A cell-based assay for aggregation inhibitors as therapeutics of polyglutamine-repeat disease and validation in Drosophila

Abstract

The formation of polyglutamine-containing aggregates and inclusions are hallmarks of pathogenesis in Huntington's disease that can be recapitulated in model systems. Although the contribution of inclusions to pathogenesis is unclear, cell-based assays can be used to screen for chemical compounds that affect aggregation and may provide therapeutic benefit. We have developed inducible PC12 cell-culture models to screen for loss of visible aggregates. To test the validity of this approach, compounds that inhibit aggregation in the PC12 cell-based screen were tested in a Drosophila model of polyglutamine-repeat disease. The disruption of aggregation in PC12 cells strongly correlates with suppression of neuronal degeneration in Drosophila. Thus, the engineered PC12 cells coupled with the Drosophila model provide a rapid and effective method to screen and validate compounds.

Figure 1

Expression of inducible Htt transgenes in PC12 cells. (A) Expression constructs. All constructs contain a carboxyl-terminal EGFP epitope tag, and some constructs contain an amino-terminal FLAG (F) tag as indicated. Htt(Q_n)-EGFP, a truncated exon 1 encoding the first 17 aa of Htt with 25, 103, 191, 250, or 300 Qs; Htt(Q_n)P-EGFP, the region of Htt encoded by exon 1 with 25, 47, or 103 Qs containing the proline-rich region (P); F-Htt(Q_n)P-EGFP, exon 1 protein (25 or 103 Qs) with an amino-terminal FLAG tag; F-Htt(Q_n) 584-EGFP, an amino-terminal FLAG-tagged construct encoding 584 aa of Htt [numbering is consistent with GenBank accession no. P42858 with 23 Qs and including consensus caspase cleavage sites (52)], with 25, 103, or 144 Qs. (B and C) Western blots of total cell lysates. Proliferating cells were induced with 5 μM PA for 48 h, and 100 μg of total protein was electrophoresed on 10% SDS gels, blotted, and incubated with CAG53b antibody. The apparent higher level of the Htt103Q polypeptide may be due to the fact that the antibody preferentially recognizes proteins containing longer polyQ stretches. (B) Htt(Q_n)-EGFP cells with 25 or 103 Qs plus and minus PA induction. (C) Pools of cells containing the indicated Htt constructs were induced with 5 μM PA for 48 h. Molecular mass markers are shown to the right of the figure.

Figure 2

Characterization of aggregation in inducible PC12 cells expressing Htt transgenes with fluorescent microscopy. (A) Diffuse EGFP fluorescence in pools of proliferating cells expressing normal-length polyQs tagged with EGFP (Htt25Q-EGFP). (B) Aggregates in proliferating cells expressing Htt103Q-EGFP. Cells have dim, diffuse fluorescence of soluble polyQs and bright EGFP fluorescent aggregates. The images shown in A and B were taken of live cells at ×10 magnification. (C) Aggregates in cytoplasm and neuropils and in the nuclei (Inset) of differentiated Htt103Q-EGFP. The images shown in C were taken from fixed cells at high magnification (×40). Aggregation is expressed as the percentage of cells with aggregates versus the total number of EGFP-positive cells. Nuclei were visualized by 4′,6-diamidino-2-phenylindole staining (blue), and cytoplasm (red) was visualized by probing with anti-α-tubulin antibodies as described in Materials and Methods. (D and E) Time course of aggregate appearance in proliferating (D) and differentiated (E) cells. ♦, Htt14A2.5; ▪, F-Htt103QP EGFP; ●, F-Htt103QP EGFP. (F) Kinetics of aggregation in proliferating cells using high-resolution video microscopy. The nucleation site of aggregation became evident after 20 min of filming (frame 20), followed by rapid polymerization, which led to complete aggregation in 30 min (frames 30, 40, and 50). Frames were taken at 10-min intervals. (B, C, and F) Htt14A2.6 clonal line. (D and E) Htt14A2.5 clonal line. The arrows indicate aggregates. np, neuropil.

Figure 3

Chemical compound screen in inducible PC12 cells expressing Htt103Q-EGFP. Aggregation in PC12 cells expressing Htt103Q-EGFP (Htt14A2.5) in the presence of DMSO (A) or treated with 10 μM Congo red in DMSO (B) is shown. The altered morphology of aggregates in the presence of Congo red is indicated with arrows. (Scale bar, 50 μm.) (C) Quantification of aggregation in the Htt14A2.5 cell line with Congo red (10 μM), minocycline hydrochloride (50 μM), and cystamine bitartrate (50 μM). Expression of truncated exon 1 was induced for 48 h with 1 μM MA, and compounds were added to cells simultaneously with an inducer.

Figure 4

Chemical compound screen in Q48-expressing Drosophila. (A and B) Quantitation of the number of rhabdomeres per ommatidium in the presence of 100 μM cystamine (A) or 250 μM Congo red (B) in Q48-expressing flies. (C) Photographs of ommatidia from flies with no transgene expression (normal eye) or Q48 expression alone and from flies fed 250 μM Congo red or 100 μM cystamine. (D) Dose response for Congo red. Flies were fed 2.5–2,500 mM Congo red. Percent rescue was calculated as (percent surviving − percent surviving on solvent alone)/(1 − percent surviving on solvent alone).