Pharmacological promotion of inclusion formation: a therapeutic approach for Huntington's and Parkinson's diseases

Abstract

Misfolded proteins accumulate in many neurodegenerative diseases, including huntingtin in Huntington's disease and alpha-synuclein in Parkinson's disease. The disease-causing proteins can take various conformations and are prone to aggregate and form larger cytoplasmic or nuclear inclusions. One approach to the development of therapeutic intervention for these diseases has been to identify chemical compounds that reduce the size or number of inclusions. We have, however, identified a compound that promotes inclusion formation in cellular models of both Huntington's disease and Parkinson's disease. Of particular interest, this compound prevents huntingtin-mediated proteasome dysfunction and reduces alpha-synuclein-mediated toxicity. These results demonstrate that compounds that increase inclusion formation may actually lessen cellular pathology in both Huntington's and Parkinson's diseases, suggesting a therapeutic approach for neurodegenerative diseases caused by protein misfolding.

Fig. 1.

The compound B2 increases polyQ–EGFP reporter inclusions and overall fluorescence. (A) Schematic representation of the reporter construct used in high-throughput screen. An ecdysone-inducible promoter drives expression of a fusion of the first 17 aa of Htt plus 103 glutamines and EGFP. (B) Distribution of hits from high-throughput screen of 37,000 chemical compounds. A total of 116 compounds decreased the EGFP signal by ≥35% and 5 compounds increased the signal by ≥50% (black bar). (C) Structures of B2, its analogs B21 and B22, and B5. (D) Reporter readout from a range of doses of B2 or its analogs B21 and B22. B2 causes a dose-dependent increase in polyQ–EGFP reporter. Analog B21 is less effective than B2, and B22 shows no activity. ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.005. (E) Fluorescence microscopy of cells from wells treated with either DMSO or 5 μM B2. Cells treated with B2 show an increase in polyQ–EGFP inclusion formation. (Magnification: ×10.)

Fig. 2.

B2 treatment prevents Htt-induced proteasome dysfunction. (A) Schematic representation of constructs stably transfected into CHO-K1 cells. CHO-K1 cells contained a heterodimeric ecdysone receptor, a CMV promoter-driven proteasome reporter (EGFP fused to the degradation peptide CL1), and an ecdysone-inducible fusion protein of the first exon of Htt (with 97Q) and mRFP. (B) Treatment of proteasome reporter cells with compounds. Ponasterone (pon) induction of mutant Htt leads to a build-up of proteasome reporter (EGFP–CL1). B2 treatment rescues the accumulation of proteasome substrate, in a dose-dependent fashion. B5 exhibits a similar, but weaker, effect. ∗, P < 0.05; ∗∗∗, P < 0.005. (C) Treatment of proteasome reporter lines with B2 vs. analogs of B2. As in the polyQ–EGFP reporter assay shown in Fig. 1C, modifications to B2 led to a decrease in activity in B21 and an elimination of activity in B22. ∗∗∗∗, P < 0.0005. (D) Fluorescence microscopy of cells from proteasome reporter line. Fewer cells exhibit build-up of EGFP–CL1 proteasome reporter when treated with B2. (Magnification: ×20.)

Fig. 3.

B2 treatment of cells produces more α-synuclein inclusions and lowers α-synuclein toxicity. (A) Transient transfection of CHO-K1 cells with synT construct. Cells were transfected with synT and simultaneously treated with either 10 μM B2 or an equivalent amount of DMSO for 48 h, then fixed and exposed to anti-α-synuclein antibody. Treatment with B2 nearly doubled the percentage of cells that are strongly immunofluorescent for α-synuclein; the remaining cells have numerous light cytoplasmic α-synuclein aggregates. (Magnification: ×10.) (B) Quantification of transfection shown in A. Brightly α-synuclein-positive cells and total DAPI-positive cells were counted in six random fields for each treatment. ∗∗∗, P < 0.0001. (C) α-Synuclein toxicity testing. B2 shows a dose-dependent prevention of toxicity from α-synuclein (measured by adenylate kinase release) when transiently transfected into H4 cells. ∗, P < 0.05.

Fig. 4.

B2 does not alter chaperone levels or activity. (A) Western blot for levels of chaperones in H4 cells transfected with vector or α-synuclein and treated with B2 or DMSO. Hsp70, Hsp27, and DJ-1 are not significantly altered by B2 treatment. (B) Refolding of heat-denatured firefly luciferase protein in rabbit reticulocyte lysate, in the presence of DMSO, B2, or Hsp90 inhibitor geldanamycin (GA). GA decreases the rate of refolding by >50% in this assay, whereas DMSO and B2 have no effect. Values are the mean of two assays, and error bars show SD.