Distinct conformations of in vitro and in vivo amyloids of huntingtin-exon1 show different cytotoxicity

Abstract

A hallmark of polyglutamine diseases, including Huntington disease (HD), is the formation of beta-sheet-rich aggregates, called amyloid, of causative proteins with expanded polyglutamines. However, it has remained unclear whether the polyglutamine amyloid is a direct cause or simply a secondary manifestation of the pathology. Here we show that huntingtin-exon1 (thtt) with expanded polyglutamines remarkably misfolds into distinct amyloid conformations under different temperatures, such as 4 degrees C and 37 degrees C. The 4 degrees C amyloid has loop/turn structures together with mostly beta-sheets, including exposed polyglutamines, whereas the 37 degrees C amyloid has more extended and buried beta-sheets. By developing a method to efficiently introduce amyloid into mammalian cells, we found that the formation of the 4 degrees C amyloid led to substantial toxicity, whereas the toxic effects of the 37 degrees C amyloid were very small. Importantly, thtt amyloids in different brain regions of HD mice also had distinct conformations. The thermolabile thtt amyloid with loop/turn structures in the striatum showed higher toxicity, whereas the rigid thtt amyloid with more extended beta-sheets in the hippocampus and cerebellum had only mild toxic effects. These studies show that the thtt protein with expanded polyglutamines can misfold into distinct amyloid conformations and, depending on the conformations, the amyloids can be either toxic or nontoxic. Thus, the amyloid conformation of thtt may be a critical determinant of cytotoxicity in HD.

Fig. 1.

thtt protein with expanded polyglutamines misfolds into distinct amyloid conformations in vitro. (A) EM images of thttQ42-4 °C and thttQ42-37 °C amyloids. (Scale bar, 100 nm.) (B) CD spectra of thttQ42-4 °C (thin, blue), thttQ42-37 °C (thin, red), thttQ62-4 °C (bold, blue), and thttQ62-37 °C (bold, red) amyloids. (C) FT-IR spectra of thttQ42-4 °C (thin, blue), thttQ42-37 °C (thin, red), thttQ62-4 °C (bold, blue), and thttQ62-37 °C (bold, red) amyloids. (D) Thermal stability of thttQ42-4 °C, thttQ42-37 °C, thttQ42-4 °C and thttQ42-37 °C amyloids. The bands indicate monomeric thttQ42/62 solubilized from thttQ42/62 amyloids by the heat treatment. (E) The band intensity in (D) was plotted against temperature for thttQ42-4 °C (thin, blue), thttQ42-37 °C (thin, red), thttQ62-4 °C (thick, blue), and thttQ62-37 °C (thick, red) amyloids. (F) Reactivity of thttQ42-4 °C and thttQ42-37 °C amyloids with various antibodies. A filter trap assay was performed in the presence of 0.5% Triton X-100 (Left) or 1% SDS (Right) and processed for immunoblotting against a 1C2, 3B5H10, or anti-htt antibody. Coomassie Brilliant Blue (CBB) staining is also shown below. Values show relative intensities of the spots.

Fig. 2.

Different conformations of thtt amyloids show distinct cytotoxicity in neuro2a cells. (A) Significant acceleration of thttQ150-GFP aggregation by introduction of in vitro thttQ42 amyloids. Buffer alone (Upper) or in vitro thttQ42 amyloids (Lower) were introduced into thttQ150-GFP stable neuro2a cells. Shown are fluorescent (Left) and DIC (Right) images of the thttQ150-GFP cells after 15 h of thttQ150-GFP expression and cell differentiation. (Scale bar, 250 μm.) (B) Buffer alone, GSTthttQ42 monomer, thttQ42 amyloids, GSTthttQ62 monomer, thttQ62 amyloids, or BSA aggregates were introduced into stable thttQ150-GFP neuro2a cells. The number of cells with thttQ150-GFP foci was counted after 15 h of the thttQ150-GFP expression and cell differentiation. Values are mean ± SD. (C) Thermal stability of the thttQ60-GFP amyloids formed in the presence of in vitro thttQ42-4 °C (thttQ60-GFP[thttQ42-4 °C]) or thttQ42-37 °C amyloid “seeds” (thttQ60-GFP[thttQ42-37 °C]). (D) The band intensity of thttQ60-GFP[thttQ42-4 °C] (blue) or thttQ60-GFP[thttQ42-37 °C] (red) amyloids in C was plotted against temperature. (E, F) Buffer alone, in vitro thttQ42-4 °C amyloids, thttQ42-37 °C amyloids, GSTthttQ42 monomer, or BSA aggregates were introduced into thttQ16-, Q60-, or Q150-GFP neuro2a cells under a regulatable promoter. The thtt expression and cell differentiation started after 3 h of the in vitro amyloid transduction. The number of cells with thtt-GFP foci was counted after 15 h (E) and cell viability was examined by MTT assay after 4 days of the thtt expression and cell differentiation (F). *, P < 0.01. Values are mean ± SD.

Fig. 3.

thtt amyloids in different brain regions from R6/2 mice show distinct conformations. (A and B) Aggregation profile is shown of in vitro thttQ42 in the presence of thtt amyloids from R6/2 mice (A) or corresponding insoluble fractions from wild-type mice (B). Black, red, blue, yellow, and green lines show in vitro thttQ42 fibrillization in the absence (black) or presence of in vivo aggregates from cerebral cortex, striatum, hippocampus, and cerebellum, respectively. (C) Thermal stability of in vitro thttQ42 amyloids formed in the absence or presence of thtt amyloids from different brain regions of R6/2 mice. (D) The band intensity of thtt amyloids in different brain regions of R6/2 mice in C was plotted against temperature. (E and F) Structural analysis of thtt amyloids in different brain regions of R6/2 mice. (E) FT-IR spectra of in vitro thttQ42 amyloids formed in the absence or presence of thtt amyloids from different brain regions of R6/2 mice. (F) Difference FT-IR spectra obtained by subtraction of a spectrum of spontaneously formed thttQ42 (without seeds) from those of thttQ42 amyloids in different brain regions of R6/2 mice.

Fig. 4.

Distinct conformations of thtt amyloids in different brain regions of R6/2 mice show distinct cytotoxicity in neuro2a cells. (A and B) Buffer alone or in vitro thttQ42 amyloids formed in the presence of thtt amyloids from different brain regions of R6/2 or WT mice were introduced into stable thttQ150-GFP neuro2a cells. The number of cells with thttQ150-GFP foci was counted after 15 h of thttQ150-GFP expression and cell differentiation (A), and cell viability was examined after 4 days by MTT assay (B). Tg, WT, CTX, ST, HP, and CBL denote R6/2 mice, wild-type mice, cerebral cortex, striatum, hippocampus, and cerebellum, respectively.*, P < 0.05. Values are mean ± SD. (C) A proposed mechanism of conformation-dependent cytotoxicity of thtt amyloid.