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. 2000 Jun 6;97(12):6739-44.
doi: 10.1073/pnas.110138997.

Inhibition of huntingtin fibrillogenesis by specific antibodies and small molecules: implications for Huntington's disease therapy

V Heiser  1 , E Scherzinger, A Boeddrich, E Nordhoff, R Lurz, N Schugardt, H Lehrach, E E Wanker
Affiliations

Inhibition of huntingtin fibrillogenesis by specific antibodies and small molecules: implications for Huntington's disease therapy

V Heiser et al. Proc Natl Acad Sci U S A. .

Abstract

The accumulation of insoluble protein aggregates in intra and perinuclear inclusions is a hallmark of Huntington's disease (HD) and related glutamine-repeat disorders. A central question is whether protein aggregation plays a direct role in the pathogenesis of these neurodegenerative diseases. Here we show by using a filter retardation assay that the mAb 1C2, which specifically recognizes the elongated polyglutamine (polyQ) stretch in huntingtin, and the chemical compounds Congo red, thioflavine S, chrysamine G, and Direct fast yellow inhibit HD exon 1 protein aggregation in a dose-dependent manner. On the other hand, potential inhibitors of amyloid-beta formation such as thioflavine T, gossypol, melatonin, and rifampicin had little or no inhibitory effect on huntingtin aggregation in vitro. The results obtained by the filtration assay were confirmed by electron microscopy, SDS/PAGE, and MS. Furthermore, cell culture studies revealed that the Congo red dye at micromolar concentrations reduced the extent of HD exon 1 aggregation in transiently transfected COS cells. Together, these findings contribute to a better understanding of the mechanism of huntingtin fibrillogenesis in vitro and provide the basis for the development of new huntingtin aggregation inhibitors that may be effective in treating HD.

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Figures

Figure 1
Figure 1
Inhibition of HD exon 1 fibrillogenesis by antibodies. (A) Effect of increasing concentrations of 1C2 antibody or mouse IgG 2a on mycHD51 aggregation. Aggregate formation was detected by the filter retardation assay. (B) Quantitative analysis of dot-blot results shown in A. The relative amount of aggregate for each sample was quantified on a Fuji-Imager (LAS 2000). For each experiment, the signal intensity obtained from the samples without added antibodies was arbitrarily set as 100. Values shown are the average of triplicate determinations ± SE. (C) Electron micrographs of mycHD51 protein aggregates after antibody treatment. Proteolytically cleaved GST-mycHD51 fusion protein was incubated for 16 h at 37°C in the presence or absence of the indicated antibodies. Proteins were negatively stained with 1% uranyl acetate and viewed by electron microscopy. (Bar = 200 nm.)
Figure 2
Figure 2
Inhibition of HD exon 1 aggregation by small molecules. (A) Structure of the chemical compounds examined for their ability to inhibit HD exon 1 fibrillogenesis. (B) Effect of various concentrations of the indicated chemical compounds on HD exon 1 aggregation as monitored by the filter retardation assay. GST-HD51 fusion protein at a conc. of 2.5 μM was predigested for 30 min at 37°C with trypsin at an enzyme/substrate ratio of 1:10 (wt/wt). Then chemical compounds were added to the cleavage reactions to give the indicated final concentration. Reaction mixtures were incubated for an additional 18 h at 37°C and insoluble polyQ-containing HD51 aggregates were detected by the filter retardation assay. (C) Quantification of the dot blot results obtained with Congo red, thioflavine S, and thioflavine T. The signal intensity obtained from the sample without added chemical compound was arbitrarily set as 100. Values shown are the average of triplicate incubations ± SE. (D) Electron micrographs of HD51 fibrils formed in the presence or absence of the indicated chemical compounds. Trypsin-digested GST-HD51 protein at 2.5 μM was incubated at 37°C for 24 h either without added chemical compound (control) or with Congo red (final conc. 2.5 μM), thioflavine T, and thioflavine S (final conc. 40 μM each). Representative samples of fibrillar structures are shown. (Bar = 200 nm.)
Figure 3
Figure 3
Time course of HD exon 1 aggregation in the presence or absence of Congo red. (A) Western blot analysis of aggregation reactions. GST-HD51 fusion protein at 2.5 μM was incubated at 37°C with trypsin. After 30 min of incubation, Congo red was added to a final conc. of 2.5 μM and incubation was continued for 24 h at 37°C. Samples corresponding to 200 ng of fusion protein were removed from the aggregation reactions at the indicated times and analyzed by SDS/PAGE and immunoblotting using the AG51 antibody. →, The origin of electrophoresis. (B) Analysis of aggregation by the filter retardation assay. Captured aggregates were detected by incubation with the HD1 antibody. (C) Quantitative analysis of the dot-blot results shown in B. The dot with the highest signal intensity was arbitrarily set as 100.
Figure 4
Figure 4
Effect of Congo red on HD exon 1 aggregation as monitored by MALDI-TOF-MS. GST-HD51ΔP fusion protein at 2.5 μM was incubated for 24 h at 37°C with trypsin (10 ng/μl) in the absence (control) or presence of Congo red (final conc. 2.5 μM). Reaction aliquots (0.5 μl) were taken at different time points and analyzed by MALDI-TOF-MS. (A) Undigested GST-HD51ΔP fusion protein. 2+, Doubly-charged molecular-ion signal of GST-HD51ΔP. (B) GST-HD51ΔP after 45 min incubation with trypsin resulting in release of a peptide, HD51ΔP, with the sequence SF[Q]51PPPPLERPHRD. *, Tryptic digestion products of GST. To confirm the identity of HD51ΔP, the isotopic pattern of HD51ΔP was resolved in high-resolution acquisition mode (Inset, right spectrum) enabling determination of the monoisotopic mass of 8,074.74 Da (expected: 8,074.78 Da). (C) GST-HD51ΔP after 24 h of incubation with trypsin. In the presence of Congo red, nonaggregated HD51ΔP is still detectable whereas in absence of the dye it is not. (D) Samples analyzed in C after 30 min of additional incubation with 10 ng/μl proteinase K (PK). The HD51ΔP monomers stabilized by Congo red are degraded by PK, demonstrating that these molecules are readily accessible for proteolytic digestion. a.i., arbitrary intensity.
Figure 5
Figure 5
Structure and IC50 values of lipid-soluble Congo red analogs. IC50 values were determined as described in Fig. 2 by using the filter retardation assay. GST-HD51 fusion protein at 2.5 μM was predigested for 30 min at 37°C with 10 ng/μl trypsin and chemical compounds at various concentrations (0.15–40 μM) were added to the cleavage reactions. After incubation for 18 h at 37°C formation of aggregates was quantified by using the filter retardation assay. Dose-response profiles for each chemical compound were generated, and IC50 values were calculated from data sets of four independent experiments.
Figure 6
Figure 6
Inhibition of HD exon 1 aggregation in COS cells. (A) Western blot analysis of soluble and insoluble fractions of transfected COS-1 cells after Congo red treatment. COS-1 cells grown for 1 wk in the presence or absence of various concentrations of Congo red (0.003–30 μM) were transfected with the pTL-CAG51 construct. Cell extracts were prepared and fractionated into soluble and insoluble fractions as described in Materials and Methods. Proteins were separated by SDS/PAGE and analyzed by immunoblotting using the HD1 antibody. (B) Filter retardation assay performed on the insoluble fraction of the transfected cell extracts. The SDS-insoluble protein aggregates retained on the filter were detected with the HD1 antibody. (C) Quantitative analysis of the dot blot results shown in B. The dot corresponding to the control experiment without added Congo red was arbitrarily set as 100.
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