Dietary restriction and activation of RIM interneurons enhance learning.

<p>(A) Effects of various durations of conditioning on the learning index of wild-type animals. <i>n</i> = 6, *<i>p</i> < 0.05, ***<i>p</i> < 0.001 by 1-way ANOVA (Tukey). (B) Animals mutant in <i>odr-1</i> or with nonfunctional AWC neurons fail to learn in response to butanone. <i>n</i> = 6, ***<i>p</i> < 0.001 by one-way ANOVA (Tukey). (C) Chronic dietary restriction (DR) (50%–90% of ad libitum food levels for animals’ entire lives) enhance learning. <i>n</i> = 3, *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001 by 1-way ANOVA (Tukey). (D) Acute DR (DR for 1 hour) as well as a 1-hour fast enhance learning. <i>n</i> = 3–6, ***<i>p</i> < 0.001 by 1-way ANOVA (Tukey). (E) Effects of various durations of fasting prior to a 1-hour conditioning period on learning. <i>n</i> = 3–10, *<i>p</i> < 0.05, ***<i>p</i> < 0.001 by 1-way ANOVA (Tukey). (F) Animals mutant in glutamatergic receptors fail to learn. <i>n</i> = 6–10, ***<i>p</i> < 0.001 by 1-way ANOVA (Tukey). (G) Reconstitution of <i>nmr-1</i> in only the RIM neurons is sufficient to restore learning capacity to <i>nmr-1</i> mutants. Learning index values for <i>nmr-1</i> mutants with <i>nmr-1</i> reconstituted under its own promoter, an RIM-specific promoter, or an AVA-specific promoter are shown. tg denotes transgenic animals; non-tg denotes non-transgenic siblings. <i>n</i> = 3, *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001 by 1-way ANOVA (Tukey). (H) Average intensity of spontaneous GCaMP transients in RIM from the 250-second imaging window aligned to a −5-second to 20-second time axis. (I) Average total intensity of RIM GCaMP fluorescence over the entire 250-second imaging window shows that conditioning significantly increases transient intensity, and fasting before conditioning has an even greater effect. <i>n</i> = 6–10, *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001 by 1-way ANOVA (Tukey). (J) Chronic activation of RIM by using a constitutively active protein kinase C (PKC) encoded by <i>pkc-1(gf)</i> promotes learning even in animals fed ad libitum while silencing of RIM using an overactive potassium channel endcoded by <i>unc-103(gf)</i> abolishes learning capacity. tg denotes transgenic animals; non-tg denotes non-transgenic siblings. <i>n</i> = 4–9, *<i>p</i> < 0.05, ***<i>p</i> < 0.001 by 1-way ANOVA (Tukey). Animals in panels (F), (G), and (J) were fed ad libitum and conditioned for 1 hour. All data are represented as mean ± SEM. Underlying data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002032#pbio.2002032.s009" target="_blank">S1 Data</a>.</p

The beneficial effects of dietary restriction on learning are distinct from its effects on longevity and mediated by depletion of a neuroinhibitory metabolite

<div><p>In species ranging from humans to <i>Caenorhabditis elegans</i>, 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 <i>C</i>. <i>elegans</i>, 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.</p></div

Genetic and pharmacological manipulations that mimic dietary restriction (DR) enhance learning by depleting kynurenic acid (KYNA).

<p>(A) RNAi interference (RNAi)-mediated reductions in the insulin receptor (<i>daf-2</i>), the mechanistic target of rapamycin (mTOR) kinase (<i>let-363</i>), Raptor (<i>daf-15</i>), Rictor (<i>rict-1</i>), and a negative regulator of autophagy (<i>mx1-3</i>), as well as animals treated with an activator of AMP-activated protein kinase (AMPK) (phenformin), have enhanced learning capacity even when fed ad libitum. <i>n</i> = 3–6, *<i>p</i> < 0.05, ***<i>p</i> < 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. <i>n</i> = 3, *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<u><i>p</i></u> < 0.001 by 2-way ANOVA (Bonferroni). (C) Learning index values for additional mutants in various neural nutrient sensing pathways: <i>eat-2</i> mutants have a pharyngeal pumping defect, <i>tph-1</i> mutants do not produce serotonin, <i>flp-18</i> mutants lack a neuropeptide Y-like peptide, <i>tdc-1</i> mutants do not produce tyramine or octopamine, <i>tbh-1</i> mutants do not produce octopamine, and <i>dbl-1</i> mutants lack a transforming growth factor β (TGFβ) ligand. <i>n</i> = 3–6, *<i>p</i> < 0.05, ***<i>p</i> < 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. <i>n</i> = 6–10, *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001 by 1-way ANOVA (Tukey). (E) Learning index values for mutants with high KYNA exposed to genetic and pharmacological DR mimetics. <i>n</i> = 3, *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001 by 2-way ANOVA (Bonferroni). (F) Learning index values for wild-type and <i>nkat-1</i> 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. <i>n</i> = 3, ***<i>p</i> < 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. <i>n</i> = 5–18, *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001 by 1-way ANOVA (Tukey). (H) HPLC measurements of steady-state KYNA levels for wild-type and <i>daf-2(e1370)</i> mutant animals. <i>n</i> = 2, *<i>p</i> < 0.05 by 2-tailed Student <i>t</i> 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 <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002032#pbio.2002032.s009" target="_blank">S1 Data</a>. n.s., not significant.</p

Median lifes pans of animals on DR mimetics.

<p>Median lifespans of animals given dietary restriction (DR) mimetics are not affected by <i>kmo-1</i> or <i>nkat-1</i> mutations. Significance was measured by logrank tests. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002032#pbio.2002032.s005" target="_blank">S5</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002032#pbio.2002032.s006" target="_blank">S6</a> Figs for full survival curves.</p

Kynurenic acid (KYNA) depletion enhances learning only in paradigms that require N-methyl D-aspartate receptors (NMDARs).

<p>(A) NaCl aversion short-term training: learning index values for animals conditioned with high NaCl without food for 3 hours. <i>n</i> = 3–6, *<i>p</i> < 0.05, ***<i>p</i> < 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. <i>n</i> = 3–6, *<i>p</i> < 0.05, ***<i>p</i> < 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. <i>n</i> = 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. <i>n</i> = 3, significance measured by 1-way ANOVA (Tukey). All data are represented as mean ± SEM. Underlying data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2002032#pbio.2002032.s009" target="_blank">S1 Data</a>. n.s., not significant.</p

Three lysines in NHR-25 are necessary for the interaction with SMO-1.

<p>(A) NHR-25 fused to the Gal4 DNA binding domain (DB) interacted with wild type (WT) SMO-1 fused to the Gal4 activation domain (AD). No interaction was seen with empty vector (No insert), SMO-1 with the terminal di-glycine residues deleted (ΔGG), or SMO-1 with a β-sheet mutation (V31K). (B) The NHR-25 3KR (K165R K170R K236R) allele specifically blocked interaction with SMO-1, as both NHR-25 and NHR-25 3KR interacted with NHR-91. (C) Schematic of NHR-25 domain structure illustrating the DNA binding domain (DBD), hinge region, and ligand binding domain (LBD). The candidate SUMO acceptor lysines (K165, K170, K236) are indicated. (D) Mutating the indicated SUMO acceptor lysines to arginine in NHR-25 only abolished the interaction when all three were mutated (K165R K170R K236R). We note the non-reciprocality of our Y2H interactions: DB-NHR-25 interacted with AD-SMO-1 and AD-NHR-25 interacted with DB-NHR-91. Switching the Gal4 domains did not result in an interaction, as sometimes occurs in Y2H interactions <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003992#pgen.1003992-Thompson1" target="_blank">[69]</a>. β-galactosidase (LacZ) reporters were assayed in A, B, and D.</p

Sumoylated NHR-25/NR5A Regulates Cell Fate during<i>C. elegans</i> Vulval Development

<div><p>Individual metazoan transcription factors (TFs) regulate distinct sets of genes depending on cell type and developmental or physiological context. The precise mechanisms by which regulatory information from ligands, genomic sequence elements, co-factors, and post-translational modifications are integrated by TFs remain challenging questions. Here, we examine how a single regulatory input, sumoylation, differentially modulates the activity of a conserved <i>C. elegans</i> nuclear hormone receptor, NHR-25, in different cell types. Through a combination of yeast two-hybrid analysis and <i>in vitro</i> biochemistry we identified the single <i>C. elegans</i> SUMO (SMO-1) as an NHR-25 interacting protein, and showed that NHR-25 is sumoylated on at least four lysines. Some of the sumoylation acceptor sites are in common with those of the NHR-25 mammalian orthologs SF-1 and LRH-1, demonstrating that sumoylation has been strongly conserved within the NR5A family. We showed that NHR-25 bound canonical SF-1 binding sequences to regulate transcription, and that NHR-25 activity was enhanced <i>in vivo</i> upon loss of sumoylation. Knockdown of <i>smo-1</i> mimicked NHR-25 overexpression with respect to maintenance of the 3° cell fate in vulval precursor cells (VPCs) during development. Importantly, however, overexpression of unsumoylatable alleles of NHR-25 revealed that NHR-25 sumoylation is critical for maintaining 3° cell fate. Moreover, SUMO also conferred formation of a developmental time-dependent NHR-25 concentration gradient across the VPCs. That is, accumulation of GFP-tagged NHR-25 was uniform across VPCs at the beginning of development, but as cells began dividing, a <i>smo-1</i>-dependent NHR-25 gradient formed with highest levels in 1° fated VPCs, intermediate levels in 2° fated VPCs, and low levels in 3° fated VPCs. We conclude that sumoylation operates at multiple levels to affect NHR-25 activity in a highly coordinated spatial and temporal manner.</p></div

Overexpression of unsumoylated NHR-25 causes multivulva induction.

<p>(A) Table providing scoring of overall multivulva (Muv) induction in the indicated strains/genotypes, as well as induction in individual VPCs. Number of animals (n) scored for each strain genotype is provided. Use of brackets denotes transgenic genotypes. (B) Graphical representation of the overall percentage of animals for each strain that display Muv induction of any VPC.</p

NHR-25::GFP (OP33) expression during vulval development.

<p>Expression in 1-cell stage Pn.p cells (A), in 2-cell stage Pn.px cells (B) and 4-cell stage Pn.pxx cells (C) in wild type and in <i>smo-1(RNAi)</i> animals (E). Higher levels and ectopic expression of NHR-25 were seen in P4.px and P8.px(x) in a <i>smo-1(RNAi)</i> background (E). Expression at the bell stage in wild type and <i>smo-1(RNAi)</i> animals (D,F). Ectopic expression in the AC observed in <i>smo-1(RNAi)</i> animals. Arrowheads indicate the position of the AC, red asterisk indicates the position of the invaginated vulva. Colored bars indicate 1° (red), 2° (yellow), and 3° (blue) lineages, as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003992#pgen-1003992-g002" target="_blank">Figure 2</a>.</p

<i>In vitro</i> sumoylation of NHR-25.

<p><i>In vitro</i> sumoylation reactions were resolved by SDS-PAGE and analyzed by either Coomassie staining (A,C) or immunoblotting with anti-NHR-25 antibody (B). (A and B) used recombinant human sumoylation enzymes (hE1, hE2, hSUMO1, hSENP1 SUMO protease), (C) used recombinant <i>C. elegans</i> CeUBC-9 and CeSMO-1 with hE1 and hSENP1. Substrates were recombinant 6×His-MBP-NHR-25 (amino acids 161–541; A,C), and the same construct <i>in vitro</i> transcribed and translated (B). In (B) an MBP control was <i>in vitro</i> transcribed and translated, as were the NHR-25 alleles 3KR (K165 K170R K236R) and 3EA(E167A E172A E238A). The positions of NHR-25, sumoylated NHR-25 and AOS1 (part of E1 heterodimer) are indicated. Size markers in kilodaltons (kDa) are provided.</p