In species ranging from humans to Caenorhabditis elegans, dietary restriction (DR) grants numerous benefits, including enhanced learning. The precise mechanisms by which DR engenders benefits on processes related to learning remain poorly understood. As a result, it is unclear whether the learning benefits of DR are due to myriad improvements in mechanisms that collectively confer improved cellular health and extension of organismal lifespan or due to specific neural mechanisms. Using an associative learning paradigm in C. elegans, we investigated the effects of DR as well as manipulations of insulin, mechanistic target of rapamycin (mTOR), AMP-activated protein kinase (AMPK), and autophagy pathways—processes implicated in longevity—on learning. Despite their effects on a vast number of molecular effectors, we found that the beneficial effects on learning elicited by each of these manipulations are fully dependent on depletion of kynurenic acid (KYNA), a neuroinhibitory metabolite. KYNA depletion then leads, in an N-methyl D-aspartate receptor (NMDAR)-dependent manner, to activation of a specific pair of interneurons with a critical role in learning. Thus, fluctuations in KYNA levels emerge as a previously unidentified molecular mechanism linking longevity and metabolic pathways to neural mechanisms of learning. Importantly, KYNA levels did not alter lifespan in any of the conditions tested. As such, the beneficial effects of DR on learning can be attributed to changes in a nutritionally sensitive metabolite with neuromodulatory activity rather than indirect or secondary consequences of improved health and extended longevity.

Learning capacity is known to decline with age, and similar effects are also associated with several neurodegenerative diseases. Regulation of insulin signaling by dietary restriction (DR) modulates lifespan in many organisms, and it has been also shown to enhance learning and memory. However, the underlying mechanisms of these processes are largely unknown due to the difficulty in disentangling the systemic effects of DR from any potentially brain-specific effects. Here, we have analyzed the molecular effects of dietary restriction in C. elegans and show that associative learning is enhanced by reducing production of the tryptophan metabolite kynurenic acid (KYNA). KYNA is an antagonist of glutamatergic signaling in neurons, and we find that its depletion in the nervous system upon DR allows for increased activation of an interneuron that is both necessary and sufficient to mediate learning. Furthermore, we show that genetic or pharmacological modulation of diverse pathways known to be involved in the physiological responses to DR also enhance learning by reducing KYNA production, likely via the activation of a specific transcription factor. Finally, we demonstrate that KYNA levels have no effect on organismal lifespan, indicating that the effects of this KYNA-mediated response to dietary restriction is truly specific to brain function and not a secondary consequence of improved health or longevity. As altered KYNA levels are associated with neurodegenerative and psychiatric diseases, our results suggest that this component may be an important modulator of learning and memory in humans as well.

Funding: NIH (grant number R01AG011816). KA. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Glenn/AFAR Aging Research Scholarship (grant number). MV. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. NIH (grant number R01AG046400). KA. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Genentech Foundation Predoctoral Fellowship (grant number). MV. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Hillblom Foundation Graduate Student Fellowship (grant number). MV. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Using the butanone association assay, we investigated the effects of DR and perturbations of molecular mechanisms that change upon restricting nutritional intake—hereafter referred to as DR mimetics—on the learning capacity of C. elegans. Specifically, we investigated the effects of reductions in either insulin or mTOR signaling pathways, as well as the effects of pharmacological and genetic interventions that lead to activations of AMPK and autophagy. Here, we show that DR and these DR mimetics each result in learning enhancements. Despite their wide-ranging cellular and organismal effects, we find that the beneficial effects of each of these interventions on learning are fully dependent on reductions in kynurenic acid (KYNA), a tryptophan-derived metabolite that can be a competitive antagonist of NMDARs [ 14 ]. We identify the neuronal sites of KYNA production and KYNA-responsive, NMDAR-expressing neurons required for learning. Although each of DR, insulin, mTOR, AMPK, and autophagy has been under intensive investigation, their effects on the kynurenine pathway (KP) have remained largely unknown. We show that each of these interventions modulate transcription of a gene encoding key enzyme of the pathway and thereby provide a potential explanation of how molecular mechanisms that function in the periphery of the animal can affect levels of a neuronal metabolite. Finally, we show that changes in KYNA levels do not alter lifespans in the context of any of the dietary, genetic, or pharmacological interventions tested, suggesting that the effects of DR and its mimetics on learning can be disentangled from their broad effects on cellular maintenance and lifespan.

C. elegans have also been used to investigate molecular underpinnings of learning and memory with paradigms for both short-term and long-term memory [ 9 – 12 ]. Many of the key molecular components implicated in mammalian learning are evolutionarily conserved in C. elegans, including N-methyl D-aspartate receptors (NMDARs), which are required for spatial memory and long-term potentiation in mice [ 13 ] and certain forms of associative learning in C. elegans [ 10 ]. One paradigm for studying short-term, associative learning in C. elegans is to pair food with butanone, an odorant that in naïve animals is only mildly attractive. This pairing results in subsequent attraction to butanone, which can be scored in a chemotaxis assay [ 11 , 12 ]. The proportion of animals attracted to butanone is calculated as a chemotaxis index, and a learning index is the difference between the chemotaxis index of conditioned animals and that of naïve animals that have not previously been exposed to butanone, which allows for normalized comparisons between treatments or strains that may differ in innate responses to butanone [ 11 , 12 ].

Caenorhabditis elegans provides an opportunity for investigating the connections between metabolism, aging, and learning. As in mammals, in C. elegans, various forms of dietary and caloric restriction extend lifespan [ 2 , 8 ]. And many of the findings that helped solidify the beneficial effects of reduced insulin signaling on lifespan extension initially emerged from studies on C. elegans [ 2 ]. Similarly, other manipulations that recapitulate some of the physiological responses to reduced food intake—for example, reductions in certain components of mechanistic target of rapamycin (mTOR) signaling and activation of AMP-activated protein kinase (AMPK), as well as activation of autophagy—promote longevity even when C. elegans have unlimited access to food [ 2 ].

Aging and various neurodegenerative disorders are characterized by progressive declines in learning capacity. There are well-established but poorly understood connections between molecular mechanisms that regulate longevity and those that influence learning and memory [ 1 ]. For example, reductions in insulin signaling are associated with lifespan extensions in many species [ 2 , 3 ]. In addition to its metabolic effects, dysregulation of insulin signaling has been implicated in cognitive defects such as Alzheimer disease [ 4 ]. In turn, dietary restriction (DR), a dietary intervention that is known to reduce insulin signaling and extend lifespan, is associated with enhancements in learning and memory and delays in cognitive decline, even in the context of neurodegenerative disorders [ 1 , 5 – 7 ]. However, given that DR or reductions in insulin signaling affect a wide range of cellular and organismal processes that can collectively promote longevity, it is unclear whether the beneficial effects elicited by these manipulations are due to direct effects on mechanisms of learning or due to myriad indirect consequences of lifespan extension and general improvements in neural maintenance and survival [ 7 , 8 ].

Results

KYNA also modulates NMDAR-dependent aversive learning behaviors nmr-1 and nmr-2 are known to mediate aversive learning when C. elegans are exposed to high NaCl concentrations without food [10]. As with attractive butanone learning, the ability of animals to learn the aversive signal of high NaCl was diminished with excess levels of KYNA but promoted upon KYNA depletion (Fig 3A). Moreover, extending the conditioning period could enhance learning of wild-type animals to the level of nkat-1 mutants but had no effect on nmr-1 or nmr-2 mutants (Fig 3B). Fasting animals in a normal NaCl environment to deplete KYNA and subsequently conditioning them with high NaCl also enhanced learning in wild-type and kmo-1 animals to the level of nkat-1 mutants with no effect on nmr-1 or nmr-2 mutants (S4 Fig). Thus, KYNA-mediated modulation of learning is independent of the sensory modality or valence of the paradigm: elevated KYNA dampens learning and reduced KYNA enhances learning, regardless of whether the stimulus is olfactory or gustatory and whether the learned association is attractive or aversive. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 3. Kynurenic acid (KYNA) depletion enhances learning only in paradigms that require N-methyl D-aspartate receptors (NMDARs). (A) NaCl aversion short-term training: learning index values for animals conditioned with high NaCl without food for 3 hours. n = 3–6, *p < 0.05, ***p < 0.001 by 1-way ANOVA (Bonferroni). (B) NaCl aversion long-term training: learning index values for animals conditioned with high NaCl without food for 6 hours. n = 3–6, *p < 0.05, ***p < 0.001 by 1-way ANOVA (Bonferroni). (C) NaCl attraction short-term training: learning index values for animals conditioned with high NaCl with food for 6 hours. n = 3–6, significance measured by 1-way ANOVA (Tukey). (D) Diacetyl short-term training: learning index values for animals conditioned with the odor diacetyl with food. n = 3, significance measured by 1-way ANOVA (Tukey). All data are represented as mean ± SEM. Underlying data can be found in S1 Data. n.s., not significant. https://doi.org/10.1371/journal.pbio.2002032.g003 Further supporting an NMDAR-dependent mechanism for effects of KYNA on learning, we found that altering KYNA or QA levels had no effect on learning in 2 paradigms in which NMDAR activity is not required: learned attraction when food was paired with either high NaCl concentrations or the odor diacetyl [25,26] (Fig 3C and 3D). Thus, KYNA-mediated modulation of learning is a general phenomenon that occurs in NMDAR-dependent paradigms.

Genetic and pharmacological manipulations that mimic aspects of DR enhance learning While many molecular components underlying learning are known and many mutations that impair learning have been studied, relatively few genetic manipulations are known to improve learning. One such manipulation is impairment of insulin signaling [11,27]. Consistent with this, we found down-regulation of the C. elegans insulin receptor daf-2 via RNAi was associated with improved learning in ad libitum fed animals with no further improvements upon fasting (Fig 4A). We reasoned that manipulating other nutritionally sensitive pathways might similarly identify molecular manipulations that improve learning. The mTOR1 and mTOR2 complexes sense nutrient availability and regulate processes ranging from fat storage and protein synthesis to development and lifespan [28]. We found that RNAi-mediated inactivations of let-363 (encoding the C. elegans mTOR homolog), daf-15 (encoding raptor), and rict-1 (encoding rictor) each led to elevated levels of learning in ad libitum fed animals (Fig 4A). This finding was reminiscent of mammalian studies reporting that chronic mTOR inhibition has procognitive effects [29,30]. Similarly, we found improved learning upon inactivation of the transcription factor mxl-3, which leads to autophagy and lipolysis in intestinal cells in response to nutrient deprivation [31], or upon treatment with phenformin, a biguanide compound that activates AMPK, a master regulator of energy homeostasis [32] (Fig 4A). None of these enhanced learning capacities were further improved by fasting (Fig 4A). All of these learning enhancements, however, required nmr-1 (Fig 4B). PPT PowerPoint slide

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larger image TIFF original image Download: Fig 4. Genetic and pharmacological manipulations that mimic dietary restriction (DR) enhance learning by depleting kynurenic acid (KYNA). (A) RNAi interference (RNAi)-mediated reductions in the insulin receptor (daf-2), the mechanistic target of rapamycin (mTOR) kinase (let-363), Raptor (daf-15), Rictor (rict-1), and a negative regulator of autophagy (mx1-3), as well as animals treated with an activator of AMP-activated protein kinase (AMPK) (phenformin), have enhanced learning capacity even when fed ad libitum. n = 3–6, *p < 0.05, ***p < 0.001 by 2-way ANOVA (Bonferroni). (B) The elevated learning capacities of genetic and pharmacological mimetics of DR are dependent on N-methyl D-aspartate receptor (NMDAR) signaling. n = 3, *p < 0.05, **p < 0.01, ***p < 0.001 by 2-way ANOVA (Bonferroni). (C) Learning index values for additional mutants in various neural nutrient sensing pathways: eat-2 mutants have a pharyngeal pumping defect, tph-1 mutants do not produce serotonin, flp-18 mutants lack a neuropeptide Y-like peptide, tdc-1 mutants do not produce tyramine or octopamine, tbh-1 mutants do not produce octopamine, and dbl-1 mutants lack a transforming growth factor β (TGFβ) ligand. n = 3–6, *p < 0.05, ***p < 0.001 by 1-way ANOVA (Tukey). (D) Average total intensity of RIM GCaMP fluorescence over the entire 250-second imaging window in animals exposed to genetic and pharmacological DR mimetics. n = 6–10, *p < 0.05, **p < 0.01, ***p < 0.001 by 1-way ANOVA (Tukey). (E) Learning index values for mutants with high KYNA exposed to genetic and pharmacological DR mimetics. n = 3, *p < 0.05, **p < 0.01, ***p < 0.001 by 2-way ANOVA (Bonferroni). (F) Learning index values for wild-type and nkat-1 animals given DR mimetics. To ensure that effects of DR mimetics in the context of KYNA depletion could be observed, animals were conditioned for only 15 minutes. n = 3, ***p < 0.001 by 2-way ANOVA (Bonferroni). (G) High-performance liquid chromatography (HPLC) measurements of steady-state KYNA levels for animals exposed to genetic and pharmacological DR mimetics. n = 5–18, *p < 0.05, **p < 0.01, ***p < 0.001 by 1-way ANOVA (Tukey). (H) HPLC measurements of steady-state KYNA levels for wild-type and daf-2(e1370) mutant animals. n = 2, *p < 0.05 by 2-tailed Student t test. Animals in panels (B), (C), (E), and (F) were ad libitum fed and conditioned. All data are represented as mean ± SEM. Underlying data can be found in S1 Data. n.s., not significant. https://doi.org/10.1371/journal.pbio.2002032.g004 Enhanced learning capacity was not a general feature of manipulating food-related signaling pathways, as mutations in various biogenic amine or peptidergic signaling pathways implicated in other food-related plasticity behaviors had minor or no effects on learning. These included mutations in genes required for synthesis of serotonin (tph-1), that of a neuropeptide Y-like molecule (flp-18), octopamine (tbh-1), both tyramine and octopamine (tdc-1), and synthesis of a transforming growth factor β (TGFβ) superfamily member ligand (dbl-1). Each of these pathways have been implicated in various food-related behaviors: for example, reductions in serotonin are associated with reduced food levels or increased population density [33,34]. Similarly, tyramine, octopamine, and neuropepetide Y-like signaling pathways are thought to be active when C. elegans are food deprived [18,35,36]. Moreover, while DR or changes in insulin, mTOR, AMPK, and autophagy can extend lifespan, not all long-lived mutants had enhanced learning. Interestingly, eat-2 mutants, which are frequently used as a model of DR because of their defects in food intake and prolonged lifespan [37–39], failed to learn (Fig 4C). The reason for this failure is not known; however, given that eat-2 mutants have defects in cholinergic signaling [37], it is possible that cholinergic signaling plays roles in the development or function of the learning circuit in addition to its role in promoting pharyngeal neuromuscular contractions required for feeding. Of note, eat-2 mutants were previously shown to have defective long-term memory but normal learning capacity [11]. The reason for the discrepancy between our results and the previous publication is not known, although both studies indicate that mutations in eat-2 do not confer enhancements on short-term learning.

The elevated learning capabilities of DR mimetics are KYNA dependent We next sought to better understand the relationship of KYNA to learning enhancements caused by DR mimetics. As in fasting or depletion of KYNA, the learning enhancements seen in DR mimetics even in the presence of plentiful food supplies correlated with increases in Ca2+ transient intensity in RIM similar to control animals that had been fasted before conditioning (Fig 4D and S4 Fig). In each case, elevation of KYNA levels using either haao-1 or kmo-1 mutants caused a significant reduction in learning capacity (Fig 4E). We next exposed nkat-1 mutants to various DR mimetic treatments. However, given that nkat-1 mutants already have elevated learning, we decreased the time animals were conditioned with butanone to avoid being confounded by a ceiling in our ability to measure learning. Under these conditions, wild-type animals did not learn, but fasted or KYNA-deficient animals still did, albeit to a lesser degree than when the conditioning was for the standard 1-hour period used elsewhere in this study (Fig 4F). Treatment of nkat-1 mutants with fasting or exposure to any of the DR mimetics did not lead to further improvements in learning (Fig 4F), suggesting that DR mimetics and nkat-1 mutants function in the same pathway to enhance learning. We next employed direct biochemical measurements of KYNA levels extracted from populations of whole animals exposed to each of the DR mimetics. As in the case of fasted animals, we found that with each of the DR mimetics, KYNA levels were already depleted, even under ad libitum fed conditions (Fig 4G). While there was a trend of decreased KYNA levels in animals exposed to daf-2 RNAi, the reduction was not statistically significant. Since these metabolite measurements were conducted on extracts from populations of animals and we could not be certain of the efficacy of daf-2 RNAi throughout the population, we used daf-2(e1370) mutants. KYNA levels of ad libitum fed daf-2 mutants were substantially reduced compared to those of wild-type animals (Fig 4H).

DR mimetics alter expression levels of KP genes Although each of insulin, mTOR, AMPK, and autophagy are extensively studied, mechanisms through which these manipulations could result in changes in the KP have not been established. Moreover, while molecular components of insulin, mTOR, and AMPK signaling pathways are broadly expressed in C. elegans, the transcriptional regulator MXL-3 acts in the intestine [31], yet its inactivation leads to reduced KYNA levels and enhanced learning. Thus, there must be mechanisms that regulate KYNA levels even when working at sites distant from the neurons involved in KYNA production. To better understand mechanisms that regulate flux through the KP, we first considered sites of expression of key enzymes that are required for generation or utilization of kynurenine, the substrate from which KYNA is produced. It has been previously reported that the tdo-2 gene encoding the first enzyme in the KP is expressed in the body wall muscle and skin-like epidermis of C. elegans [24]. Indeed, using a transcriptional fusion of the tdo-2 promoter to green fluorescent protein (GFP), we observed robust expression in the epidermis (Fig 5A and 5C). We found that kmo-1 shows epidermal expression as well (Fig 5B). Neither tdo-2 nor kmo-1 appeared to be expressed in the neurons that express nkat-1 (Fig 5B and 5C). This suggests that kynurenine must be transported across issues in order for RIM to produce KYNA. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 5. kmo-1 and nkat-1 have distinct tissue expression patterns and exhibit distinct transcriptional patterns of regulation. (A) Cartoon representation of the anterior portion of adult C. elegans comparable to images in panels (B) and (C). Labeled anatomical structures correspond to those in panels (B) and (C). (B) Five-μm thick z-projection of an adult animal with mCherry under control of the kmo-1 promoter and green fluorescent protein (GFP) under control of the nkat-1 promoter. Left: anterior portion of the animal. Right: magnified view of the nerve ring showing no overlap of GFP and mCherry. Asterisk indicates pharynx; arrowhead indicates epidermis; arrow indicates intestine. (C) 5-μm thick z-projection of an adult animal with mCherry under control of the tdo-2 promoter and GFP under control of the nkat-1 promoter. Left: anterior portion of the animal. Right: magnified view of the nerve ring showing no overlap of GFP and mCherry. Asterisk indicates pharynx; arrowhead indicates epidermis; arrow indicates intestine. (D) Change in transcript levels of kynurenine pathway (KP) genes in animals treated with dietary restriction (DR) or DR mimetics as determined by real-time quantitative PCR (qPCR). Data are represented as fold change compared to ad libitum fed animals on vector (RNA interference [RNAi]). n = 3 biological replicates, *p < 0.05, **p < 0.01, ***p < 0.001 by 1-way ANOVA (Tukey). (E) Learning index values for hlh-30 mutants on let-363 and mxl-3 RNAi. n = 3, significance measured by 2-way ANOVA (Bonfferoni). (F) Learning index values for daf-16 mutants on DR mimetics. n = 3, **p < 0.01, ***p < 0.001 measured by 2-way ANOVA (Bonferroni). (G) Change in kmo-1 transcript levels in daf-16 mutants as determined by real-time qPCR. Data are represented as fold change compared to wild-type. n = 3 biological replicates, *p < 0.05, ***p < 0.001 by 1-way ANOVA (Tukey). All data are represented as mean ± SEM. Underlying data can be found in S1 Data. https://doi.org/10.1371/journal.pbio.2002032.g005 We next examined tdo-2, kmo-1, and nkat-1 transcripts via quantitative PCR (qPCR) in animals given DR mimetics. Compared to ad libitum fed animals on vector RNAi, fasting and DR mimetics caused no significant changes in either tdo-2 or nkat-1 expression. In contrast, each of the mimetics resulted in a significant increase in kmo-1 expression (Fig 5D). Given coincident tissue expressions of tdo-2 and kmo-1 in a large tissue such as the epidermis, these data indicate that up-regulation of kmo-1 could compete with KYNA production by shunting kynurenine, the common substrate between NKAT-1 and KMO-1, down a different branch of the KP, resulting in reduced KYNA levels (Fig 2A). To further understand the relationship between kmo-1 expression and DR, we set out to investigate the effects of several transcription factors with key roles in DR on kmo-1 expression. It is well established that insulin signaling causes functional inactivation the FOXO transcription factor DAF-16 and that many of the consequences of reduced insulin signaling seen in daf-2 mutants require daf-16 [3]. Similarly, mTOR and AMPK signals can be transduced via the NRF2 master regulator SKN-1 [3]. Additionally, mTOR activity affects HLH-30, a TFEB orthologue that competes with MXL-3 for binding sites and exerts opposing effects, and fasting increases hlh-30 expression [31,40]. Finally, cross-talk among many of these pathways has been shown. Thus, we sought to determine the effects of daf-16, skn-1, and hlh-30 reduction-of-function mutations on learning. We found that skn-1 mutants failed to chemotax, so they could not be properly assayed. Although other phenotypes resulting from loss of let-363 and mxl-3 are known to involve hlh-30 [31], hlh-30 mutants had no learning phenotype (Fig 5E), suggesting it may not play a role in regulating KYNA production relevant for this behavior. In contrast, daf-16 mutants had a learning defect that blocked the enhancements of fasting (Fig 5F). Moreover, loss of daf-16 not only blocked the enhanced learning of insulin-deficient animals, it abrogated the enhanced learning of various DR mimetics (Fig 5F), raising the possibility that insulin signaling pathway serves as a major link between various nutritionally sensitive pathways and the KP. Consistent with this, we found that, compared to wild-type, daf-16 mutants had significantly reduced kmo-1 transcript levels (Fig 5G). It is currently unknown whether kmo-1 is a direct target of DAF-16.