Significance Neurodegenerative diseases such as Alzheimer’s (AD), Parkinson’s (PD), and Huntington’s (HD) present a significant and increasing burden on society. Perturbations in the kynurenine pathway (KP) of tryptophan degradation have been linked to the pathogenesis of these disorders, and thus manipulation of this pathway may have therapeutic relevance. Here we show that genetic inhibition of two KP enzymes—kynurenine-3-monooxygenase and tryptophan-2,3-dioxygenase (TDO)—improved neurodegeneration and other disease symptoms in fruit fly models of AD, PD, and HD, and that alterations in levels of neuroactive KP metabolites likely underlie the beneficial effects. Furthermore, we find that inhibition of TDO using a drug-like compound reverses several disease phenotypes, underscoring the therapeutic promise of targeting this pathway in neurodegenerative disease.

Abstract Metabolites of the kynurenine pathway (KP) of tryptophan (TRP) degradation have been closely linked to the pathogenesis of several neurodegenerative disorders. Recent work has highlighted the therapeutic potential of inhibiting two critical regulatory enzymes in this pathway—kynurenine-3-monooxygenase (KMO) and tryptophan-2,3-dioxygenase (TDO). Much evidence indicates that the efficacy of KMO inhibition arises from normalizing an imbalance between neurotoxic [3-hydroxykynurenine (3-HK); quinolinic acid (QUIN)] and neuroprotective [kynurenic acid (KYNA)] KP metabolites. However, it is not clear if TDO inhibition is protective via a similar mechanism or if this is instead due to increased levels of TRP—the substrate of TDO. Here, we find that increased levels of KYNA relative to 3-HK are likely central to the protection conferred by TDO inhibition in a fruit fly model of Huntington’s disease and that TRP treatment strongly reduces neurodegeneration by shifting KP flux toward KYNA synthesis. In fly models of Alzheimer’s and Parkinson’s disease, we provide genetic evidence that inhibition of TDO or KMO improves locomotor performance and ameliorates shortened life span, as well as reducing neurodegeneration in Alzheimer's model flies. Critically, we find that treatment with a chemical TDO inhibitor is robustly protective in these models. Consequently, our work strongly supports targeting of the KP as a potential treatment strategy for several major neurodegenerative disorders and suggests that alterations in the levels of neuroactive KP metabolites could underlie several therapeutic benefits.

The kynurenine pathway (KP), the major catabolic route of tryptophan (TRP) metabolism in mammals (Fig. 1), has been closely linked to the pathogenesis of several brain disorders (1). This pathway contains several neuroactive metabolites, including 3-hydroxykynurenine (3-HK), quinolinic acid (QUIN) and kynurenic acid (KYNA) (2). QUIN is a well-characterized endogenous neurotoxin that specifically activates N-methyl-D-aspartate (NMDA) receptors, thereby inducing excitotoxicity (3, 4). The metabolites 3-HK and QUIN are also neurotoxic via the generation of free radicals and oxidative stress (5, 6). Conversely, KYNA—synthesized by kynurenine aminotransferases (KATs)—is neuroprotective through its antioxidant properties and antagonism of both the α7 nicotinic acetylcholine receptor and the glycine coagonist site of the NMDA receptor (7⇓⇓⇓⇓⇓–13). Levels of these metabolites are regulated at two critical points in the KP: (i) the initial, rate-limiting conversion of TRP into N-formylkynurenine by either tryptophan-2,3-dioxygenase (TDO) or indoleamine-2,3-dioxygenase 1 and 2 (IDO1 and IDO2); and (ii) synthesis of 3-HK from kynurenine by the flavoprotein kynurenine-3-monoxygenase (KMO) (1).

Fig. 1. Consequences of KP manipulation. KP metabolites and enzymatic steps are indicated in black, whereas the key KP enzymes TDO, KMO, and KATs are indicated in purple. The metabolites 3-HK and QUIN are neurotoxic (as indicated by red arrows), whereas KYNA and TRP are neuroprotective (as indicated by green arrows). Inhibition of TDO results in increased TRP levels, and either TDO or KMO inhibition leads to a reduction in the 3-HK/KYNA ratio (highlighted in blue). The enzyme 3-hydroxyanthranilic acid dioxygenase is not present in flies, and thus QUIN is not synthesized.

Alterations in levels of the KP metabolites have been observed in a broad range of brain disorders, including both neurodegenerative and psychiatric conditions (14). In neurodegenerative diseases such as Huntington’s (HD), Parkinson’s (PD), and Alzheimer’s (AD), a shift toward increased synthesis of the neurotoxic metabolites QUIN and 3-HK relative to KYNA may contribute to disease (1). Indeed, in patients with HD and HD model mice, 3-HK and QUIN levels are increased in the neostriatum and cortex (15, 16). Moreover, KYNA levels are reduced in the striatum of patients with HD (17). Several studies have also found perturbation in KP metabolites in the blood and cerebrospinal fluid of patients with AD, with decreased levels of KYNA correlating with reduced cognitive performance (18, 19). Similarly, in the basal ganglia of patients with PD, a reduction in KYNA levels combined with increased 3-HK has been observed (20, 21).

Drosophila melanogaster has provided a useful model for interrogation of the KP in both normal physiology and in neurodegenerative disease (22, 23). In fruit flies, TDO and KMO are encoded by vermillion (v) and cinnabar (cn), respectively, and both are implicated in Drosophila eye color pigmentation and brain plasticity (24, 25). In flies, TDO is the sole enzyme that catalyzes the initial step of the KP, as IDO1 and IDO2 are not present (Fig. 1), and so provides a distinctive model for examining the role of this critical step in the pathway. Moreover, we have previously found that downregulating cn and v gene expression significantly reduces neurodegeneration in flies expressing a mutant huntingtin (HTT) fragment—the central causative insult underlying HD (22). We also observed that pharmacological manipulations that reduced the 3-HK/KYNA ratio were always associated with neuroprotection. Notably, reintroduction of physiological levels of 3-HK in HD flies that lacked this metabolite due to KMO inhibition was sufficient to abolish neuroprotection (22). Furthermore, in a Caenorhabditis elegans model of PD, genetic down-regulation of TDO ameliorates α-synuclein (aSyn) toxicity (26). This effect appeared to be independent of changes in the levels of serotonin or KP metabolites but was correlated with increased TRP levels. Supplementing worms with TRP also suppressed aSyn-dependent phenotypes (26). The present study was designed to further define the mechanism(s) that underlies the neuroprotection conferred by TRP treatment and TDO inhibition and to extend our analyses of the neuroprotective potential of the KP to fruit fly models of AD and PD.

Discussion Impairments in KP metabolism have been linked to several neurodegenerative disorders, and in particular to the pathogenesis of HD (37). Notably, increased levels of 3-HK and QUIN have been measured in the neostriatum and cortex of patients with early stage HD (15), and these changes are associated with an up-regulation of IDO1 transcription (38) and a reduction in the activity of KAT, which is critical for KYNA synthesis (17). These data in patients with HD are supported by observations in HD mice, which show increased cerebral KMO activity (39). We previously found that either genetic or pharmacological inhibition of KMO is protective in HD flies and leads to a corresponding increase in KYNA levels relative to 3-HK (22). Furthermore, we reported that KYNA treatment reduced neurodegeneration in these flies. Here, we have extended this work by generating transgenic flies that overexpress hKAT and thereby synthesize ∼20-fold more KYNA than control flies. This increased formation of KYNA reduced neurodegeneration and eclosion defects in HD model flies. Furthermore, KMO inhibition by RNAi revealed beneficial effects in several behavioral and disease-relevant outcome measures, including larval crawling, longevity, climbing, and rhabdomere degeneration, in AD and PD model flies. These results strongly support the notion that KMO inhibition has relevance as a treatment strategy in a broad range of neurodegenerative diseases. In addition, these data also suggest that the design of small molecules capable of increasing KAT activity could have therapeutic relevance for neurodegenerative disorders. The present results, demonstrating that both genetic and pharmacological inhibition of TDO provides robust neuroprotection in fly models of AD and PD, also confirmed and extended the results of our previous study, which had identified TDO as a candidate drug target in HD flies (22). These protective effects are associated with a decrease in the 3-HK/KYNA ratio, i.e., a shift toward increased KYNA synthesis. Work in C. elegans has revealed that TDO inhibition is also protective in models of proteotoxicity, although amelioration of the phenotypes occurred independently of changes in the levels of KP metabolites and was instead associated with elevated TRP levels (26). Although the underlying mechanism remained unclear, the favorable effects of high TRP levels in the nematode were substantiated by the fact that TRP treatment conferred robust protection from disease-related phenotypes (Fig. 1). In the present study, too, TRP supplementation of the diet was effective, ameliorating rhabdomere degeneration and eclosion defects in HD flies. However, TRP feeding was also associated with a reduction in the 3-HK/KYNA ratio, suggesting that the protective effects of the amino acid may be linked to an increase in the production of the neuroprotective metabolite KYNA (Fig. 1). Indeed, partial inhibition of KYNA synthesis in TDO-deficient flies proved sufficient to completely reverse neuroprotection. In addition, restoration of physiological 3-HK levels in TDO-deficient HD flies did not reverse neuroprotection, in contrast to KMO-deficient flies (22). In primary neurons, 3-HK toxicity is dependent upon its uptake via neutral amino acid transporters, and coapplication of TRP can block this toxicity by competing for the same transporters (6). Thus, it is possible that the vast excess of TRP observed in the heads of HTT93Q v−/− flies (approximately eightfold versus controls) competes with 3-HK for rhabdomere uptake, thereby requiring hyperphysiological levels of 3-HK to reverse TDO-dependent neuroprotection. A similar mechanism may also contribute to the neuroprotection observed with TRP treatment in general. Herein, we have also found that RNAi knockdown of either cn or v does not increase TRP levels, and thus the neuroprotection observed in the AD and PD flies strongly correlates with a decrease in the 3-HK/KYNA ratio. The mechanism causing TRP treatment to favor KYNA synthesis over the formation of 3-HK in Drosophila, as well as the unexpected qualitative differences in the effects of TDO inhibition and TRP administration on KP metabolism between fruit flies and nematodes, clearly requires further investigation. Interestingly, we found that QUIN—which is not normally synthesized in fruit flies (30)—potentiated neurodegeneration in HD flies, and reversed the protective effects of KMO inhibition. As the same QUIN treatment did not cause neuron loss in wild-type flies, mutant HTT may potentiate vulnerability by enhancing NMDA receptor function (40, 41) and/or by increasing susceptibility to toxic free radicals (42), i.e., by augmenting the two major mechanisms known to be involved in QUIN-induced neurotoxicity (43). If verified in mammals, a reduction in brain QUIN levels—along with a decrease in 3-HK levels—relative to KYNA could therefore be especially promising in the treatment of HD (44). Our observation of increased levels of QUIN in HTT93Q versus WT flies is enigmatic, but may be due to altered feeding behavior, increased permeability of the blood–brain barrier (45, 46), or differences in KP metabolism, and would be interesting to explore in future studies. In conclusion, the present set of experiments further validates the hypothesis that KP metabolism is causally linked to neuronal viability and that modulation of the KP constitutes a promising therapeutic strategy for a variety of major neurodegenerative disorders. Notably, we provide the first genetic evidence to our knowledge that KMO inhibition is protective in animal models of PD and AD and that pharmacological targeting of TDO is also neuroprotective. We have clarified the mechanism underlying the protective effects of TDO inhibition, which will stimulate efforts to target this step of the KP in neurodegenerative disease. These results, together with supportive studies in flies (47) and rodents (48), raise the possibility that inhibition of TDO and KMO—or combinatorial treatment—may offer therapeutic advantages. The availability of new TDO inhibitors (49, 50), and access to the crystal structures of both TDO (51) and KMO (52), should allow further testing of these hypotheses in the near future.

Materials and Methods Fruit flies were maintained on standard maize food at 25 °C in a light/dark cycle of 12:12 h. The elavGAL4 [c155], w; +; UASaSyn (8146), w; +; UASAβ 42 (32037), w; +; UASAβ 42Arc (33774), cn3, and v36f null fly stocks were obtained from the Bloomington Drosophila Stock Center. The c164GAL4 driver line was a gift from Juan Botas, Baylor College of Medicine, Houston. HTT93Q exon 1 flies (27) were a gift from Larry Marsh and Leslie Thompson, University of California, Irvine. cn and v RNAi lines are part of the phiC31 RNAi Library (KK) and were obtained from the Vienna Drosophila RNAi Center (53). The gene encoding kynurenine aminotransferase (hKAT) was amplified from a human fetal cDNA library (54) and cloned into the pJFRC2 vector (55)—a gift from Gerald Rubin (Addgene plasmid no. 26214)—by standard methods. The resulting construct was injected by BestGene into attP40 Drosophila strains (56). Pseudopupil analysis, eclosion analysis, feeding experiments, measurement of KP metabolites, behavioral assays, longevity analysis, and statistical analyses are described in detail in SI Appendix, Materials and Methods. Measurement of KP metabolites in treated flies was performed at either 0 or 7 d posteclosion.

Acknowledgments We thank J. Lawrence Marsh, Leslie Thompson, and Juan Botas for their transgenic fly lines and BestGene for the generation of transgenic lines. C.B. was supported by grants from the CHDI Foundation and Parkinson’s UK (to F.G. and C.P.K.). F.G. and C.P.K. also acknowledge grants from the Medical Research Council and the Biotechnology and Biological Sciences Research Council for valuable infrastructure supporting this work. Work in the R.S. laboratory was supported by NIH Grant R01-NS057715.

Footnotes Author contributions: C.B., E.W.G., C.P.K., R.S., and F.G. designed research; C.B., K.V.S., S.S.I., F.M.N., J.G.E., G.G.L.M., and E.W.G. performed research; C.B., K.V.S., F.M.N., E.W.G., and F.G. analyzed data; and C.B., C.P.K., R.S., and F.G. wrote the paper.

The authors declare no conflict of interest.

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