We then studied the five adult and four juvenile cases listed in Supporting Information Table S1. Among the adult cases, a fraction of the amide I bands of cytoplasm of case 955 had a right shoulder. Second derivative spectra showed that elevated contributions of components at 1627 cm−1, 1639 cm−1, 1682 cm−1, and 1693 cm−1 accounted for the differences observed. PCA analysis partially separated both categories of spectra across the PC1 axis, which was most strongly influenced by variability around 1627 cm−1 (Supporting Information Figure S3). Both the [β-sheet/unordered]/α-helix ratio and amide I bandwidth of

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DISCUSSION The polymorphic nature of protein aggregates has precedents in R6/2 mice, which express a highly expanded htt allele and develop a disease resembling HD.29 Nekooki and collaborators have studied by FT-IR prion-like amplified htt aggregates of R6/ 2 mice. They have obtained circumstantial evidence indicating that htt aggregates adopt distinct amyloid conformations.17 The fact that a protein can form distinct amyloid structures has been reported for the β amyloid protein.30,31 We find a remarkable constancy in the position of components indicative of β-sheet and β-sheet/unordered structures. For all patients examined, the median peak heights were exactly at 1627.7 cm−1 (β sheet) and 1639.2 cm−1 (β sheet/unordered). This shows a nearly perfect uniformity in the arrangement of the β-sheet and β-sheet/unordered structures and probably in the underlying pattern of intra/inter residue interactions. We observed that the enrichment in the β-sheet varied substantially among amyloid inclusions, even in the same patient. This variation may result from the recruitment of variable amounts of proteins other than expanded huntingtin. Transcriptional regulators, heat shock proteins, and ubiquitin/proteasome components have been shown to be trapped in htt inclusions.5 The secondary structure of proteins aggregated in HD also differs according to brain region, subcellular compartment, and polyQ length. We hypothesize that the length of the polyQ as well as factors specific to subcellular compartments and/or brain regions32 modulate aggregation and promote the formation of polymorphic inclusions. Proteolytic activities may differ among brain regions or neuronal cell compartments and thus fragment htt differently. Our results suggest that HD brain contains large amounts of oligomers with both antiparallel β sheets and β-sheet/unordered structures. Because of their small size and transitory nature, it has proven difficult to obtain detailed structural data about these intermediate assemblies. The antiparallel organization of β sheets in oligomeric α-synuclein, β-amyloid peptide, and αB-Crystallin 3772



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has been described.26,33,34 Antiparallel β sheets have also been shown to compose the amyloid fibrils formed by mutated HET-s prion protein domains and to confer neurotoxicity to these fibrils.35

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CONCLUSION The toxicity of htt/polyQ inclusions has been a matter of intense debate because of contradictory data suggesting that aggregates are harmful, neutral, or even protective.5,36,37 The structural polymorphism of htt inclusions described here may reconcile these seemingly opposite views. We propose that the inclusions lacking any structural rearrangement constitute nontoxic amorphous aggregates,38,39 whereas the amyloid inclusions enriched in both β-sheet and β-sheet/unordered are highly neurotoxic, as they are always associated with the most severe form of the disease and found in the most affected brain regions (Figure 5).

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +33142862272. Fax: +33142605537. E-mail: guylaine. hoff[email protected] (G.H.); philippe.djian@ parisdescartes.fr (P.D.). Author Contributions §

Co-last author.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the Harvard Brain Bank and the Laboratoire de Neuropathologie R. Escourolle (Hôpital de la Pitié-Salpêtrière, Paris) for providing brain samples. The study was supported by CNRS, INSERM, and Université Paris Descartes.

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REFERENCES

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