Aggregation of Expanded Huntingtin in the Brains of Patients with Huntington Disease

Abstract

Huntingtin containing an expanded polyglutamine causes neuronal death and Huntington disease. Although expanded huntingtin is found in virtually every cell type, its toxicity is limited to neurons of certain areas of the brain, such as cortex and caudate/putamen. In affected areas of the brain, expanded huntingtin is not found in its intact monomeric form. It is found instead in the form of N-terminal fragments, oligomers and polymers, all of which accumulate in the cortex. Whereas the oligomer is mostly soluble, the polymers and the fragments associate with each other and with other proteins to form the insoluble inclusions characteristic of the disease. It is likely that the aggregates containing expanded huntingtin are toxic to neurons, but it remains to be determined whether the oligomer or the inclusion is the toxic species.

Acknowledgements

Guylaine Hoffner was supported by the Bettencourt-Schueller Foundation and the Region Ile de France, and research was financed by CNRS and the Institut des Maladies Rares. We are grateful to the Proceedings of the National Academy of Science, the Journal of Neurochemistry, The International Society for Neurochemistry and Blackwell Publishing for permission to reproduce the figures.

Figures and Tables

Figure 1 Aggregation of Q₂₀₅ in PC12 cells. (A) A 24 h induction by ponasterone A was suffisant to induce the formation of large aggregates containing EGFP-Q₂₀₅ in the cell line PC12-EGFP-Q₂₀₅. Each cell contained a small number (one to three usually) of large aggregates and showed a very low level of diffuse cytoplasmic fluorescence. This suggested that most of the soluble recombinant protein had been recruited to large aggregates. However, a number of cells displayed either a multitude of small aggregates, or a high level of diffuse cytoplasmic fluorescence with no evidence of aggregation. These differences are probably related to differences in the time-course of aggregation in different cells. (B) Purified inclusions from the cell line PC12-Q₂₀₅ were either treated or not with formic acid (FA), boiled in SDS/2-ME and submitted to electrophoresis through 7.5% polyacrylamide. After transfer to nitrocellulose, the proteins were stained with the 1C2 antibody. In the absence of formic acid treatment, no signal was detected. After treatment with 90% formic acid for one hour at 37°C, electrophoresis revealed a strong band corresponding to a monomer (mnQ₂₀₅) with an apparent molecular mass of ∼100 kDa; the deduced molecular mass is 36,340, but expanded polyQ reduces mobility in gel electrophoresis. A second band with a considerably higher molecular mass was clearly stained and corresponded to an oligomer (oligQ₂₀₅). Reproduced from Iuchi et al.30

Figure 2 State of expanded huntingtin in Huntington disease. (A) Crude extracts of cortex (Cx) and cerebellum (Cb) were pre-incubated in formic acid for 1 h at 37°C. Western blots prepared after electrophoresis on a 4% polyacrylamide gel were stained consecutively with the anti-N-terminal and the 1C2 antibody. All samples of cortex produced a broad oligomeric band of expanded huntingtin. No such band was found in the cerebellum. In contrast, all of the cerebellar samples, but none of the cortical ones, yielded a clear band of monomeric expanded huntingtin. (B) Iodixanol-purified nuclei, either treated or not with formic acid, were analyzed by electrophoresis through an 8% polyacrylamide gel. After transfer to nitrocellulose, proteins were stained with the 1C2 antibody. In the absence of formic acid pre-treatment, no resolved band is present. The only detectable signal consist of a weakly stained band at the top of the 4% stacking gel. After treatment with formic acid, electrophoresis reveals a strongly stained polymer (pol.) at the well, a compressed oligomer (olig.) band whose mean mobility corresponds to that of the free cytosolic oligomer in A, and a broad band of fragments (frag.) with molecular masses ranging from ∼50 to 150 kDa. We may conclude that the nuclei contain insoluble polymer, oligomer, and fragments, all of which possess expanded polyQ. Reproduced from Iuchi et al.30

Figure 3 Polymeric state of expanded huntingtin in Huntington disease. Purified nuclei of cortex and cerebellum of Huntington disease patient 3815 and of controls were extracted with concentrated formic acid. The suspension was diluted 10-fold in Tris/SDS/2-ME, boiled for five minutes, and immediately passed through cellulose acetate filters. The retained material was visualized with antibody 1C2. Control SALDAV 080010 was a lymphoblast line possessing a huntingtin with 79 glutamine residues. Rat cortex, whose huntingtin possesses Q₈, should not be stainable by 1C2. Cortex 3815: 20,000 nuclei gave a strong signal. The corresponding cerebellum, SALDAV 080010, and rat cortex gave weak signals. Cortex of patient 3123: 120,000 nuclei produced a weaker signal than 20,000 nuclei of cortex 3815, in keeping with a number of inclusions smaller in case 3123 than in case 3815. Arrow indicates control without nuclei. Resistance to formic acid of the cortical polymers of the two patients strongly suggests stabilization of the polymers by covalent bonds. Reproduced from Iuchi et al.30

Figure 4 State of expanded huntingtin in purified aggregates of patients with Huntington disease. (A) Inclusions were purified from cortex of patient 3815. Samples at the various stages of purification were preincubated or not in formic acid and analyzed by Western blotting with the 1C2 antibody: (Cr) Crude extract, (1) after dialysis, (2) after low speed centrifugation, (3) final pellet of purified inclusions. In the absence of formic acid treatment, only a faint signal was detected in the well. After treatment with 90% formic acid for one hour at 37°C, electrophoresis of the cortical extract revealed an oligomer band (olig.), a polymer (pol.) and a broad range of fragments (frag.) with molecular masses ranging from ∼50 to 150 kDa. No such material was released from the cerebellum. α-tubulin, an extremely abundant neuronal protein, was not detectable after purification. (B) Crude extracts (Cr.) and purified inclusions (Inc.) from cortex (Co) of patient 3815 were either treated or not with formic acid. Similarly treated cerebellar extracts (Cr. Ce and Inc. Ce) were used as control. Crude extracts loaded on the gel contained 100 µg of protein; inclusions were purified from 1 mg of crude extract. Proteins were resolved through an 8% polyacrylamide gel and analyzed by Western blotting, using the 1C2 antibody. A smear of water-soluble fragmented huntingtin was detected in the absence of formic acid treatment in cortex, but not in cerebellum. Treatment with formic acid increased the intensity of the smear, presumably because further fragments were released from the purified inclusions. The smear of fragments released from the purified inclusions of patient 3815 overlapped with that present in the untreated extract, but extended to lower molecular weights. The absence of fragmented huntingtin in cerebellum is likely to explain the absence of inclusions in this region of the brain. (C) Inclusions purified from the brain of two patients (3815 and 3825) were incubated in the presence of formic acid and stained sequentially with the 4C8 and the 1C2 antibody. The 4C8 antibody failed to detect the lower part of the smear of fragmented huntingtin detected by the 1C2 antibody; the staining was abruptly interrupted at ∼60–70 kDa presumably because fragments smaller than 70 kDa did not contain the more C-terminal epitope recognized by 4C8. The signal was stronger for case 3825, because four times more material was loaded on the gel. (D) Inclusions were purified as described in (26) except that centrifugation was at 300 000 g rather than 100 000 g. After the high-speed centrifugation, the pellet containing the inclusions and the supernatant were submitted to electrophoresis through a 0.75% agarose gel and stained with the 1C2 antibody. A polymer was present in the cortical (Co) inclusions (pellet) (Inc.) of patient 3815 and 3123, but not in the supernatant (Sup.). The polymer was therefore entirely associated with the inclusions. No such polymer was found in the cerebellum (Ce). Reproduced from Hoffner et al.26