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