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Published in final edited form as: Curr Biol. 2011 Jun 7;21(11):961–966. doi: 10.1016/j.cub.2011.04.028

The Kynurenine Pathway Modulates Neurodegeneration in a Drosophila Model of Huntington’s Disease

Susanna Campesan 1, Edward W Green 1, Carlo Breda 1, Korrapati V Sathyasaikumar 2, Paul J Muchowski 3, Robert Schwarcz 2, Charalambos P Kyriacou 1, Flaviano Giorgini 1,*
PMCID: PMC3929356  NIHMSID: NIHMS550938  PMID: 21636279

Summary

Neuroactive metabolites of the kynurenine pathway (KP) of tryptophan degradation have been implicated in the pathophysiology of neurodegenerative disorders, including Huntington’s disease (HD) [1]. A central hallmark of HD is neurodegeneration caused by a polyglutamine expansion in the huntingtin (htt) protein [2]. Here we exploit a transgenic Drosophila melanogaster model of HD to interrogate the therapeutic potential of KP manipulation. We observe that genetic and pharmacological inhibition of kynurenine 3-monooxygenase (KMO) increases levels of the neuroprotective metabolite kynurenic acid (KYNA) relative to the neurotoxic metabolite 3-hydroxykynurenine (3-HK) and ameliorates neurodegeneration. We also find that genetic inhibition of tryptophan 2,3-dioxygenase (TDO), the first and rate-limiting step in the pathway, leads to a similar neuroprotective shift toward KYNA synthesis. Importantly, we demonstrate that the feeding of KYNA and 3-HK to HD model flies directly modulates neurodegeneration, underscoring the causative nature of these metabolites. This study provides the first genetic evidence that inhibition of KMO and TDO activity protects against neurodegenerative disease in an animal model, indicating that strategies targeted at two key points within the KP may have therapeutic relevance in HD, and possibly other neurodegenerative disorders.

Results

The kynurenine pathway (KP), responsible for >95% of the degradation of tryptophan in mammals, contains three metabolites shown to be neuroactive: kynurenic acid (KYNA), 3-hydroxykynurenine (3-HK), and quinolinic acid (QUIN) (Figure 1A) [1]. 3-HK and QUIN are neurotoxic by distinct mechanisms— 3-HK is a potent free-radical generator [1, 3, 4], whereas QUIN is an excitotoxic N-methyl-D-aspartate (NMDA) receptor agonist [5, 6]. KYNA, on the other hand, has neuroprotective properties as an antagonist of excitatory amino acid receptors and a free-radical scavenger [7–10]. Fruit flies do not synthesize QUIN [11], providing a unique genetic model to study the roles of 3-HK and KYNA in neurodegeneration (Figure 1A). Levels of these metabolites can be modulated by agents that inhibit the first step in the KP (catalyzed in mammals by tryptophan 2,3-dioxygenase [TDO], indoleamine 2,3-dioxygenase [IDO], and indoleamine 2,3-dioxygenase 2) or by inhibiting kynurenine 3-monooxygenase (KMO), which lies at the branching point between 3-HK and KYNA synthesis. Both IDO and KMO inhibitors have shown promise in preclinical studies of a variety of human disorders [12]. In flies, KMO and TDO are encoded by the genes cinnabar (cn) and vermillion (v), respectively, which are expressed in the eye and have been extensively characterized for their role in eye color pigmentation [13]. The role of the KP in eye function is conserved from flies to humans, where the pathway plays a central role in the formation of ultraviolet irradiation (UV) filters in the lens [14]. Notably, fly mutants that perturb the KP have been found to alter brain plasticity and modulate life span [15, 16]. To date, the therapeutic potential of KMO specifically, and the KP in general, has not been explored in an animal model of neurodegeneration, and the role of KP metabolites in this disease process has not been clarified. This study interrogates these questions using both genetic and pharmacological tools in a fruit fly model of Huntington’s disease (HD).

Figure 1. Schematic Representation of the Kynurenine Pathway in Drosophila melanogaster.

Figure 1

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(A) Kynurenine pathway metabolites are essential for formation of the ommochromes, biological pigments required for wild-type eye color in the fly. Unlike yeast and mammalian counterparts, the neurotoxic metabolite quinolinic acid (QUIN) is not present in Drosophila, making the fly a unique system to model the effects of 3-hydroxykynurenine (3-HK) and kynurenic acid (KYNA) on neurodegeneration.

(B) Flies expressing Htt93Q exhibit increased 3-HK/KYNA ratios at day 1 and day 7 relative to UAS controls.

(C and D) cn3/cn3 (C) and v36f/v36f (D) flies show a significant decrease in 3-HK/KYNA at day 1 versus +/+ genotypes.

(E) elav-Gal4 RNAi knockdown of cn and v KK lines leads to a decrease in 3-HK/KYNA at day 1 versus +/+ genotypes. RNAi knockdown of cn and v was confirmed by qPCR analysis (Figure S2). Statistical comparisons by analysis of variance (ANOVA) with Newman-Keuls post hoc tests. n = 4–8 samples per genotype. (*p < 0.05; **p < 0.01; ***p < 0.001; ns = not significant).

Data are shown as means ± standard errors of the mean (SEMs).

HD Model Flies Exhibit Perturbed Flux through the KP

Increased flux through the central KP is correlated to pathology in HD patients, as well as in mouse and yeast models of HD [1]. To examine whether the pathway is dysfunctional in a similar fashion in HD model flies, we analyzed 3-HK and KYNA in fly heads and determined the 3-HK/KYNA ratio, an established measure of flux through the central KP [17]. We employed the bipartite GAL4/UAS system with the elav-GAL4 driver to direct panneuronal expression of either a mutant huntingtin (htt) exon 1 fragment (Htt93Q) or a nonexpanded htt exon 1 fragment (Htt20Q) [18]. Expression of Htt93Q in flies provides a well-characterized model of HD and presents several disease-relevant phenotypes, including degeneration of photoreceptor neurons (rhabdomeres)—a robust readout for neurodegeneration. We found an ~2- to 3-fold increase in the 3-HK/KYNA ratio in HD flies as compared to controls at day 1 and day 7 posteclosion (Figure 1B; see also Table S1 available online), supporting a pathogenic role for the KP in this model. A similar effect was also observed in flies expressing Htt20Q (Figure S1). We then analyzed 3-HK and KYNA levels in putative null mutant flies lacking either KMO (cn3/cn3) or TDO (v36f/v36f). Synthesis of 3-HK was essentially eliminated in KMO-deficient flies, whereas KYNA levels increased 3.3- to 5.8-fold versus controls, leading to a dramatic decrease in the 3-HK/KYNA ratio (Figure 1C). Similarly, in TDO-deficient flies, 3-HK was much reduced (~97%), and the remaining KP metabolism was shifted toward KYNA synthesis, resulting in a significant ~70%–95% reduction in the 3-HK/KYNA ratio (Figure 1D). The presence of KP metabolites suggests that the v36f mutation may not be a complete amorph, but a strong hypomorph, and that some TDO activity remains in these flies, albeit at much-reduced levels.

cinnabar and vermillion Mutations are Neuroprotective in HD Model Flies

The results above highlight the value of KMO- and TDO-deficient flies for testing the role of 3-HK and KYNA in mutant htt toxicity in vivo. We therefore generated elav-GAL4>UASHtt93Q (Htt93Q-expressing) flies carrying cn3, v36f, or their respective UAS-RNA interference (RNAi) transgenes and assayed neurodegeneration as well as 3-HK/KYNA ratios. In both cn and v mutants expressing Htt93Q, as well as in the RNAi lines, metabolites were robustly shifted toward KYNA synthesis (Figures 1C–1E), an environment predicted to be more neuroprotective. As expected, control flies exhibited seven visible rhabdomeres, whereas robust degeneration of these neurons was observed in Htt93Q flies. We then examined rhabdomere loss in the eyes of cn3/cn3 and v36f/v36f flies expressing Htt93Q compared to controls and observed a significant ~26% rescue of neurons in cn3/cn3 flies and an ~43% rescue in v36f/v36f adults at day 1, as well as neuroprotection, albeit reduced, at day 7 (~15% and ~29%, respectively) (Figures 2A–2C). Furthermore, we found that panneuronal RNAi knockdown of cn and v conferred similar or better levels of rescue of rhabdomeres at both day 1 (~65% and ~67%, respectively) and day 7 (~55% and ~54%) (Figures 2D and 2E). Htt93Q-expressing flies heterozygous for cn3 (cn3/+) neither exhibited significantly increased rhabdomere numbers (Figure 2B) nor showed any difference in 3-HK/KYNA ratio compared to Htt93Q flies (Figure 1C). Conversely, Htt93Q v36f/+ flies exhibited both partial rescue of neurodegeneration (Figures 2B and 2C) and a significant decrease in 3-HK/KYNA ratio (Figure 1D). Taken together, these genetic data reveal an association between increased 3-HK/KYNA ratios in Htt93Q flies and neurodegeneration.

Figure 2. Inhibition of cn and v Ameliorates Neurodegeneration in the Fly.

Figure 2

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(A) Pseudopupil images from wild-type flies, HD flies, and HD flies carrying either cn3 or v36f amorphic alleles at day 1. In wild-type and in cn and v flies not expressing Htt93Q, seven rhabdomeres are visible per ommatidium.

(B and D) Quantification of mean rhabdomeres (± SEM) per ommatidium in Htt93Q, Htt93Q cn3/cn3, Htt93Q cn3/+, Htt93Q v36f/v36f, Htt93Q v36f/+, Htt93Q cn RNAi, and Htt93Q v RNAi flies at day 1 and day 7 after eclosion. Statistical comparisons by ANOVA and post hoc tests versus Htt93Q flies. n = 11–29 flies per genotype (*p<0.05; **p < 0.01; ***p < 0.001). ns = not significant.

(C and E) Percent rescue of neurodegeneration in Htt93Q cn3/cn3, Htt93Q cn3/+, Htt93Q v36f/v36f, Htt93Q v36f/+, Htt93Q cn RNAi, and Htt93Q v RNAi flies at day 1 and day 7.

(F) Rhabdomere neurodegeneration at day 7 is ameliorated by treatment with the KMO inhibitor UPF 648 (doses in mM) relative to PBS control. n = 10–13 flies per treatment (***p < 0.001, ANOVA and post hoc tests).

(G) Treatment with 100 µM UPF 648 shifts KP pathway flux toward synthesis of KYNA (**p < 0.01, Student’s t test). Flies were tested at day 7. n = 5 samples per treatment.

Data are shown as means ± standard errors of the mean (SEMs).

KMO Inhibitors Ameliorate Neurodegeneration in HD Model Flies

Having shown that genetic disruption at two key steps in the KP ameliorates an HD-relevant phenotype, we tested the efficacy of a well-characterized KMO inhibitor, UPF 648 [19], as a potential therapeutic modulator of the KP. We selected newly eclosed adult flies and placed them on media containing one of three different doses of UPF 648 (30 µM, 100 µM, 300 µM) or vehicle (phosphate-buffered saline [PBS]). Flies were transferred to new media every day, and rhabdomeres were scored on day 7. UPF 648 treatment led to robust protection (up to ~90% rescue of neurodegeneration, Figure 2F) and a significant shift toward KYNA synthesis (Figure 2G). To further support the view that neuroprotection is due to inhibition of KMO, we tested two additional KMO inhibitors: JM6, a novel KMO inhibitor that is protective in mouse models of HD and Alzheimer’s disease [20], and Ro 61-8048 [21], a compound we previously found protective in HD model yeast [1]. We observed similar neuroprotective results for these inhibitors (Figures S3A and S3B). To test whether off-target effects were contributing to the observed neuroprotection, we treated Htt93Q cn flies with 30 µM Ro 61-8048. Strikingly, we found that Ro 61-8048 conferred no added protection to Htt93Q cn flies (Figure S3C).Thus, either genetic orpharmacological inhibition of KMO resulted in a similar level of neuroprotection—clearly showing that these beneficial effects are due to specific inhibition of KMO and not to off-target effects of Ro 61-8048. These studies reveal that pharmacological as well as genetic inhibition of KMO is protective in Htt93Q-expressing flies.

3-HK and KYNA Modulate Neurodegeneration

In order to directly test the causative role of 3-HK in Htt93Q toxicity, we then fed 3-HK to Htt93Q and Htt93Q cn3/cn3 flies. To ensure that flies were being fed the metabolite at physiological levels, we took advantage of previous work describing restoration of wild-type eye pigmentation in cn3 flies fed 3-HK as larvae [22]. Flies were mated so that eggs of the desired genotype were deposited directly onto media containing 3-HK, and thus emerging larvae were fed the metabolite throughout development. We tested a series of doses and found that 0.8 mg/ml was the minimum sufficient to restore wild-type eye color to newly emerged cn3 flies (Figure 3A). We therefore fed this dose of 3-HK, as well as a higher one of 1.4 mg/ml, to Htt93Q and Htt93Q cn3/cn3 larvae and scored rhabdomeres in newly eclosed adult flies. 3-HK feeding led to a dose-dependent restoration of neurodegeneration in Htt93Q cn3/cn3 flies (Figure 3B). Notably, at the highest dose of 3-HK, no significant rhabdomere rescue in Htt93Q cn3/cn3 flies was observed compared to Htt93Q (p = 0.15), suggesting that 3-HK treatment eliminates the protective effect of KMO inhibition. Even at the highest dose, 3-HK levels in Htt93Q cn3/cn3 flies did not exceed levels in untreated control Htt93Q flies (Figure S4A), indicating that restoration of 3-HK within the physiological range is sufficient to eliminate cinnabar-dependent protection. In flies fed 3-HK, the 3-HK/ KYNA ratio also showed a significant, dose-dependent shift away from protective metabolite levels (Figure 3C). 3-HK feeding significantly potentiated the toxicity observed in Htt93Q flies (Figure 3B; p < 0.001 for 1.4 mg/ml treatment), indicating that maximal levels of KP-dependent toxicity are not achieved in this model. Whereas 3-HK treatment of wild-type larvae resulted in a several-fold increase of 3-HK in the head of newly eclosed flies, we did not observe any rhabdomere neurodegeneration (Figures S4A and S4B).

Figure 3. 3-HK and KYNA Directly Modulate Neurodegeneration in Htt93Q Flies.

Figure 3

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(A) Representative images of newly emerged (day 0) cn3 flies fed 3-HK, showing restoration of wild-type (WT) eye color. Doses of 0.4, 0.6, 0.8, 1.1, or 1.4 mg/ml 3-HK were fed to cn3 and control flies (0.6 and 1.1 mg/ml not shown).

(B) 3-HK abrogates cn3 protection in day 0 Htt93Q flies (ns = not significant, p = 0.15). n = 8–12 flies per treatment. Comparisons were made between genotypes with the same treatment.

(C) 3-HK feeding leads to significantly increased 3-HK/KYNA ratios in newly emerged Htt93Q and Htt93Q cn3 flies versus untreated controls. n = 3–6 samples per treatment.

(D) KYNA feeding at 1 mg/ml (**p < 0.01) and 5 mg/ml (*p < 0.05) increases rhabdomere number at day 0. n = 12 flies per treatment. Statistical comparisons by ANOVA and post hoc tests for (B), (C), and (D) (*p < 0.05; **p < 0.01; ***p < 0.001).

(E) Feeding of 5 mg/ml KYNA decreases the 3-HK/KYNA ratio in Htt93Q flies at day 0 (*p = 0.05, Student’s t test). n = 4–5 samples per condition.

Data are shown as means ± standard errors of the mean (SEMs).

We subsequently interrogated KYNA in similar feeding experiments with Htt93Q flies, testing four doses over a wide range of concentrations (0.1 mg/ml to 5 mg/ml). We found that the two highest doses (1 mg/ml and 5 mg/ml) conferred a significant but modest increase in rhabdomere number of Htt93Q flies (Figure 3D), and examination of metabolites revealed a correlated decrease in flux through the central KP in these flies (Figure 3E). Supporting our observations, a recent study with a synthetic compound similar in structure to KYNA has shown some protective effects in HD model mice [23].

Genetic Manipulation of the KP Modulates Neurodegeneration

We also tested an additional KP mutation in the cardinal gene (cd1) known to increase levels of 3-HK [24] (Figure 1A). First, we found a 1.8-fold increase in 3-HK levels in cd1 versus control flies (Figure 4A), validating the previous study. We therefore hypothesized that cd1/cd1 flies expressing Htt93Q would show increased levels of neurodegeneration in the eye. Surprisingly, we found that the cd1 mutation was neuroprotective (Figure 4B). When 3-HK and KYNA were examined in Htt93Q cd1/cd1 flies, we observed no change in 3-HK levels and an ~3-fold increase in KYNA levels compared to controls, leading to a significant reduction in the 3-HK/KYNA ratio (Figures 4A, 4C, and 4D). Although the mechanism underlying this surge in KYNA levels remains to be clarified, our observation provides further strong and unanticipated support for the central role of 3-HK and KYNA dynamics in mutant htt toxicity. Supporting our 3-HK feeding observations, cd1/cd1 control flies do not exhibit rhabdomere neurodegeneration (Figure S4D), indicating that flies expressing Htt93Q are specifically sensitive to perturbations in 3-HK. This was confirmed with an analysis of Htt20Q flies, which exhibit increased KP flux but an absence of neurodegeneration (Figures S1 and S4D).

Figure 4. Htt93Q Flies Carrying the cd1 Mutation Exhibit Reduced Neurodegeneration.

Figure 4

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(A) Day 1 cd1/cd1 flies in the control elav-GAL4 background exhibit a significant increase in 3-HK, whereas 3-HK levels in Htt93Q cd1/cd1 flies are unchanged (n = 4–5).

(B) Rhabdomeres in Htt93Q cd1/+ and Htt93Q cd1/cd1 flies are significantly increased versus Htt93Q flies at day 1 and day 7 after eclosion (n = 12–13). In cd control flies, seven rhabdomeres are visible per ommatidium (see Figure S4D).

(C) Day 1 Htt93Q cd1/cd1 flies exhibit an ~3-fold increase in KYNA levels versus Htt93Q flies, whereas KYNA levels in the elav-GAL4 background are unchanged (n = 4–5 samples per genotype).

(D) cd1/cd1 flies in the control elav-GAL4 background exhibit a significant increase in 3-HK/ KYNA ratios, whereas 3-HK/KYNA ratios in Htt93Q cd1/cd1 flies are significantly reduced compared to Htt93Q flies (n = 4–5). ANOVA with Newman-Keuls post hoc tests, except for (C), Student’s t test (*p < 0.05; **p < 0.01; ***p < 0.001).

Data are shown as means ± standard errors of the mean (SEMs).

Discussion

We have observed that both genetic and chemical inhibition of KMO ameliorate neurodegeneration in HD model flies, supporting our previous observations that inhibition of KMO is protective in a yeast model of mutant htt toxicity [25, 26]. We also found that this neuroprotection is correlated with decreases in 3-HK relative to KYNA. Importantly, this protection can be abrogated by 3-HK feeding, showing the causative nature of this metabolite. Furthermore, a KYNA feeding regime that forces a neuroprotective ratio of these metabolites also reduces neurodegeneration in HD flies. Therefore, for the first time, we show that KP metabolites directly modulate neurodegeneration in an animal model of neurodegenerative disease. Finally, our genetic dissection of the KP with vermillion and cardinal mutants further underscores the critical relationship between imbalances in the KP and neurodegeneration.

3-HK generates free radicals by auto-oxidation [3, 4], and this mechanism is believed to underlie 3-HK-dependent toxicity observed in neuronal cell lines and in primary neurons [1]. It is therefore likely that 3-HK is contributing to neurodegeneration in our studies by generation of free radicals. At endogenous concentrations in the mammalian brain, KYNA functions as a competitive inhibitor of the glycine coagonist site of the NMDA receptor and a potent, noncompetitive inhibitor of the α7 nicotinic acetylcholine receptor [27]. KYNA treatment results in dose-dependent behavioral changes in rodents, mimicking properties of NMDA receptor antagonists [7]. Moreover, KYNA is neuroprotective against excitotoxic lesions and inhibits the release of glutamate at concentrations in the physiological range [8, 9]. In a study parallel to ours, Muchowski and colleagues found, by using a novel KMO-inhibitor, that glutamate reduction by KYNA is the likely mechanism for neuroprotection in the central nervous system (CNS) of HD mice [20]. Because flies express orthologs of both NMDA and α7 nicotinic acetylcholine receptors [28, 29], KYNA could be conferring neuroprotection by antagonizing these receptors and decreasing glutamate-dependent excitotoxicity, as well as by scavenging free radicals [10].

Interestingly, we find that increased 3-HK relative to KYNA is only neurotoxic in the presence of Htt93Q, suggesting that general cellular dysfunction due to mutant htt expression [30] confers a sensitized background for KP modulation. In a related fashion, we find that expression of Htt20Q is sufficient to induce increased 3-HK/KYNA ratios but does not confer neurodegeneration—providing further support that additional Htt93Q-dependent cellular defects are required to uncover KP-dependent toxicity. A similar observation was made by Romero and colleagues with flies expressing full-length htt constructs [31]. These authors found that whereas expression of either an expanded construct (128QhttFL) or a nonexpanded construct (16QhttFL) led to elevated resting synaptic Ca2+ levels, this perturbation was associated with pathogenesis only in the case of 128QhttFL flies.

Our discovery that inhibition of TDO is protective in the fly validates this protein as a novel therapeutic target for HD. TDO and IDO, which are both expressed in the brain [1], have distinct structural and biochemical characteristics [32], and selective inhibitors are being actively explored as potential therapeutic compounds [12]. It is now imperative that such compounds be tested in animal models of HD, and perhaps other neurodegenerative disorders, to characterize their efficacy and therapeutic potential.

In summary, our results are consistent with the view that modulation of the KP is central to mutant htt toxicity [1]. Our observations provide genetic and pharmacological support for the “kynurenine hypothesis,” underscoring the important role that Drosophila plays in the understanding and possible development of therapy for human neurodegenerative disorders. Our study favors a model in which increased flux toward KYNA synthesis is neuroprotective in HD, provides unequivocal evidence that 3-HK, independent of QUIN, is pathogenic, and shows that reduction of 3-HK relative to KYNA is therapeutic. Based upon these observations and a new study from our group in which KMO inhibition is also beneficial to HD model mice [20], it seems timely to consider testing the efficacy of KMO inhibitors, and potentially inhibitors of other KP enzymes, in HD patients.

Acknowledgments

This study was supported by grants from the Huntington’s Disease Association and CHDI Foundation, Inc. to F.G. and C.P.K., who also acknowledge grants from the Biotechnology and Biological Sciences Research Council for valuable infrastructure supporting this work. R.S. and P.J.M. are recipients of grants from the National Institutes of Health. We gratefully acknowledge the gift of the htt exon 1 fly lines from J. Lawrence Marsh and Leslie Thompson. We also thank Mariaelena Repici for comments on the manuscript and Marian Thomas for technical assistance. This study is dedicated to the memory of Dr. Paolo Guidetti, a great scientist and friend.

Footnotes

Supplemental Information

Supplemental Information includes four figures, one table, and Supplemental Experimental Procedures and can be found with this article online at doi:10.1016/j.cub.2011.04.028.

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