A genomic screen in yeast implicates kynurenine 3-monooxygenase as a therapeutic target for Huntington disease

Abstract

Huntington disease is a fatal neurodegenerative disorder caused by expansion of a polyglutamine tract in the protein huntingtin (Htt), which leads to its aggregation in nuclear and cytoplasmic inclusion bodies. We recently identified 52 loss-of-function mutations in yeast genes that enhance the toxicity of a mutant Htt fragment. Here we report the results from a genome-wide loss-of-function suppressor screen in which we identified 28 gene deletions that suppress toxicity of a mutant Htt fragment. The suppressors are known or predicted to have roles in vesicle transport, vacuolar degradation, transcription and prion-like aggregation. Among the most potent suppressors was Bna4 (kynurenine 3-monooxygenase), an enzyme in the kynurenine pathway of tryptophan degradation that has been linked directly to the pathophysiology of Huntington disease in humans by a mechanism that may involve reactive oxygen species. This finding is suggestive of a conserved mechanism of polyglutamine toxicity from yeast to humans and identifies new candidate therapeutic targets for the treatment of Huntington disease.

Figure 1

Suppression of Htt103Q toxicity in yeast gene deletion strains. a, Cell viability (spotting) assays are shown for the library parental strain, BY4741, and 6 gene deletion strains that suppress Htt103Q-mediated toxicity. Shown are five-fold serial dilutions starting with equal numbers of cells. Growth on media containing GAL induces expression of Htt103Q. b, Fluorescence microscopy of the localization of Htt103Q in IBs (GFP labeling in green). A large number of small IBs form in the parental yeast strain (BY4741) expressing Htt103Q-GFP under inducing conditions (+GAL). The majority of suppressor strains also contained Htt103Q IB's under inducing conditions. A sampling of these includes ecm37Δ, mgt1Δ, paf1Δ, and ymr082cΔ.

Figure 2

Genetic analysis of the kynurenine pathway on Htt103Q-mediated toxicity. a, Schematic representation of the kynurenine pathway in yeast and mammals. Arrows represent enzymatic steps in the pathway, with the respective enzymes listed to the right. The yeast genes encoding the enzymes are listed below each respective enzyme. Gene deletions that enhance or suppress toxicity of Htt103Q are noted with bold italics. b, Analysis of Htt103Q toxicity by cell viability (spotting assays) in the parental yeast strain (BY4741) and the kynurenine pathway gene deletion strains. pYES2 is the empty vector control. Shown are five-fold serial dilutions starting with equal numbers of cells. Growth on media containing GAL induces expression of the Htt103Q.

Figure 3

Htt103Q toxicity is mediated in part by 3HK and QUIN. a, Changes in levels of 3HK and QUIN in parental, bna1Δ, and bna4Δ cells expressing Htt103Q (*P < 0.001, **P < 0.01). b, Changes in 3HK and QUIN levels in aro9Δ and npt1Δ strains expressing Htt103Q (*P < 0.001). c, 3HK and QUIN levels with Ro 61-8048 treatment (*P < 0.001, **P = 0.003, ***P < 0.01) d, Growth with Ro 61–8048 treatment; 1 μM (** P = 0.015) and 100 μM (**P < 0.001). All statistical comparisons were performed using Student's t-test.

Figure 4

Htt103Q toxicity is mediated in a manner that involves ROS. a, ROS is visualized as red fluorescence while Htt103Q aggregates were visualized by GFP fluorescence. Parental cells expressing Htt103Q showed high levels of ROS, while in bna4Δ cells expressing Htt103Q ROS are not detected. b, Changes in levels of ROS (*P = 0.001, **P = 0.002). c, Ro 61-8048 decreases levels of ROS in cells expressing Htt103Q (*P = 0.011). All statistical comparisons were performed using Student's t-test.

Figure 5

Model depicting the non-cell autonomous contribution by microglia to neuronal dysfunction in Huntington's disease (HD). In this model, cell-autonomous expression of mutant Htt in microglia causes dysfunction, perhaps by interactions with mitochondria or the mitochondrial membrane protein KMO, leading to upregulation of 3HK and QUIN synthesis, and thereby increased levels of ROS. The combined effects of ROS and NMDA receptor-mediated excitotoxicity by QUIN contribute to the dysfunction of neurons expressing mutant Htt. Other functional categories represented in our genomic screen, and previously implicated in HD, are highlighted in the dysfunctional neuron.