2 Mice Are Not Amyloid and ... - ACS Publications

Subscriber access provided by University of Newcastle, Australia

Article

The inclusions of R6/2 mice are not amyloid and differ structurally from those of Huntington disease brain William André, Christophe Sandt, Isabelle Nondier, Philippe Djian, and Guylaine Hoffner Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b04199 • Publication Date (Web): 11 Apr 2017 Downloaded from http://pubs.acs.org on April 14, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

19 March 2017

The inclusions of R6/2 mice are not amyloid and differ structurally from those of Huntington disease brain

William Andréa,b, Christophe Sandtb, Isabelle Nondiera, Philippe Djiana* and Guylaine Hoffnera*

a

Centre National de la Recherche Scientifique/Université Paris Descartes, UMR

8118, Laboratoire de physiologie cérébrale, Paris, France. b

Synchrotron SOLEIL, 91192 Gif-sur-Yvette, France.

* Corresponding authors: Centre Universitaire des Saints-Pères, 45 rue des SaintsPères, 75006 Paris, France, Tel.: +33142862272. E-mail: [email protected], [email protected]

1 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 30

ABSTRACT R6/2 mice contain an N-terminal fragment of human huntingtin with an expanded polyQ and develop a neurological disease resembling Huntington disease. Although the brain of R6/2 mice contains numerous inclusions, there is very little neuronal death. In that respect, R6/2 mice differ from patients with Huntington disease whose striatum and cerebral cortex develop inclusions associated with extensive neuronal loss.

We have previously demonstrated

using

synchrotron-based

infrared

microspectroscopy that the striatum and the cortex of patients with Huntington disease contained inclusions specifically enriched in amyloid β-sheets. We had concluded that the presence of an amyloid motif conferred toxicity to the inclusions. We demonstrate here by synchrotron based infrared microspectroscopy in transmission and attenuated total reflectance mode that the inclusions of R6/2 mice possess no detectable amyloid and are composed of proteins whose structure is not distinguishable from that of the surrounding soluble proteins. The difference in structure between the inclusions of patients affected by Huntington disease and those of R6/2 mice might explain why the former but not the latter cause neuronal death.


Page 3 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


INTRODUCTION Huntington disease is the most frequent of nine neurological diseases caused by polyglutamine (polyQ) expansion

1-12

. Huntington disease is caused by expansion of

the polyQ-encoding polyCAG found in the first exon of the huntingtin gene. The disease occurs when the polyQ expands beyond 35-40 repeats. A polyQ between about 35 and 60 glutamines produces the adult form of Huntington disease (AHD), whereas a polyQ longer than 60 Qs produces the rare juvenile form of the disease (JHD). Huntington disease is characterized by the progressive destruction of neurons in the striatum and to a lesser extent in the cortex

13

, and by the formation of

inclusions in the affected regions of the brain. Inclusions are predominantly cytoplasmic in adult Huntington disease and nuclear in the juvenile form

14

. The

inclusions contain N-terminal fragments of the huntingtin bearing the expanded polyQ 14-16, co-aggregated with other proteins 17, 18. Abnormal conformation and aggregation of the expanded polyQ are thought to be key pathogenic events in polyQ diseases, but the precise mechanisms by which expanded polyQ causes neuronal toxicity has not been established 19-25. In 1994, Max Perutz postulated that, beyond a critical length, polyQ acquires a β-sheet conformation and forms aggregates stabilized by hydrogen bonds formed between the main-chain and side-chain amides of anti-parallel β-sheets

26

. Subsequent studies

showed that expanded polyQ undergoes a conformational transition leading to the formation of fibrils with high β-sheet content 27-32.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 30

Using synchrotron-based Fourier Transform Infrared microspectroscopy (sFTIR), we have demonstrated that the proteins aggregated in the inclusions of Huntington disease brain adopt polymorphic secondary structures, which vary according to the clinical presentation of the disease, the subcellular location of the inclusions and the affected brain region. One of the amyloid structures appears to be particularly toxic to neurons as it is associated with the most severe forms of the disease and found in the most affected areas of the brain 33. R6/2 mice are transgenic mice that contain the N-terminal region of human huntingtin with 150 Qs. Although R6/2 mice develop a severe neurological disease and possess numerous inclusions in their brain, they show very little neuronal loss 34. We demonstrate here by sFTIR analysis that the inclusions of R6/2 mice differ from those of Huntington disease brain by their lack of detectable amyloid structure. We suggest that the absence of altered conformation in the inclusions of R6/2 mice explains why very little neuronal loss is observed in R6/2 mice.

EXPERIMENTAL SECTION Preparation of samples Breeding pairs of R6/2 mice were purchased from The Jackson Laboratory (stock 002810, Bar Harbor, ME). Genotype was confirmed by polymerase chain reaction. Frozen samples from cortex, striatum, cerebellum, liver and muscle of two R6/2 mice and two wild-type (wt) littermates were prepared. Two male R6/2 mice were studied (G8, sacrificed at 11 months of age and G20 sacrificed at 13 months). 4 ACS Paragon Plus Environment

Page 5 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


The two wt littermates were male G6 and female G47, both sacrificed when they were 11-month-old. Frozen brain samples from AHD patients were obtained from the Harvard Brain Tissue Center (Belmont, USA) and the Hôpital de la Pitié-Salpêtrière (Laboratoire de Neuropathologie R.Escourolle, France). Detailed characteristics of the patients are given in Table S-1. Frozen tissues were anchored to the sample holder using small amounts of OCT (Tissue Tek) as previously described 35. 12-µm thick frozen sample sections were prepared using a cryostat (Cryocut 1080, Leica) before being deposited on 1-mm thick CaF2 slides (Crystal GmbH).

Immunohistochemistry We performed immunological staining in order to locate the inclusions. Sections were fixed with a 10% buffered formalin solution (about 4% formaldehyde, SigmaAldrich) and then incubated in phosphate buffer saline (PBS) containing 5% bovine serum albumin (BSA) to prevent nonspecific binding. We used a rabbit anti-Nterminal huntingtin antibody (Hoffner et al., 2002) coupled with a goat secondary anti-rabbit antibody bearing Alexa-488. Sections were incubated with the primary antibody (1/400 dilution) for 2 h and then with the secondary antibody (1:100 dilution) for 1 hr. After each incubation, sections were washed three times for 5 min each with PBS containing 0.1% Igepal CA-630 (Fluka, Sigma-Aldrich). Nuclei were counterstained for 5 min with a solution of Hoechst 33258 (Molecular Probe) or TOPRO-3 (ThermoFisher Scientific) diluted in BSA/PBS. Sections were then washed



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 30

three times with PBS/Igepal and once with bi-distilled water before being air-dried and stored at -80° until analysis.

sFTIR and ATR coupled to sFTIR microspectroscopy IR microspectroscopy was performed at the SMIS beamline of the SOLEIL synchrotron (Gif-sur-Yvette, France). This beamline exploits the bending and edge radiation of a bending magnet to supply near, mid and far infrared (IR). We used a ThermoNicolet Continuµm XL microscope equipped with 32x magnification / 0.6 numerical aperture Schwarzschild objective and condenser, a 50x50 µm2 MCT A detector, a XYZ motorized stage and a motorized aperture. The microscope was coupled to a Nicolet 5700 spectrometer equipped with a Michelson interferometer and a KBr beamsplitter (Thermo). An Olympus fluorescence accessory equipped with U-MWU, U-MWB and U-MWIBBP cubes for excitation in the UV, blue and green, and emission in the blue, green and red was coupled to the IR microscope and a Basler Scout camera. In order to eliminate most of the diffraction from the microscope aperture and the sample, microscope was operated in dual aperture mode, which means that the beam passes twice through the aperture. In order to improve the spatial resolution above the diffraction limit, we used Attenuated Total Reflection (ATR) mapping with a truncated ZnSe hemisphere. Using a ZnSe hemispheric Internal Reflection Element (IRE) for ATR improves the spatial resolution by a factor equal to the refractive index of the ATR element: 2.42 at 6-µm wavelength (location of the β-sheet peak) for ZnSe. When measuring in ATR 6 ACS Paragon Plus Environment

Page 7 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


mode, a perfect contact is needed between the sample and the IRE, however depositing the brain section directly on the IRE limits the field of view to a small fraction of the millimeter-wide sample. Therefore in order to examine the entire sample, the sections were deposited on a 1-mm thick ZnSe window, and the 5-mm diameter ZnSe hemisphere truncated to 4 mm in thickness was then applied on the window. The window complemented the hemisphere so that total reflection occurred on the sample at the surface of the window. Perdeuterated hexadecane oil of refractive index matching that of the ATR element was used to ensure optical continuity between the window and the hemisphere. With this set-up, it was possible to move the hemisphere freely across the sample and to search for inclusions with the fluorescence accessory since ZnSe is transparent in the visible (even if yellow).

Data acquisition We collected all spectra using the OMNIC software (Thermo Scientific) in transmission mode with a 6 x 6 µm2 aperture size. Data acquisition was performed in the 4000-800 cm-1 mid-infrared range at a resolution of 6 cm-1 with 250 co-added scans. Background spectra were collected from clean areas of CaF2 windows carrying samples with 500 co-added scans. We eliminated spectra with visible Mie scattering or edge effect. For ATR experiments, spectra were collected in reflection mode with a 10x10 µm² aperture size giving a resolution of 4.1x4.1 µm² on sample.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 30

IR spectral analysis IR spectra and chemical maps were processed using the OMNIC software. Analyses were performed either on original spectra or on their second derivatives, in order to increase the number of discriminative features present in spectra. A baseline correction was applied from 1450 cm-1 to 1735 cm-1 for all spectra. Derivation of the spectra to the second order was achieved by using a Savitsky-Golay algorithm with a seven-points filter and a polynomial order three 36. Maps were collected on pre-defined areas encompassing fluorescently labeled cytoplasmic or nuclear inclusions, with an aperture of 6 x 6 µm2 and step sizes of 3 to 4 µm. They were processed to display relative protein secondary structure by calculating the area ratio of a range attributed to β-sheet (1625-1631 cm-1) to one assigned to α-helix (1654-1660 cm-1), with a linear baseline of 1300-1800 cm-1.

Statistical analysis We used the multivariate data analysis software Unscrambler (Camo, Norway) to perform Principal Component Analysis (PCA). PCA allows a simple exploration of numerous spectral data and extracts spectral information necessary to classify the spectra. It was necessary to process our data prior to analysis in order to eliminate variations due to physical effects such as sample thickness and Mie scattering. Offset Correction was applied to subtract baseline drift from 1450 to 1735 cm-1. Unit vector normalization was applied to compensate for intensity changes due to nonhomogeneous sample thickness. This also eliminated any increase in absorption due 8 ACS Paragon Plus Environment

Page 9 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


to the inclusions being potentially denser than the surrounding tissue. PCA was performed on the amide I range (from 1580 to 1720 cm-1) using the Nonlinear Iterative Partial Alternating Square (NIPALS) algorithm. Results were displayed in the Score Plot, which revealed the distribution of spectra in the variance space and in the Loading Plot, which showed the spectral information behind the Score Plot.

RESULTS R6/2 mice possess inclusions in virtually all brain regions as well as in peripheral organs such as liver and muscle

37, 38

. The structure of the nuclear and

cytoplasmic inclusions (Nis and Cis, respectively) found in brain, liver and muscle of R6/2 mice was investigated by using sFTIR and ATR coupled to sFTIR. Inclusions were visualized by immunofluorescence labeling using an antibody directed against the N-terminal region of huntingtin

39

. IR spectra of the inclusions and the

surrounding tissue were collected. The protein secondary structure was determined from the amide I protein band. The assignment of spectroscopic IR bands to protein secondary structures is well established: IR bands located between 1648 cm-1 and 1660 cm-1 are assigned to α-helix, 1640-1648 cm-1 to unordered structures, 16231641 and 1670-1695 cm-1 to β-sheets

40

. An increase between 1615 cm-1 and 1630

cm-1 is characteristic of amyloid aggregates 41.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 30

sFTIR analysis does not detect any structural rearrangement in R6/2 inclusions Immunostaining carried out on frozen sections of R6/2 mice showed that the inclusions were nuclear in cortex, striatum and liver, cytoplasmic in muscle, and both nuclear and cytoplasmic in cerebellum. The diameter of the inclusions was 1-2 µm in all tissues examined. Therefore the mouse inclusions were relatively small and quite uniform in size (Figure 1A). In Huntington disease brain, Nis possess a diameter comparable to that of R6/2 inclusions, while in contrast Cis are much larger (average diameter of 4-5 µm), and very variable in size, (diameter between 1 and 10 µm) 15. Brain, liver and muscle of two R6/2 mice (G8 and G20) were submitted to sFTIR analysis. For each of the two mice, three sections of each tissue (cortex, striatum, cerebellum, liver and muscle) were examined. One spectrum was collected for each inclusion studied. One spectrum was also obtained from each of the inclusion-free cytoplasmic and nuclear samples used as controls. The total number of spectra collected is given in Table S-2. As the results obtained with the two R6/2 mice were nearly identical, only analyses of mouse G20 are shown (Figure 1B). Spectra of Nis and of nuclei free of inclusion were acquired and analyzed in cortex, striatum, cerebellum and liver. We were surprised to discover that the amide I bands of Nis were always superimposable with those of inclusion-free nuclei. Similarly, the spectra of cerebellar and muscle Cis closely resembled those of the corresponding cytoplasm (Figure 1B, left panels). We confirmed these observations by PCA analysis. Neither the spectra of Nis nor those of Cis could be separated from those of nuclei or cytoplasm, respectively 10 ACS Paragon Plus Environment

Page 11 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(Figure 1B, middle and right panels). As the samples were immunostained prior to data collection, we wondered whether antibodies and labels added to the FTIR contribution. Fluorescent labels have very high quantum yields and can thus be detected at very low concentrations. Therefore the concentration of antibodies and fluorescent labels attached to the inclusions were probably too low to be detected by FTIR microspectroscopy. In addition, IgGs are known to possess high amounts of βsheets in their secondary structures, up to 70% 42. The presence of detectable amount of IgG in the inclusions would thus increase the β-sheet contribution in the inclusion spectra. Since we detected no increase in β-sheet structure in the inclusions, an effect of the presence of IgG can be ruled out. We may conclude that the lack of spectral differences between the R6/2 inclusions and the cellular compartment where they were localized showed that R6/2 inclusions lacked structural rearrangement and did not possess an amyloid structure detectable by sFTIR.

sFTIR detects the amyloid nature of small Huntington disease inclusions The fact that we did not detect structural rearrangements in R6/2 inclusions could have resulted from the inability of sFTIR to probe such small structures. However, this seemed unlikely since we had previously detected an elevated peak at 1627 cm-1 in the JHD Nis whose diameter is comparable to that of the mouse inclusions 33.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 30

In order to determine whether we could detect structural rearrangement in small cytoplasmic inclusions as well, we acquired spectra of 1- to 2-µm diameter cortical Cis and of their surrounding cytoplasm in adult Huntington disease cases 1029 and 955. Results are shown in Figure 2. In both patients, the amide I bands of the inclusions differed from those of their surrounding cytoplasm by an increased absorbance in the 1600-1640 cm-1 region (left panels). Second derivatives revealed that the increased absorbance resulted from the increased contribution of a peak at 1627 cm-1 (right panels). A small increase in the contribution of a peak at 1693 cm-1 was also observed in case 955. These results demonstrated that the small cortical Cis of AHD patients were enriched in β sheets and were amyloid, just as are the larger inclusions found in these patients

33

. We concluded that the lack of rearrangement

observed in R6/2 inclusions was genuine and was not due to lack of sensitivity of the method. To further confirm these results, we generated chemical maps representing the β-sheet/α-helix ratio. For this, we photographed areas encompassing fluorescently labeled Nis in cortical sections of R6/2 mouse G20. We then acquired matching IR spectra and processed them to create the maps. The representative map of an R6/2 Nis is illustrated in Figure 3A. The immunolabeled area (upper-left panel) did not correspond with a cytoplasmic region showing high β-sheet/α-helix ratio (uppermiddle and upper-right panels). This was confirmed by the fact that the spectrum of the Nis closely paralleled that of the surrounding cytoplasm (lower-left panel). As a consequence, the corresponding second derivative spectra showed only negligible 12 ACS Paragon Plus Environment

Page 13 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


differences (lower-right panel). The absence of an elevated peak at 1627 cm-1 in R6/2 Nis confirmed that they were nonamyloid. The results obtained for R6/2 inclusions contrasted those derived from small Huntington disease Cis (Figure 3B) since in the latter the immunolabeled area (upperleft panel) specifically corresponded with a region of very high β-sheet/α-helix ratio (upper middle and upper-right panels). The amide I band of the human inclusions differed from that of the surrounding cytoplasm by an increased β-sheet contribution and by a left enlargement (lower-left panel). Second derivative spectra revealed that the β-sheet contribution resulted from a large increase in the peak at 1627 cm-1, whereas the left enlargement resulted from elevated peaks at around 1681 cm-1 and 1693 cm-1 (lower-right panel). These results confirmed that small Huntington disease Cis were highly enriched in β-sheets and possessed an amyloid structure.

sFTIR analysis outside of the inclusions shows the absence of differences between R6/2 and control tissues In a previous study by sFTIR, we had demonstrated that in some Huntington disease cases, the amide I band outside of the inclusions showed increased absorbance in the β-sheet region. This had suggested that, in addition to inclusions, Huntington disease brain contains large amounts of free oligomers rich in β-sheets (1627 cm-1 and 1693 cm-1) and β-sheet/unordered structures (1639 cm-1) 33. In order to determine whether R6/2 cells were also enriched in β-sheets and β-sheet/unordered structures outside of the inclusions, we undertook a comparison of the amide I bands 13 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 30

of two R6/2 mice (G8 and G20) with those of two control mice (G6 and G47) in inclusion-free nuclei and cytoplasm of brain, muscle and liver. Results for nuclear inclusions are illustrated in Figure S1, those for cytoplasmic inclusions in Figure S2. Average amide I bands of R6/2 nuclei were virtually superimposable (cortex), marginally narrower (striatum and muscle) or slightly diminished at their right side (cerebellum and liver) compared to those of wt nuclei. Amide I bands of R6/2 nuclei never showed an increased β-sheet contribution, when compared to those of wildtype nuclei (Figure S-1, left panels). PCA analyses only partially separated both categories of spectra across the two first PCs (Figure S-1, middle and right panels). In cytoplasm, average amide I bands of R6/2 mice differed very little from those of the wild-type mice and these small differences could never be ascribed to a β-sheet contribution in R6/2 spectra (Figure S-2A, left panels), except for the cortex, where the right side of the average amide I band of R6/2 was slightly enlarged. However, analysis of the corresponding second derivative spectra did not show that the right enlargement in cortex was associated with increased peaks at either 1627 or 1639 cm-1 (Figure S-2B). PCA analysis could either not separate at all R6/2 from wild-type spectra or only separated them partially (Figure S-2A, middle and right panels). We conclude that nuclei and cytoplasm of R6/2 brain, liver and muscle were not enriched in either β-sheets or β-sheet/unordered structures, and therefore that in contrast to patients with Huntington disease, R6/2 mice do not contain detectable oligomers outside of the inclusions 33.


Page 15 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ATR-sFTIR confirmed the nonamyloid nature of R6/2 inclusions We then analyzed the mouse inclusions by ATR coupled to sFTIR, a technique that provides excellent quality IR data and improves resolution. All the mouse spectra illustrated until now in this study were obtained in transmission mode. This was necessary in order to have a faithful comparison with our previously published human spectra, all of which were obtained in transmission mode 33. As the absence of detectable β-sheets in mouse inclusions could be have been ascribed to lack of sensitivity and/or spatial resolution, it appeared necessary to confirm our results using the ATR mode. Figure 4A illustrates the immunofluorescence labeling of Nis in the hippocampus of R6/2 mouse G8 as it can be visualized through the ATR-system. Numerous Nis with a diameter between 1 and 2 µm were visible. Because of the ATR system, the image is somewhat fuzzy. We acquired by ATR-sFTIR spectra of Nis and of inclusion-free nuclei from the cortex of the G8 mouse (Figure 4B). We observed that both the amide I bands (upperleft panel) and the second derivative spectra (upper-right panel) of Nis were only marginally different from those of nuclei. The difference was due to a minor increase in the contribution of a peak at 1639 cm-1 in the inclusions. The assignment of the 1639 cm-1 wavenumber is equivocal because it is located in a region of overlap between affiliations to β-sheets and affiliations to unordered structures. The 1639 cm-1 wavenumber 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 33. In spite of this difference, the spectra of Nis could not be separated from 15 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 30

those of nuclei by PCA analysis (lower panels). In a second series of experiments we studied the hippocampus of the G20 mouse. As in cortex, we found a small increase in the inclusions at 1639 cm-1, but no separation by PCA (Figure 4C). In summary, we detected through the use of ATR-sFTIR minor structural differences in R6/2 brain between Nis and inclusion-free nuclei. These differences, observed in the two brain regions investigated, resulted from the fact that the inclusions showed a modest increase in the “β-sheet/unordered” peak at 1639 cm-1. However no increase in the contribution of the amyloid peak at 1627 cm-1 was detected. We concluded that, although mouse inclusions might contain small amounts of β-sheet structures, they did not contain amyloid structures. These results were in agreement with those obtained by transmission sFTIR, as they showed that R6/2 inclusions were nonamyloid.

DISCUSSION Alzheimer’s disease and prion diseases have both been linked to misfolding of disease-specific amyloidogenic proteins. These proteins form toxic amyloid fibrils, which self-replicate in vitro and in vivo 43-45, acting as pathogenic seeds for amyloidplaque formation. In Huntington disease also, evidence has been provided as to the toxic nature of amyloid aggregates of the expanded polyQ 33, 46. We demonstrate here by sFTIR that the proteins aggregated in the inclusions of R6/2 mice lack structural rearrangements and thus are nonamyloid. Therefore these inclusions differ from most of those found in the brain regions affected by Huntington disease, which possess 16 ACS Paragon Plus Environment

Page 17 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


amyloid β-sheets of different kinds

33

. Although the brain of R6/2 mice develops

numerous inclusions, it shows little neuronal death

47-50

. This is in contrast to the

cortex and the striatum of patients with Huntington disease, where the presence of numerous inclusions correlates with extensive neuronal loss 14 and the severity of the disease

51, 52

. We propose that difference in secondary structure explains why R6/2

inclusions do not cause cell death. Nearly 20 diseases, both neurological and non-neurological, are characterized by the deposition of amyloid aggregates 53. These protein deposits are thought to be the cause of the diseases

54

. In the non-neurological diseases, large quantities of

aggregates form in the extracellular space and physically impair the functioning of the organs in which they occur 55. In diseases of the central nervous system, such as Huntington disease, the aggregates are intracellular and presumably disrupt the normal function of the cell

56

. In contrast to the proteins of the unstructured

aggregates that we have found in the neurons of the R6/2 mouse, the proteins in amyloid aggregates undergo a change in secondary structure and expose hydrophobic side-chains that are normally not exposed. These side-chains might interact inappropriately with cellular components thus causing neuronal dysfunction and death 56. Although protein misfolding and aggregation are recognized as critical events in Huntington disease pathogenesis, the role of inclusions has long been controversial. The controversy springs mainly from the existence of inclusions without neuronal death and of neuronal death without inclusions

57-59

. The various experimental 17

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 30

conditions that have been used have presumably resulted in the formation of inclusions with various structures, which in turn have have mild, severe, neutral or even beneficial effects on cell viability. The results presented in this paper provide an explanation for the lack of correlation between inclusions and neuronal death in R6/2 mice, and lend further support to the notion that only certain structural changes in the protein aggregates are linked to neuronal death 33, 46, 60. Inclusions are thought to assemble from submicroscopic aggregates such as oligomers and protofibrils. These forms of aggregation are detected as elevated βsheet structures located outside of the inclusions. We did not find any elevated βsheet structures in R6/2 brain whether inside or outside of the inclusions. NekookiMachida et al. have reported the presence of polyQ-containing amyloid aggregates in the brain of R6/2 mice 46. The material studied by these authors was greatly amplified prior to detection by FTIR, and therefore might have originated from amyloid oligomers present in such limited amounts that we could not directly detect them in our study. There is mounting evidence that the conformation of huntingtin aggregates largely depends on the context in which they are formed and is influenced by factors such as experimental conditions, subcellular localization, age, length of the polyQ, and sequence surrounding the polyQ

33, 46, 59, 61

. We propose that the structural

differences observed between R6/2 and Huntington disease inclusions originate from the length of the polyQ and from the protein context. R6/2 mice contain a short fragment of huntingtin with approximately 150 Qs. In contrast, the huntingtins of the 18 ACS Paragon Plus Environment

Page 19 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


patients that we studied contained 63 to 97 glutamine residues

33

within huntingtin

fragments much larger than that encoded by the transgene of the R6/2 mouse 15. We hypothesize that the mechanism of aggregation of longer huntingtin fragments with shorter polyQs differs from the mechanism at work in R6/2 mice. Morton and colleagues have studied the formation of inclusions in the brain of mice bearing very long polyQ expansions (from 170 to 450 Qs). These mice contain cytoplasmic inclusions that are very large and ultrastructurally different from those of R6/2 mice 49. It is clear from this example that the length of the polyQ has an influence on the nature of the inclusions. It would be interesting to determine whether the inclusions formed by very long polyQ expansions and in knock-in mice differ in structure from the inclusions of R6/2 mice.

CONCLUSION R6/2 mice have been widely used for testing drugs aimed at preventing or improving the course of Huntington disease. Reduction in the number of inclusions has been correlated with clinical improvement in R6/2 mice and has been used as a measure of drug efficacy 62. However, the fact that R6/2 inclusions differ in structure from Huntington disease inclusions raises questions as to the validity of the R6/2 model in the study of the mechanisms of polyQ aggregation. Future work should unravel the structure of inclusions in other animal models of Huntington disease, to determine if they reproduce more adequately the characteristics of the aggregates



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 30

found in patients with Huntington disease. It would be of particular interest to determine the structure of inclusions in knock-in mice such as YAC128 63, 64.

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

CONFLICT OF INTEREST DISCLOSURE The authors declare no competing financial interest.

REFERENCES 1. David, G.; Abbas, N.; Stevanin, G.; Dürr, A.; Yvert, G.; Cancel, G.; Weber, C.; Imbert, G.; Saudou, F.; Antoniou, E.; Drabkin, H.; Gemmill, R.; Giunti, P.; Benomar, A.; Wood, N.; Ruberg, M.; Agid, Y.; Mandel, J.; Brice, A. Nat Genet 1997, 17, 65-70. 2. The Huntington's Disease Collaborative Research Group. Cell 1993, 72, 971-983. 3. La Spada, A.; Wilson, E.; Lubahn, D.; Harding, A.; Fischbeck, K. Nature 1991, 352, 77-79. 4. Koide, R.; Ikeuchi, T.; Onodera, O.; Tanaka, H.; Igarashi, S.; Endo, K.; Takahashi, H.; Kondo, R.; Ishikawa, A.; Hayashi, T. Nat Genet 1994, 6, 9-13. 5. Imbert, G.; Saudou, F.; Yvert, G.; Devys, D.; Trottier, Y.; Garnier, J.; Weber, C.; Mandel, J.; Cancel, G.; Abbas, N.; Dürr, A.; Didierjean, O.; Stevanin, G.; Agid, Y.; Brice, A. Nat Genet 1996, 14, 285-291. 6. Kawaguchi, Y.; Okamoto, T.; Taniwaki, M.; Aizawa, M.; Inoue, M.; Katayama, S.; Kawakami, H.; Nakamura, S.; Nishimura, M.; Akiguchi, I. Nat Genet 1994, 8, 221-228. 7. Orr, H.; Chung, M.; Banfi, S.; Kwiatkowski, T. J.; Servadio, A.; Beaudet, A.; McCall, A.; Duvick, L.; Ranum, L.; Zoghbi, H. Nat Genet 1993, 4, 221-226. 8. Pulst, S.; Nechiporuk, A.; Nechiporuk, T.; Gispert, S.; Chen, X.; Lopes-Cendes, I.; Pearlman, S.; Starkman, S.; Orozco-Diaz, G.; Lunkes, A.; DeJong, P.; Rouleau, G.; Auburger, G.; Korenberg, J.; Figueroa, C.; Sahba, S. Nat Genet 1996, 14, 269-276. 9. Sanpei, K.; Takano, H.; Igarashi, S.; Sato, T.; Oyake, M.; Sasaki, H.; Wakisaka, A.; Tashiro, K.; Ishida, Y.; Ikeuchi, T.; Koide, R.; Saito, M.; Sato, A.; Tanaka, T.; Hanyu, S.; Takiyama, Y.; Nishizawa, M.; Shimizu, N.; Nomura, Y.; Segawa, M.; Iwabuchi, K.; Eguchi, I.; Tanaka, H.; Takahashi, H.; Tsuji, S. Nat Genet 1996, 14, 277-284. 10. Zhuchenko, O.; Bailey, J.; Bonnen, P.; Ashizawa, T.; Stockton, D.; Amos, C.; Dobyns, W.; Subramony, S.; Zoghbi, H.; Lee, C. Nat Genet 1997, 15, 62-69. 20 ACS Paragon Plus Environment

Page 21 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


11. Koide, R.; Kobayashi, S.; Shimohata, T.; Ikeuchi, T.; Maruyama, M.; Saito, M.; Yamada, M.; Takahashi, H.; Tsuji, S. Hum Mol Genet 1999, 8, 2047-2053. 12. Zuhlke, C.; Hellenbroich, Y.; Dalski, A.; Kononowa, N.; Hagenah, J.; Vieregge, P.; Riess, O.; Klein, C.; Schwinger, E. Eur J Hum Genet 2001, 9, 160-164. 13. Vonsattel, J.; Myers, R.; Stevens, T.; Ferrante, R.; Bird, E.; Richardson, E. J. J Neuropathol Exp Neurol 1985, 44, 559-577. 14. DiFiglia, M.; Sapp, E.; Chase, K.; Davies, S.; Bates, G.; Vonsattel, J.; Aronin, N. Science 1997, 277, 1990-1993. 15. Hoffner, G.; Island, M.; Djian, P. J Neurochem 2005, 95, 125-136. 16. Landles, C.; Sathasivam, K.; Weiss, A.; Woodman, B.; Moffitt, H.; Finkbeiner, S.; Sun, B.; Gafni, J.; Ellerby, L.; Trottier, Y.; Richards, W.; Osmand, A.; Paganetti, P.; Bates, G. J Biol Chem 2010, 285, 8808-8823. 17. Hoffner, G.; Djian, P. Biochimie 2002, 84, 273-278. 18. Wanderer, J.; Morton, A. Histochem Cell Biol 2007, 127, 473-484. 19. Perutz, M.; Windle, A. Nature 2001, 412, 143-144. 20. Nagai, Y.; Popiel, H. Curr Pharm Des 2008, 14, 3267-3279. 21. Legleiter, J.; Lotz, G.; Miller, J.; Ko, J.; Ng, C.; Williams, G.; Finkbeiner, S.; Patterson, P.; Muchowski, P. J Biol Chem 2009, 284, 21647-21658. 22. Saunders, H.; Bottomley, S. Protein Eng Des Sel 2009, 22, 447-451. 23. Thakur, A.; Jayaraman, M.; Mishra, R.; Thakur, M.; Chellgren, V.; Byeon, I.; Anjum, D.; Kodali, R.; Creamer, T.; Conway, J.; Gronenborn, A.; Wetzel, R. Nat Struct Mol Biol 2009, 16, 380-389. 24. Legleiter, J.; Mitchell, E.; Lotz, G.; Sapp, E.; Ng, C.; DiFiglia, M.; Thompson, L.; Muchowski, P. J Biol Chem 2010, 285, 14777-14790. 25. Takahashi, T.; Katada, S.; Onodera, O. J Mol Cell Biol 2010, 2, 180-191. 26. Perutz, M.; Johnson, T.; Suzuki, M.; Finch, J. Proc Natl Acad Sci U S A 1994, 91, 53555358. 27. Scherzinger, E.; Lurz, R.; Turmaine, M.; Mangiarini, L.; Hollenbach, B.; Hasenbank, R.; Bates, G.; Davies, S.; Lehrach, H.; Wanker, E. Cell 1997, 90, 549-558. 28. Lathrop, R.; Casale, M.; Tobias, D.; Marsh, J.; Thompson, L. Proc Int Conf Intell Syst Mol Biol 1998, 6, 105-114. 29. Bevivino, A.; Loll, P. Proc Natl Acad Sci U S A 2001, 98, 11955-11960. 30. Chen, S.; Berthelier, V.; Hamilton, J.; O'Nuallain, B.; Wetzel, R. Biochemistry 2002, 41, 7391-7399. 31. Poirier, M.; Li, H.; Macosko, J.; Cai, S.; Amzel, M.; Ross, C. J Biol Chem 2002, 277, 41032-41037. 32. Takahashi, T.; Kikuchi, S.; Katada, S.; Nagai, Y.; Nishizawa, M.; Onodera, O. Hum Mol Genet 2008, 17, 345-356. 33. André, W.; Sandt, C.; Dumas, P.; Djian, P.; Hoffner, G. Anal Chem 2013, 85, 3765-3773. 34. Mangiarini, L.; Sathasivam, K.; Seller, M.; Cozens, B.; Harper, A.; Hetherington, C.; Lawton, M.; Trottier, Y.; Lehrach, H.; Davies, S.; Bates, G. Cell 1996, 87, 493-506. 35. Choo, L. P.; Wetzel, D. L.; Halliday, W. C.; Jackson, M.; LeVine, S. M.; Mantsch, H. H. Biophys J 1996, 71, 1672-1679. 36. Susi, H.; Byler, D. M. Biochem Biophys Res Commun 1983, 115, 391-397. 37. Woodman, B.; Butler, R.; Landles, C.; Lupton, M. K.; Tse, J.; Hockly, E.; Moffitt, H.; Sathasivam, K.; Bates, G. P. Brain Res Bull 2007, 72, 83-97. 38. Moffitt, H.; McPhail, G. D.; Woodman, B.; Hobbs, C.; Bates, G. P. PLoS One 2009, 4, e8025. 39. Hoffner, G.; Kahlem, P.; Djian, P. J Cell Sci 2002, 115, 941-948. 40. Barth, A. Biochim Biophys Acta 2007, 1767, 1073-1101. 41. Nilsson, M. R. Methods 2004, 34, 151-160. 42. Richardson, J. S. Adv Protein Chem 1981, 34, 167-339. 21 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 30

43. Petkova, A. T.; Leapman, R. D.; Guo, Z.; Yau, W. M.; Mattson, M. P.; Tycko, R. Science 2005, 307, 262-265. 44. Lu, J. X.; Qiang, W.; Yau, W. M.; Schwieters, C. D.; Meredith, S. C.; Tycko, R. Cell 2013, 154, 1257-1268. 45. Xiao, Y.; Ma, B.; McElheny, D.; Parthasarathy, S.; Long, F.; Hoshi, M.; Nussinov, R.; Ishii, Y. Nat Struct Mol Biol 2015, 22, 499-505. 46. Nekooki-Machida, Y.; Kurosawa, M.; Nukina, N.; Ito, K.; Oda, T.; Tanaka, M. Proc Natl Acad Sci U S A 2009, 106, 9679-9684. 47. Tobin, A. J.; Signer, E. R. Trends Cell Biol 2000, 10, 531-536. 48. Davies, S.; Turmaine, M.; Cozens, B.; DiFiglia, M.; Sharp, A.; Ross, C.; Scherzinger, E.; Wanker, E.; Mangiarini, L.; Bates, G. Cell 1997, 90, 537-548. 49. Morton, A. J.; Glynn, D.; Leavens, W.; Zheng, Z.; Faull, R. L.; Skepper, J. N.; Wight, J. M. Neurobiol Dis 2009, 33, 331-341. 50. Yu, Z. X.; Li, S. H.; Evans, J.; Pillarisetti, A.; Li, H.; Li, X. J. J Neurosci 2003, 23, 21932202. 51. Becher, M.; Kotzuk, J.; Sharp, A.; Davies, S.; Bates, G.; Price, D.; Ross, C. Neurobiol Dis 1998, 4, 387-397. 52. Sieradzan, K. A.; Mechan, A. O.; Jones, L.; Wanker, E. E.; Nukina, N.; Mann, D. M. Exp Neurol 1999, 156, 92-99. 53. Falk, R. H.; Comenzo, R. L.; Skinner, M. N Engl J Med 1997, 337, 898-909. 54. Dobson, C. M. Philos Trans R Soc Lond B Biol Sci 2001, 356, 133-145. 55. Tan, S. Y.; Pepys, M. B., Amyloidosis. Histopathology 1994, 25, 403-414. 56. Bucciantini, M.; Giannoni, E.; Chiti, F.; Baroni, F.; Formigli, L.; Zurdo, J.; Taddei, N.; Ramponi, G.; Dobson, C. M.; Stefani, M. Nature 2002, 416, 507-511. 57. Saudou, F.; Finkbeiner, S.; Devys, D.; Greenberg, M. Cell 1998, 95, 55-66. 58. Arrasate, M.; Mitra, S.; Schweitzer, E.; Segal, M.; Finkbeiner, S., Inclusion body formation reduces levels of mutant huntingtin and the risk of neuronal death. Nature 2004, 431, 805-810. 59. Hoffner, G.; Djian, P. Brain Sci 2014, 4, 91-122. 60. Hoffner, G.; Djian, P. Mol Neurobiol 2015, 52, 1297-1314. 61. Tonoki, A.; Kuranaga, E.; Ito, N.; Nekooki-Machida, Y.; Tanaka, M.; Miura, M. Genes Cells 2011, 16, 557-564. 62. Ferrante, R. J.; Andreassen, O. A.; Dedeoglu, A.; Ferrante, K. L.; Jenkins, B. G.; Hersch, S. M.; Beal, M. F. J Neurosci 2002, 22, 1592-1599. 63. Van Raamsdonk, J. M.; Warby, S. C.; Hayden, M. R. Brain Res Bull 2007, 72, 124-131. 64. Heng, M. Y.; Detloff, P. J.; Albin, R. L. Neurobiol Dis 2008, 32, 1-9.


Page 23 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure legends. Figure 1. Analysis of spectra of Nis and Cis in brain, liver and muscle of R6/2 mice. (A) Fluorescent labeling of Nis and Cis. Frozen sections of cortex, cerebellum and liver of R6/2 mouse G20 stained with an antibody against the N-terminal region of huntingtin (green) and counterstained with TO-PRO-3 for DNA (blue). Numerous Nis are detected in cortex and liver, whereas several Cis are observed in cerebellum. Scale bar for cortex: 10 µm. Scale bars for cerebellum and liver: 5 µm. (B) Spectral analysis of inclusions in R6/2 brain, liver and muscle. Spectra of Nis and Cis were compared with those of surrounding nucleoplasm and cytoplasm, respectively. Left panels show average spectra of inclusions in red, and those of nuclei and cytoplasm in black. Middle and right panels display the results of PCA. Score plots in middle panels and loading plots in right panels. The score plots correspond to PC1 vs PC2, with spectra of inclusions in red and spectra of surrounding nucleoplasm (for Nis) or cytoplasm (for Cis) in black. Spectra of Nis and nuclei were not visually distinguishable and could not be separated by PCA, whether in brain, liver or muscle. Spectra of Cis were not distinguishable from those of surrounding cytoplasm. Absence of a peak at 1627 cm-1 demonstrated that R6/2 Nis and Cis were not amyloid. R6/2 mouse G20 was used in these analyses. Cereb, cerebellum.

Figure 2. Analysis of spectra of small Cis in the cortex of adult Huntington disease cases 1029 and 955. Left panels show spectrum of a Cis (red) and of cytoplasm around it (black). Right panels illustrate corresponding second derivative 23 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 30

spectra (with same colors). Arrows and arrowhead mark enrichments in peaks attributed to β-sheet structures at 1627 cm-1 and 1693 cm-1, respectively. Peak at 1627 cm-1 signifies an amyloid structure. Therefore, in spite of their small size (comparable to that of R6/2 inclusions), the amyloid nature of small Huntington disease Cis was readily detected.

Figure 3. Chemical maps of β-sheet content in R6/2 Nis and small Huntington disease Cis. (A) Immunofluorescent labeling and chemical map obtained from an area encompassing cortical Nis of R6/2 mouse G20. Upper-left panel shows Nis fluorescently labeled in green (white arrows). Upper-middle panel illustrates unprocessed representation of β-sheet/α-helix ratios using a color scale that goes from red (highest) to blue (nil); upper right panel represents the corresponding interpolated image. No increase in β-sheet content at the site of the two inclusions was detected in R6/2 mouse (white arrows). Lower panels show analyses of representative IR spectra acquired on map. Lower-left panel displays spectra collected on the surface of inclusions (red) and on surrounding tissue (black). Lowerright panel shows corresponding second derivative spectra with same colors. Arrow indicates the position of the 1627 cm-1 wavenumber: no peak is detected in R6/2 Nis, thus confirming their nonamyloid structure. (B) Small Cis in striatum of Huntington disease case 3701. Order of panels is the same as in (A). In contrast to R6/2 mouse, Huntington disease patient shows correspondence between inclusion and highest βsheet content (upper panels). Surface of high β-sheet content is larger than inclusion 24 ACS Paragon Plus Environment

Page 25 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


because of the lower resolution of chemical map. Arrow in lower right panel indicates a peak at 1627 cm-1 wavenumber and therefore shows amyloid nature of inclusion. Arrowheads mark enrichment in peaks at 1681 and 1693 cm-1. Scale bars, 5 µm.

Figure 4. ATR-sFTIR analysis of Nis in the brain of R6/2 mice. (A) Frozen section of the R6/2 mouse G8 was stained with an antibody against the N-terminal region of huntingtin (green) and counterstained with DAPI for DNA (blue). Numerous Nis are detected in hippocampus. Minor distortion and fuzziness resulted from the use of the ATR system. (B) Spectral analysis of cortical Nis. Upper-left panel shows average spectra of Nis and nuclei; upper-right panel, second derivative spectra; lower-left panel, score plots of PCA (PC1 vs PC2); lower-right panel, loading plots of PCA. Plots for Nis are in red and surrounding cytoplasm in black. Arrowhead and arrow show position of 1639 and 1627 cm-1 wavenumber, respectively. (C) Spectral analysis of hippocampal Nis. In both cortical and hippocampal inclusions, only minor differences distinguished spectra and second derivative spectra of Nis from those of surrounding nuclei, with a marginal increase in the peak at 1639 cm-1. Spectra of Nis and nuclei were not separated by PCA. Therefore, use of ATR-sFTIR confirmed that R6/2 inclusions possessed a nonamyloid nature. Hippoc, hippocampus.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 30

Figure 1 26 ACS Paragon Plus Environment

Page 27 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 30

Figure 3


Page 29 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4 29 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

84x47mm (300 x 300 DPI)

ACS Paragon Plus Environment

Page 30 of 30

2 Mice Are Not Amyloid and ... - ACS Publications

Recommend Documents