Inclusions of R6/2 Mice Are Not Amyloid and Differ Structurally from Those of Huntington Disease Brain

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

Structure of Inclusions of Huntington’s Disease Brain Revealed by Synchrotron Infrared Microspectroscopy: Polymorphism and Relevance to Cytotoxicity

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

Mapping and optical image of a birefringent structure.

<p>The poorly soluble foscarnet can be detected and quantified after a mapping of the biopsy thanks to the characteristic peak at 936 cm^-1.</p

Selected examples of infrared spectra from biopsies.

<p>a) Amorphous silica identified by a band at 1102 cm⁻¹, b) sodium hydrogen urate monohydrate identified by specific bands at 3600 and 1004 cm⁻¹, c) several calcium phosphates including whitlockite (peaks at 1080, 1025 cm⁻¹ and associated shoulders, d) octacalcium phosphate and carbapatite, identified by a shoulder at 1119 cm⁻¹, e) normal tissue, with signal of water (3300 cm⁻¹ and peaks around 1600 cm⁻¹) and proteins (peaks at 2900 cm⁻¹).</p

Optical image and mapping of BR165 biopsy (scale from blue to red with increasing concentration), and FT-IR spectra of crystals.

<p>a) Optical image of BR165, b) carbapatite map (done at 1030 cm^-1), c) sodium hydrogen urate monohydrate map (done at 3600 cm^-1), d) FT-IR spectra of those compounds.</p

Second derivatives of IR spectra.

<p>Spectra recorded on steatosis or non-steatotic hepatocytes were superimposed (upper panel). Second derivatives of the spectra were calculated and superimposed in the frequency domain 2600–3200 cm⁻¹ (lower panel).</p

Spectroscopic analysis of non-steatotic hepatocytes on fatty liver.

<p>Spectroscopic analyses were performed on periportal hepatocytes on tissue section from normal or fatty liver. The video image is shown (left panel) with the corresponding averaged IR spectra (right panel) and the chemical imaging of the sum of DAG (middle panel).</p

Assignment of frequency to chemical functions.

<p>From <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007408#pone.0007408-Dreissig1" target="_blank">[19]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007408#pone.0007408-Banyay1" target="_blank">[20]</a>.</p

Histological features of steatosis.

<p>Tissue sections of 6 µm thickness were performed on paraffin embedded biopsies from normal liver or from fatty liver and stained with HES (hematoxylin, eosin and safran). Normal hepatic lobule without steatosis (left panel) or fatty liver area exhibiting macrovacuolar and microvesicular steatosis (right panel) are shown. Upper panel: ×100, lower panel: ×400. PT: portal tract, BD: biliary duct, PV: portal vein, HA: hepatic artery, CLV: centrilobular vein, SV: steatotic vacuole.</p

History of patients and origin of samples.

<p>History of patients and origin of samples.</p