Identical oligomeric and fibrillar structures captured from the brains of R6/2 and knock-in mouse models of Huntington's disease

Abstract

Huntington's disease (HD) is a late-onset neurodegenerative disorder that is characterized neuropathologically by the presence of neuropil aggregates and nuclear inclusions. However, the profile of aggregate structures that are present in the brains of HD patients or of HD mouse models and the relative contribution of specific aggregate structures to disease pathogenesis is unknown. We have used the Seprion ligand to develop a highly sensitive enzyme-linked immunosorbent assay (ELISA)-based method for quantifying aggregated polyglutamine in tissues from HD mouse models. We used a combination of electron microscopy, atomic force microscopy (AFM) and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) to investigate the aggregate structures isolated by the ligand. We found that the oligomeric, proto-fibrillar and fibrillar aggregates extracted from the brains of R6/2 and HdhQ150 knock-in mice were remarkably similar. Using AFM, we determined that the nanometre globular oligomers isolated from the brains of both mouse models have dimensions identical to those generated from recombinant huntingtin exon 1 proteins. Finally, antibodies that detect exon 1 Htt epitopes differentially recognize the ligand-captured material on SDS-PAGE gels. The Seprion-ligand ELISA provides an assay with good statistical power for use in preclinical pharmacodynamic therapeutic trials or to assess the effects of the genetic manipulation of potential therapeutic targets on aggregate load. This, together with the ability to identify a spectrum of aggregate species in HD mouse tissues, will contribute to our understanding of how these structures relate to the pathogenesis of HD and whether their formation can be manipulated for therapeutic benefit.

Figure 1.

Seprion ligand quantification of aggregate load in tissues from HD mouse models. Quantification of aggregate levels in brain regions (A) and peripheral tissues (B) of R6/2 mice. (C) Power analysis indicating the number of R6/2 mice required to have an 80% chance of detecting a specific percentage reduction in aggregate load in response to a therapeutic intervention initiated at 4 weeks of age and terminated at either 8 weeks or 12 weeks of age (P > 0.05). Quantification of aggregate levels in brain regions (D) and peripheral tissues (E) of Hdh^Q150/Q150 mice. In all cases, n = 6/genotype/age and the age at which statistically significant aggregate levels can first be detected is indicated by the corresponding P-value. Black bars = R6/2 or HdhQ150, gray bars = wild-type. Ctx = cerebral cortex; Hipp = hippocampus; Str = striatum; Cerb = cerebellum; Br St = brain stem; Buf = buffer.

Figure 2.

Immuno-EM analysis of captured material from R6/2 and Hdh^Q150/Q150 cortex. Representative examples of immunogold labelling of Htt aggregates with MW8, MW1 and 3B5H10 captured from the cortex of (A) R6/2 mice aged 2 and 12 weeks of age and (B) Hdh^Q150/Q150 mice aged 2 and 22 months of age. MW8 immunolabels fibrillar structures at each age in both the R6/2 and Hdh^Q150/Q150 tissue. Oligomers and protofibrils (A) and oligomers (B) detected by MW1 and protofibrils (A) and oligomers (B) detected by 3B5H10 are shown. Scale bar = 100 nm.

Figure 3.

Variation in the oligomeric and fibrillar structures isolated from R6/2 and Hdh^Q150/Q150 brains before phenotype onset and at late-stage disease. (A) Immunolabelled ‘shadows’, (B–E) oligomeric/proto-fibrillar structures and (F–I) immature fibrils and fibrillar structures that have been consistently captured by the Seprion ligand bead-captured material as identified by transmission EM and immunogold-labelling. The right-hand table indicates the age at which a structure was identified in each of the mouse models and if present, whether it was associated with immunogold labelling with MW1, 3B5H10 or MW8. Scale bar = 100 nm.

Figure 4.

Still images of the electron tomography of oligomeric/proto-fibrillar structures captured from R6/2 and Hdh^Q150/Q150 brains for which the three-dimensional structure is shown in Supplementary Material, Figure S1. (A) Electron tomography of oligomeric structures illustrated in Figure 3D and B of the filamentous structures illustrated in Figure 3G immunolabelled with MW8.

Figure 5.

AFM analysis of nanometre globular aggregates from R6/2 and Hdh^Q150/Q150 brains. (A) AFM of Seprion ligand-captured material from both R6/2 and Hdh^Q150/Q150 cortex at the ages indicated fractionated to resolve nanometre globular oligomers. Observed aggregates were similar to those generated by the in vitro incubation of exon 1 Htt proteins with 53Q or 46Q at 2 µm for 1 h. Scale bar = 400 nm. (B) Histograms collating the height, diameter, volume and aspect ratio (longest width/shortest width) of the R6/2 and Hdh^Q150/Q150 aggregates measured at all ages. There is a remarkable similarity in the dimensions of the aggregates isolated from the R6/2 and Hdh^Q150/Q150 mice. These are comparable to those generated by the aggregation of 2 µm exon 1 Htt proteins with 53Q or 46Q in vitro for 1 h.

Figure 6.

SDS–PAGE and immunoblotting of Seprion bead-captured aggregates from R6/2 cortex. Seprion ligand bead-captured material was fractionated by 10% SDS–PAGE alongside the corresponding mouse lysates. Blots were immunoprobed with S830 (A), MW8 (B), MW1 (C,G) or 3B5H10 (D–F) antibodies. In (C) and (D) material had been captured from the same lysates, fractionated on two gels and subsequently immunoprobed with MW1 (C) and 3B5H10 (D). The blot in (C) was stripped and reprobed with 3B5H10 (F). Asterisks denote high molecular bands detected by S830, 3B5H10 and MW1 that enter the resolving gel. Arrows indicate fragments that resolve at a size similar to monomeric Htt that are differentially recognized by the Htt antibodies. T = R6/2 transgenic; Wt = wild-type; B = buffer; W = well; In = interface between the stacking and resolving gel.