Identification of combinatorial drug regimens for treatment of Huntington's disease using Drosophila

Abstract

We explore the hypothesis that pathology of Huntington's disease involves multiple cellular mechanisms whose contributions to disease are incrementally additive or synergistic. We provide evidence that the photoreceptor neuron degeneration seen in flies expressing mutant human huntingtin correlates with widespread degenerative events in the Drosophila CNS. We use a Drosophila Huntington's disease model to establish dose regimens and protocols to assess the effectiveness of drug combinations used at low threshold concentrations. These proof of principle studies identify at least two potential combinatorial treatment options and illustrate a rapid and cost-effective paradigm for testing and optimizing combinatorial drug therapies while reducing side effects for patients with neurodegenerative disease. The potential for using prescreening in Drosophila to inform combinatorial therapies that are most likely to be effective for testing in mammals is discussed.

Fig. 1.

Htt-induced neuronal degeneration is widespread. (A) Mushroom bodies of control (OK107>GFP) and polyQ flies (OK107>GFP; Httex1p Q93). The α′ and β′ (not visible) lobes are completely missing, and the γ lobe neuropil density is dramatically reduced, as are the number of Kenyon cell bodies (KCB). (B) Volumetric analysis of mushroom bodies in flies bearing the UAS>Httex1p Q93 transgene and a dunce GFP fusion protein expressed in all Kenyon cells and the mushroom body crossed to elav>Gal4. Quantification of the volume of the mushroom bodies in 10- to 14-day-old females indicates that Httex1p Q93 flies show ≈24% volume reduction in mushroom bodies compared with control (***, P < 0.001). (C) The median bundle group of neurons in central brain of OK107>GFP flies exhibits ≈50% cellular loss when Httex1p Q93 is expressed. At 13 days, controls show 20.5 ± 2.0 SE, whereas experimentals show 8.5 ± 0.4 SE. (Scale bar, 10 μm.) (D) Populations of neurons and neurites in the thoracic and abdominal regions of ventral nerve cord exhibit extensive loss of cell bodies (arrows) and loss and disorganization of neuronal projections (arrowheads). (Scale bar, 50 μm.) (E) Higher magnification of cell bodies of ventral nerve cord marked with arrows on D. (Scale bar, 10 μm.) (F) Schematic of the adult brain showing approximate location of structures discussed in A–D.

Fig. 2.

Toxicity and dosage studies. (A) Toxicity of the DMSO carrier. Httex1p Q93-expressing embryos were transferred into vials containing standard fly food supplemented with increasing concentrations of DMSO. A significant reduction in viability was observed with the highest concentration of DMSO tested. (B) Compound combinations at low concentration on adults not expressing Httex1p Q93 showed no change in survival. (C) Httex1p Q93-expressing siblings, however, showed significant rescue of lethality as compared to control DMSO food. ***, P << 0.001; **, P < 0.01.

Fig. 3.

Combination therapy suppresses photoreceptor degeneration in flies expressing Httex1p. (A) Flies expressing Httex1p Q93 were fed threshold or effective concentrations of SAHA, cystamine (cyst), and Congo red (Cr), and their eyes were scored for neurodegeneration at 7 days posteclosion using the pseudopupil technique. Percent rescue was determined as follows: 100 × (Rt - Rc)/(7 - Rc), where Rt and Rc are rhabdomeres/ommatidium in the treated and control groups, respectively. (B) Photographs from ommatidia of 7-day-old flies expressing Httex1p Q93 fed threshold concentrations of SAHA and/or cystamine (cyst) demonstrate the effect observed. (C) Combinatorial therapy improves motor function. Climbing ability of adults grown on food supplemented with threshold concentrations of SAHA, cystamine (cyst), and/or Congo red (Cr) in different combinations was evaluated at day 7 posteclosion. For each condition, the average of 20 flies was calculated. ***, P << 0.001; **, P < 0.01; *, P < 0.05, compared with DMSO.

Fig. 4.

A distinct regimen of combination therapy suppresses photoreceptor degeneration. (A) The effect of compounds singly and in combinations at threshold concentration on flies not expressing Httex1p Q93 showed no change in survival. (B) Geldanamycin (Geld) alone at a concentration of 9 μg/ml suppresses photoreceptor neuronal loss. (C) Combinations of SAHA with either geldanamycin or Y-27632 (Y) at threshold concentrations effectively reduce neuronal loss. Combinations of geldanamycin and Y-27632 do not show additional suppression of neuronal loss but do not inhibit the ability of other combinations to rescue neurons.