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. 2022 Feb 17;220(4):iyac025. doi: 10.1093/genetics/iyac025

Involvement of HECT-type E3 ubiquitin ligase genes in salt chemotaxis learning in Caenorhabditis elegans

Yasuaki Ike 1, Masahiro Tomioka 1,✉, Yuichi Iino 1,✉
Editor: E Hallem
PMCID: PMC8982016  PMID: 35176147

Abstract

The ubiquitin-proteasome system is associated with various phenomena including learning and memory. In this study, we report that E3 ubiquitin ligase homologs and proteasome function are involved in taste avoidance learning, a type of associative learning between starvation and salt concentrations, in Caenorhabditis elegans. Pharmacological inhibition of proteasome function using bortezomib causes severe defects in taste avoidance learning. Among 9 HECT-type ubiquitin ligase genes, loss-of-function mutations of 6 ubiquitin ligase genes cause significant abnormalities in taste avoidance learning. Double mutations of those genes cause lethality or enhanced defects in taste avoidance learning, suggesting that the HECT-type ubiquitin ligases act in multiple pathways in the processes of learning. Furthermore, mutations of the ubiquitin ligase genes cause additive effects on taste avoidance learning defects of the insulin-like signaling mutants. Our findings unveil the consequences of aberrant functions of the proteasome and ubiquitin systems in learning behavior of Caenorhabditis elegans.

Keywords: ubiquitin ligase, associative learning, Caenorhabditis elegans, proteasome

Introduction

Behavioral plasticity, characterized by functional and structural synaptic changes, is important for the survival of animals in various environments. One of the mechanisms controlling synaptic plasticity is the ubiquitin-proteasome system (UPS). Proteasomal inhibition results in impaired learning behavior (Lopez-Salon et al. 2001). Proteasomal degradation of synaptic proteins also regulates activity-dependent synaptic plasticity (Ageta et al. 2001; Ehlers 2003). UPS carries out ubiquitination of target proteins which is a prerequisite of proteasomal degradation. Ubiquitination is characterized by the addition of a ubiquitin molecule, which is highly conserved from yeast to humans, to the targeted protein (Kipreos 2005; Komander and Rape 2012). This reaction is involved in synaptic development and function (Schaefer et al. 2000; Wan et al. 2000; Chin et al. 2002; Yao et al. 2007; Helton et al. 2008; Xiong et al. 2012), and it requires E1 ubiquitin-activating enzyme, E2 ubiquitin conjugating enzyme, and E3 ubiquitin ligase. In particular, ubiquitin ligases are classified based on the domains they carry, such as RING, HECT, and U-box. Among them, several HECT-type ubiquitin ligases are associated with learning behavior (Camera et al. 2016; Pérez-Villegas et al. 2018; Ambrozkiewicz et al. 2020). However, it is unclear whether they function via identical or different pathways. Moreover, it remains poorly known whether other ubiquitin ligases containing the HECT domain are also involved in learning.

Caenorhabditis elegans has a simple nervous system compared to mammals; nonetheless, it exhibits behavioral plasticity similar to mammals. The C. elegans genome encodes 9 HECT-type ubiquitin ligases, all of which have mammalian orthologs. Given that C. elegans is an in vivo model that is easy to genetically manipulate and to perform learning behavior analysis, it also represents a good platform to comprehensively examine behavioral phenotypes and analyses of genetic interactions between ubiquitin ligases.

This study aimed to explore the involvement of the UPS in associative learning using C. elegans exposed to starvation and sodium chloride (hereafter referred to as taste avoidance learning) as experimental model. We examined taste avoidance learning with mutants of 9 HECT-type ubiquitin ligase genes and found defects in salt chemotaxis behavior including taste avoidance learning in 6 ubiquitin ligase mutants. Analyses of genetic interactions suggested that some of these ubiquitin ligases have redundant functions and act in the different pathways from the insulin-like pathway, which is known to contribute to taste avoidance learning (Ohno et al. 2014; Nagashima et al. 2019).

Materials and methods

Caenorhabditis elegans strains

Bristol strain N2 was used as the wild type in vivo model and Escherichia coli strain NA22 was used as food source. The animals were cultivated on nematode growth media (NGM) plates at 15, 20, or 23° according to standard methods (Brenner 1974). Double mutants were generated by crossing, and their genotypes were confirmed by polymerase chain reaction analysis. The mutants and the respective genotyping primers used in this study are listed in Supplementary Tables 1 and 2, respectively. The ubr-5(pe767) mutant, which harbors a nonsense mutation in exon 7, was generated by ethyl-methanesulfonate mutagenesis. The mutants of ubiquitin ligases were crossed with N2 multiple times. The number of crosses for each mutant is as follows.

  • Four times: uba-1(it129), eel-1(zu462), eel-1(tm1515), hecd-1(ok1437), wwp-1(gk372)

  • Five times: ubr-5(pe767), ubr-5(ok1108)

  • Six times: eel-1(ok1575), hecd-1(tm2371), wwp-1(ok1102), Y92H12A.2(tm5771), hecw-1(gk170677), oxi-1(gk185003), etc-1(tm5615), herc-1(ok1524), Y92H12A.2(ok3321)

We note that eel-1(zu462) and eel-1(tm1515) mutants were obtained by excluding unc-33(e204) and unc-17(e113) mutations from the JJ1972 and FX31520 strains, respectively.

Behavioral assays

Salt concentration learning assays were performed as described previously (Tomioka et al. 2016). Adult animals were grown on NGM plates seeded with E. coli NA22. For conditioning, adult animals were transferred onto NGM plates with 25, 50, or 100 mM NaCl, which are represented as low, middle or high salt, respectively, for 5 h. Next, about 50–200 animals were placed at the center of a test plate with a NaCl gradient and were allowed to move for 45 min. The number of animals was counted in each region of the test plates, and the chemotaxis index was calculated using the following equation:

Chemotaxis Index = (NA-NB)(NAll-NC),

where NA and NB represent the numbers of animals in the high- and low-salt regions, respectively; NAll represents the total number of animals in the test plate; and NC represents the number of animals in the center region. All assays were performed 8 times.

Bortezomib Treatment

Bortezomib (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in dimethyl sulfoxide (DMSO). Two days before the behavioral assays, bortezomib was spread on NGM plates. Plates seeded with E. coli NA22 were incubated at 37° overnight. The day before the behavioral assays, the animals were transferred onto the NGM plates supplemented with bortezomib and cultivated for 16 h. Additional bortezomib was spread on the NGM plates for conditioning and left overnight. Bortezomib was used at 10 µl per plate and final concentrations were from 0 to 40 µg/ml. Time of bortezomib treatment was determined according to the previous study (Segref et al. 2011).

Cloning of uba-1 cDNA

uba-1 cDNA was amplified from N2 total cDNA with KOD One DNA polymerase (Toyobo). Primers used for cloning were 5′-aggacccttggctagATGACTACCATCCTTGAGCTAACATCGGCCAAC-3′ and 5′-taccgtcgacgctagcTTAGAAAGAGTAGCGAATGTATGGAACTTCGACATCTTC-3′. Lower case letters indicate sequence homologous to the vector. To generate a vector containing uba-1 fused with sl2::CFP, PCR-amplified uba-1 cDNAs were inserted into the pDEST-sl2::CFP vector linearized by Nhe I using In-Fusion HD Cloning Kit (Takara). The uba-1 expression plasmids were generated by the GATEWAY system (Invitrogen).

Temperature shift

In experiments using the temperature sensitive mutant uba-1(it129) (Fig. 2), 16 h before conditioning, animals were transferred from the cultivation temperature of 20°–25°, and conditioning was performed also at 25°. Behavioral assays were then carried out at 23°.

Fig. 2.

Fig. 2.

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uba-1 acts in the nervous system in taste avoidance learning. Salt chemotaxis learning of the wild type, the uba-1(it129) mutant with a) or without b) transgenes for uba-1(+) expression in the nervous system, the ASER neuron, or the intestine under the control of the rgef-1, gcy-5, or ges-1 promoter, respectively. Salt chemotaxis learning assays were performed at the restrictive temperature unless indicated as “No temperature shift.” Error bars indicate the standard error of the mean. *P < 0.05, **P < 0.01, ***P < 0.001, n.s.: not significant. The strains carrying transgenes were compared to the corresponding transgene (−) animals. The statistical analysis was performed by one-way ANOVA with Bonferroni’s multiple comparison test.

Venus transcriptional reporter preparation

The promoters for eel-1, hecd-1, ubr-5, and wwp-1 were fused with Venus using a PCR-based approach. Genomic fragments of the ubiquitin ligases, including 5′ UTR, 1st exon and 1st intron, were amplified from N2 genomic DNA with KOD-Plus-Neo (TOYOBO). Primers used for PCR are listed in Supplementary Table 4. The first PCR amplified promoters of each ubiquitin ligase, and second PCR fused the promoter and venus sequences. A lowercase part of the primer sequences in Supplementary Table 4 depicts a part of the venus sequence.

Confocal microscopy

Fluorescent images were collected with a Leica SP5 microscope and a 10X objective. Animals were cultivated at 20°. Day 1 adult animals were placed on a 5% agarose pad and were anesthetized in M9 buffer containing 200 mM sodium azide.

Multiworm tracking

Multiworm tracking analysis was conducted as previously described with modifications (Kunitomo et al. 2013). Animals and chemotaxis assay plates were prepared as salt concentration learning assay, but sodium azide used for trapping animals was omitted. Less than 100 animals were placed on an assay plate to reduce collisions. Images of assay plates were acquired for 45 min at 1 frame per second. Fractions of animals in each of following regions on the plate were counted: A (high salt region), A’ (outer region of A), B (low salt region), B’ (outer region of B), and C (center region).

Statistical analysis

One-way analysis of variance (ANOVA) with Tukey’s, Dunnett’s, and Bonferroni’s multiple comparison tests were used to assess statistical significance of the analyses as indicated in figure legends. All data were analyzed using GraphPad Prism 5.0 software (GraphPad Software, San Diego, CA, USA). P-value < 0.05 was considered statistically significant.

Results

A proteasome inhibitor, bortezomib, causes defects in taste avoidance learning

Wild-type worms are attracted to the salt concentrations at which they were conditioned on plates with food: they are attracted to low or high salt concentrations after conditioning with low or high salt, respectively, in the presence of food. On the other hand, they avoid the salt concentrations at which they were conditioned on plates without food: they avoid low or high salt concentrations after conditioning with low or high salt in the absence of food (taste avoidance learning) (Fig. 1a) (Kunitomo et al. 2013). Hereafter, different conditions of salt conditioning are abbreviated as follows: low salt conditioning with food as “low/fed,” high salt conditioning with food as “high/fed,” low salt conditioning without food as “low/starved” and high salt conditioning without food as “high/starved” and so forth. First, the requirement of proteasomal function in taste avoidance learning was investigated by using the proteasome inhibitor, bortezomib (BTZ), because proteasomal function is enhanced by starvation (Zhao and Goldberg 2016). When worms were exposed to BTZ for 21 h before and during starvation conditioning, they showed dramatic behavioral changes in salt chemotaxis in a dose-dependent manner. In particular, when high concentrations of BTZ were supplied, the worms were attracted to the salt concentrations at which they experienced starvation, as if they were fed at those salt concentrations. On the other hand, similarly treated worms showed only weak abnormality in chemotaxis after fed conditioning (Fig. 1, b and c). We note that high concentration of BTZ causes some degree of movement defect, manifested by larger number of animals at the starting point (Supplementary Fig. 1). However, immobility alone cannot explain reversed preference in BTZ-treated animals after starvation conditioning. These data suggest that proteasomal function is involved in salt chemotaxis learning especially in starvation-induced switching from salt attraction to avoidance.

Fig. 1.

Fig. 1.

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Proteasomal inhibition affects phenotypes of taste avoidance learning. a) Schematic illustration of salt chemotaxis learning assay. After worms were conditioned on an agar plate at specific salt concentrations with food for 5 h, they are attracted to the salt concentrations. On the other hand, they avoid the salt concentrations which they experienced in the absence of food. b,c) Salt chemotaxis of wild-type worms conditioned with food b) or without food c) at 3 different salt concentrations with bortezomib (BTZ), a proteasomal inhibitor at the indicated concentrations. Error bars represent the standard error of the mean. *P < 0.05, **P < 0.01, ***P < 0.001. One-way ANOVA with Dunnett’s post hoc test compared to conditions of 0 µg/ml BTZ solution.

The ubiquitination system acts in the adult nervous system in salt chemotaxis learning

Because proteasomal degradation occurs after poly-ubiquitination of the substrate, involvement of the ubiquitination system in salt chemotaxis learning was examined next. We first investigated the function of ubiquitin-activating enzyme (E1) in salt chemotaxis learning. The genome of C. elegans encodes a single E1 gene, uba-1. We used the uba-1(it129) mutant, which has been reported as a temperature-sensitive mutant (Kulkarni and Smith 2008). The uba-1(it129) mutant showed no significant defect at permissive temperature (20°), while when it was shifted to restrictive temperature (25°) 21 h before the assay, it showed increased migration to lower salt after 5 kinds of conditioning except for low/fed compared to the wild type (Fig. 2). Pan-neuronal expression of uba-1 cDNA using the rgef-1 promoter rescued these defects, whereas expression in the ASER neuron, a taste receptor neuron important for the regulation of salt chemotaxis learning, or the intestine using the gcy-5 or the ges-1 promoter, respectively, did not rescue the learning defects, suggesting that uba-1 functions in the nervous system other than ASER or multiple neurons including ASER.

Multiple HECT-type E3 ubiquitin ligases are involved in taste avoidance learning

Since HECT-type E3 ubiquitin ligases were previously reported to be involved in learning behavior in mammals (Camera et al. 2016; Pérez-Villegas et al. 2018; Ambrozkiewicz et al. 2020), the following experiments were focused on the HECT-type ubiquitin ligases in C. elegans (Supplementary Fig. 2). Salt chemotaxis learning of 9 loss-of-function mutants of HECT-type ubiquitin ligase was examined and 6 of them were found to show abnormal phenotypes compared to the wild type (Fig. 3, a and b).

Fig. 3.

Fig. 3.

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Six out of 9 mutants of HECT E3 ubiquitin ligase genes showed abnormal learning behaviors in salt chemotaxis. a,b) Salt chemotaxis of the wild type and HECT E3 ubiquitin ligase mutants after conditioning with or without food at 3 different salt conditions. Error bars indicate the standard error of the mean. *P < 0.05, **P < 0.01, ***P < 0.001. One-way ANOVA with Dunnett’s post hoc test compared to the wild type. Experiments a) and b) were conducted independently. c) Bar graphs of part of Fig. 1, b and c.

After high/fed conditioning, eel-1(ok1575), hecd-1(tm2371), and wwp-1(ok1102) mutants showed defects in high-salt migration. These genes encode orthologs of mammalian HUWE1, HECTD1, and WWP1, respectively. The eel-1(ok1575) and wwp-1(ok1102) mutants also showed low-salt migration abnormality after low/fed conditioning.

After starved conditioning, in addition to the mutants described above, 3 other mutants, ubr-5(pe767), Y92H12A.2(tm5771), and etc-1(tm5615) mutants, showed abnormal chemotaxis compared to the wild type. The eel-1(ok1575) mutant showed defects in all kinds of starved conditioning. The hecd-1(tm2371) mutant showed reduced migration after low/starved and middle/starved conditioning, whereas the etc-1(tm5615) mutant showed enhanced migration to high salt after low/starved and middle/starved conditioning. Salt chemotaxis defects in mutants of ubr-5(pe767), an ortholog of human UBR5, and Y92H12A.2(tm5771), an ortholog of human NEDD4, were observed only after low/starved and high/starved conditioning, respectively.

Among the mutants with significant abnormality, mutants of eel-1(ok1575) and wwp-1(ok1102) showed weak chemotaxis after most kinds of conditioning. Behavioral analysis of these mutants by multiworm tracker suggests that both eel-1(ok1575) and wwp-1(ok1102) mutants have locomotion abnormality (Supplementary Fig. 3), but the learning defects were not simply caused by it; the salt preference seemed to be lost earlier compared to the wild type (Supplementary Figs. 4 and 5). In each of the eel-1, hecd-1, wwp-1, ubr-5, and Y92H12A.2 mutants, multiple alleles showed similar phenotypes in salt chemotaxis learning, suggesting that the behavioral defects were caused by the mutations of each ubiquitin ligase but not by another genetic background (Supplementary Fig. 6). The alleles except for the hecd-1(tm2371) and etc-1(tm5615) cause early stop codons and therefore are considered as null, and the hecd-1(tm2371) allele showed similar defects as the early stop allele. Transcriptional Venus reporters of eel-1, hecd-1, wwp-1, and ubr-5 were expressed in various tissues, including the nervous system, consistent with the finding that the E1 gene, uba-1, functions in the nervous system in salt chemotaxis learning (Supplementary Fig. 7).

We note that those ubiquitin ligase genes were reported to function in several biological phenomena in addition to salt chemotaxis learning observed here. eel-1 is known to be involved in GABAergic neurotransmission and Wnt signaling in C. elegans (De Groot et al. 2014; Opperman et al. 2017). wwp-1 is known to regulate lifespan and response against bacterial toxin (Carrano et al. 2009, 2014; Chen et al. 2010). hecd-1 and ubr-5 were reported to be involved in the regulation of Notch signaling in C. elegans (Chen and Greenwald 2014;Chen and Chalfie 2015; Safdar et al. 2016). It was reported that eel-1 and hecd-1 are also associated with development (Page et al. 2007; Zahreddine et al. 2010; Beard et al. 2016). The learning phenotypes of hecw-1, oxi-1, and herc-1 mutants did not differ significantly from that of the wild type. In summary, 6 HECT domain-containing E3 ubiquitin ligase mutants showed several types of defects in salt chemotaxis learning. Salt chemotaxis defects after starvation conditioning were more obvious than those after fed conditioning, when the ubiquitin ligases were mutated or proteasomal function was inhibited (Fig. 3c): 3 of 6 mutants showed defects only after starvation conditioning.

Ubiquitin ligases function in multiple pathways

Behavioral assays showed that 6 out of 9 mutants of HECT E3 ubiquitin ligases have defects in salt chemotaxis learning. To test whether some of these ubiquitin ligases could act in the same pathway, behavioral phenotypes in double mutants between them were observed. The etc-1 mutant was omitted from the analysis because of its weak defect compared to other mutants.

Genetic interactions between ubr-5, hecd-1, and wwp-1 were examined. Behavioral phenotypes of other combinations of mutants, such as eel-1 hecd-1, wwp-1; eel-1, ubr-5; eel-1 and wwp-1 Y92H12A.2, could not be tested because the double mutants showed embryonic lethal, larval arrest or sterile phenotypes (Supplementary Table 3). We also note that eel-1 and Y92H12A.2 mutants showed abnormalities in the high/starved conditioning in the analysis in Fig. 3, but in the test of genetic interactions, statistically significant phenotype was not observed for the eel-1 mutant. Therefore, we couldn’t interpret a genetic interaction between eel-1 and Y92H12A.2.

As described before, hecd-1 and ubr-5 single mutants moved to lower salt concentrations after low/starved conditioning compared to the wild type. The ubr-5; hecd-1 mutant showed an additive phenotype after low/starved, suggesting that they function in different pathways or, in the same pathway in a partially redundant manner (Fig. 4a). Furthermore, the ubr-5 mutation enhanced low-salt migration bias in the hecd-1 mutant after middle/fed, high/fed and middle/starved conditioning, after which the ubr-5 single mutant did not show significant differences compared to the wild type. These data suggest that ubr-5 and hecd-1 coordinately promote high-salt migration bias.

Fig. 4.

Fig. 4.

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Genetic interactions between ubr-5, hecd-1, and wwp-1 in salt chemotaxis learning. a) Genetic interaction between hecd-1 and ubr-5 in salt chemotaxis learning. b) Genetic interactions between hecd-1, wwp-1, and ubr-5 in salt chemotaxis learning. Error bars indicate the standard error of the mean. *,†P < 0.05, **,††P < 0.01, ***,†††P < 0.001. One-way ANOVA with Tukey’s post hoc test compared to the single mutants used to generate the double mutant. The symbols represent the results of statistical tests compared to the following mutants. a), *ubr-5, †hecd-1. b) †wwp-1, *hecd-1 or ubr-5.

Both wwp-1 and hecd-1 single mutants show significant differences from the wild type after high/fed and low/starved conditioning. The wwp-1; hecd-1 mutant showed an additive phenotype after the conditioning, suggesting that these 2 genes function in different pathways or in the same pathway redundantly to promote high-salt migration (Fig. 4b). Similar to the observation in the ubr-5; hecd-1 mutant, chemotaxis defects in hecd-1 and wwp-1 mutants after middle/starved and high/starved were enhanced by wwp-1 and hecd-1 mutations, respectively, the latter of which did not cause significant effect on chemotaxis after corresponding conditioning.

Both wwp-1 and ubr-5 single mutants show defects after low/starved conditioning. The wwp-1 ubr-5 mutant did not show significant difference compared to the wwp-1 single mutant: the ubr-5 mutation did not cause chemotaxis defect in the wwp-1 mutant background, suggesting that wwp-1 and ubr-5 function in the same genetic pathway (Fig. 4b).

In summary, those analyses of genetic interactions imply that the HECT E3 ubiquitin ligases act in multiple pathways to promote high-salt migration after salt concentration learning.

Mutations of the HECT E3 ubiquitin ligase genes enhance learning defects of the insulin pathway mutants

Our previous research revealed that the isoforms of DAF-2 that include exon 11.5, such as DAF-2c, act in the axon to control taste avoidance learning (Ohno et al. 2014). On the other hand, DAF-2 isoforms that skip exon 11.5, such as DAF-2a, regulates taste avoidance learning via the FOXO-type transcription factor DAF-16 in the cell body (Nagashima et al. 2019). To test genetic interactions between the HECT E3 ubiquitin ligases and the insulin signaling genes, phenotypes of double mutants of ubiquitin ligase and the insulin pathway genes were examined. A total of 5 mutants of the ubiquitin ligases were used in this experiment (Fig. 5a).

Fig. 5.

Fig. 5.

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Most of HECT E3 ubiquitin ligases and the insulin pathway genes act in different genetic pathways in taste avoidance learning. a) Salt chemotaxis learning of single mutants of the HECT E3 ubiquitin ligase genes. b) Effects of the HECT E3 ubiquitin ligase mutations on salt chemotaxis learning of the daf-2c(pe2722) mutant. c) Effects of the HECT E3 ubiquitin ligase mutations on salt chemotaxis learning of the daf-16(mgDf50) mutant. Error bars indicate the standard error of the mean. *,†,‡P < 0.05, **,††,‡‡P < 0.01, ***,†††,‡‡‡P < 0.001, a) and results of daf-2c and daf-16 single mutants of b) and c) were compared to the wild type by one-way ANOVA with Dunnett’s post hoc test. The other results of b) and c) were compared to the single mutants used to generate the double mutant by one-way ANOVA with Tukey’s post hoc test. The symbols represent the results of statistical tests compared to the wild type or the mutants as indicated in figures.

The deletion mutant of the daf-2c isoform, daf-2c(pe2722), showed increased migration bias toward high salt after fed conditioning (low/fed and middle/fed conditioning). The daf-2c mutants also showed significant defects in taste avoidance learning: they showed defects in high and low-salt migration after starvation conditioning (Nagashima et al. 2019) (Fig. 5b). After fed conditioning, increased high-salt migration of the daf-2c mutants was suppressed by the eel-1 and hecd-1 mutations, suggesting that EEL-1 and HECD-1 function downstream of or in parallel with the DAF-2c pathway. After starvation conditioning, mutations of eel-1, hecd-1, wwp-1, and Y92H12A.2 enhanced the defects in taste avoidance of the daf-2c mutant overall, suggesting that EEL-1, HECD-1, WWP-1, and Y92H12A.2 function in taste avoidance learning at least in part in different pathways from DAF-2c. Of these, although the eel-1 mutation altered chemotaxis of the daf-2c mutant in several conditions, it did not cause an additive effect on the low-salt migration defect of the daf-2c mutant after high/starved conditioning, implying that EEL-1 and DAF-2c may function in the same genetic pathway to promote low-salt migration after starvation conditioning. On the other hand, the ubr-5 mutation did not cause an additive effect on high-salt migration defect of the daf-2c mutant after low/starved conditioning where ubr-5 single mutants have a defect in the same direction, which suggests that UBR-5 and DAF-2c function in the same genetic pathway to promote high-salt migration after starvation conditioning (Fig. 5b).

The deletion mutant of daf-16, daf-16(mgDf50), showed no significant difference from the wild type after fed conditioning, whereas they showed abnormal learning behavior after starvation conditioning in all conditions (Fig. 5c). Mutations of all ubiquitin ligase genes we tested (eel-1, hecd-1, ubr-5, wwp-1, and Y92H12A.2) enhanced taste avoidance defects of the daf-16 mutant after starvation conditioning overall, except for hecd-1 and ubr-5 after high/starved and Y92H12A.2 after low/starved conditioning, suggesting that those ubiquitin ligase genes function in taste avoidance learning in parallel with DAF-16, at least in part.

Discussion

In this study, we have shown that functions of the proteasome, the E1 ubiquitin-activating enzyme and HECT E3 ubiquitin ligases are involved in associative learning in which salt concentrations and the presence or absence of food are associated during conditioning to cause altered chemotaxis behavior when tested later. Mutants of eel-1, hecd-1, and wwp-1 showed defects in chemotaxis after both fed and starvation conditioning, whereas ubr-5, Y92H12A.2 and etc-1 showed weak but significant defects only in taste avoidance learning, namely only after starvation. Analyses of genetic interactions suggested that the ubiquitin ligases contribute to salt chemotaxis learning via multiple pathways. On the other hand, ubr-5 was suggested to function in the same genetic pathway as wwp-1 in taste avoidance learning. Mammalian orthologs of herc-1, oxi-1, and Y92H12A.2 are associated with learning behavior and neural function (Long et al. 2013; Camera et al. 2016; Ambrozkiewicz et al. 2020). In C. elegans, Y92H12A.2, but not herc-1 or oxi-1, is involved in taste avoidance learning (Fig. 3). Caenorhabditiselegans also shows learning behaviors related to odor, temperature or pathogens responses (Colbert and Bargmann 1995; Mohri et al. 2005; Zhang et al. 2005). herc-1 and oxi-1 may function in other types of learning paradigm as mentioned above in C. elegans. Indeed, hecw-1, whose loss-of-function mutation caused no significant defects in taste avoidance learning, is associated with pathogen avoidance behavior (Chang et al. 2011). Alternatively, different types of ubiquitin ligase may be used in learning between mammals and C. elegans.

The mutations of eel-1, hecd-1, wwp-1, and Y92H12A.2 caused additive effects on defects in taste avoidance learning of daf-2c and daf-16. On the other hand, the ubr-5 mutation did not enhance the taste avoidance learning defect of the daf-2c mutant, suggesting that ubr-5 functions in the DAF-2c pathway. In addition, the eel-1 mutation did not further enhance the defect in low-salt migration after high/starved conditioning of daf-2c. Therefore, each of ubr-5 and eel-1 may function in the DAF-2c pathway to promote migration toward high and low salt concentrations after starvation conditioning, respectively. Because the eel-1 mutant shows reduced GABAergic miniature inhibitory postsynaptic current (mIPSC) frequency compared to the wild type (Opperman et al. 2017), EEL-1 may act in synaptic transmission along with the axonal insulin receptor isoform, DAF-2c. The UPS regulates a RNA-binding protein involved in alternative splicing (Zhang et al. 2015). Thus, EEL-1 and UBR-5 may function upstream of DAF-2c to promote DAF-2c production via regulation of alternative splicing of daf-2. The daf-16 mutation did not further enhance the defect in low-salt migration after middle/starved conditioning of eel-1 (Fig. 5, a and c). Considering that daf-16 regulates expression of a ubiquitin ligase gene (Chen and Chalfie 2015), eel-1 may act downstream of daf-16 after middle/starved conditioning. It was reported that mammalian orthologs of wwp-1 and Y92H12A.2 are involved in control of the amount of PTEN (Phosphatase and tensin homolog), which negatively regulates the insulin pathway (Kwak et al. 2010; Maddika et al. 2011). Therefore, WWP-1 and Y92H12A.2 may regulate taste avoidance learning via modulation of PTEN (encoded by daf-18) also in C. elegans. Considering that wwp-1 functions in parallel with daf-16 in C. elegans (Chen et al. 2010), other pathways mediated by wwp-1 may act in taste avoidance learning. It will be interesting to further examine how the ubiquitin ligases may modulate the action of the DAF-2c pathway in taste avoidance learning. Previous reports indicate that the insulin pathway acts in ASER in taste avoidance learning (Tomioka et al. 2006; Nagashima et al. 2019). We show that uba-1 may mainly act in the nervous system other than ASER (Fig. 2a). Considering that UBA-1 is the only ubiquitin-activating enzyme in C. elegans, it seems unlikely that taste avoidance learning is mainly regulated by ubiquitination of the components of the insulin pathway.

Administration of a proteasomal inhibitor BTZ 21 h before and during starvation conditioning strongly disrupted the ability of taste avoidance learning (Fig. 1, b and c). Considering that the inhibitor was applied to late L4 worms with the mature nervous system, the learning defect caused by the inhibitor was not due to developmental defects, such as defects in maturation of the neural circuits. Rather, proteasome function may be required for processes of taste avoidance learning, such as transmission of starvation information and integration of starvation and salt concentrations. This is consistent with the observation that the temperature-sensitive uba-1(it129) mutant showed learning defect after temperature shift at the adult stage.

As shown in Figs. 1 and 3, proteasomal inhibition and loss of functions of ubiquitin ligases prominently disrupted high-salt migration after low/starved conditioning: salt chemotaxis only after low/starved conditioning was reduced by administration of low concentrations of BTZ (Fig. 1c), and 4 out of 9 HECT E3 ubiquitin ligase mutants showed decreased high-salt migration after low/starved conditioning (Fig. 3). These findings imply that multiple pathways modulated by the ubiquitin ligases are required for high-salt migration after starvation conditioning.

Interestingly, pharmacological inhibition of proteasome function caused defects in learned behavior weakly or strongly after fed or starvation conditioning, respectively, whereas downregulation of the ubiquitin ligase or ubiquitin-activating enzyme function strongly disrupted learned behavior after both fed and starvation conditioning (e.g. chemotaxis defects in the ubr-5; hecd-1 and uba-1 mutants). Ubiquitination may regulate chemotaxis after fed conditioning in a proteasome system-independent manner. Indeed, it was reported that mono-ubiquitination regulates endocytosis and is predicted to control miniature excitatory postsynaptic current (mEPSC) (Haglund et al. 2003; Mosesson et al. 2003; Kikuma et al. 2019).

Cell- or tissue-specific rescue experiments using the uba-1(it129) mutants revealed that uba-1 acts in the nervous system in taste avoidance learning (Fig. 2). Because ubiquitination is mediated by a series of E1, E2, and E3 enzyme reactions, E3 ubiquitin ligases can function in the nervous system along with UBA-1, although their detailed molecular mechanisms are unclear. Considering that double mutations of HECT-type ubiquitin ligase genes caused additive effects on salt chemotaxis learning, there is possibility that distinct ubiquitin ligases act on a variety of substrates in the nervous system. Previous studies suggest that the substrate specificities for HECT-type E3 ubiquitin ligases are complex. It is known that the human NEDL1 and HERC2 ubiquitin ligases interact with the common substrates via different N-terminal domains (Li et al. 2008; Cubillos-Rojas et al. 2014). Conversely, Nedd4 family of ubiquitin ligases with the common domain structures showed different binding specificities for their substrates (Laine and Ronai 2007). In addition, it is also known that the HUWE1 and UBR5 ubiquitin ligases bind to the substrates via the catalytic HECT domains (Jiang et al. 2011; Jang et al. 2014). A recent study suggests that a flexible ring-shaped structure of Nematocida HUWE1 harbors multiple substrate-docking sites and regulates the activity of the HECT catalytic domain (Grabarczyk et al. 2021).

In C. elegans, OGT-1, an N-acetylglucosaminyltransferase, has been reported to bind EEL-1 (Giles et al. 2019). This study suggested that OGT-1 is expressed and functions in the motor circuit, but its transferase activity is not needed for its function. Another protein interacting with EEL-1 is the SKN-1 transcription factor (Page et al. 2007). However, it has not been confirmed whether OGT-1 and/or SKN-1 are ubiquitinated by the E3 activity of EEL-1. The C. elegans Kruppel-like family of transcription factor, KLF-1, is known to be a substrate of WWP-1 (Carrano et al. 2014), and is involved in dietary-restriction-induced longevity. The securing IFY-1, a proposed securin-like protein, and the cyclin B1 CYB-1 have been reported as substrates of ETC-1 and function in pharyngeal development (Wang et al. 2013). But regulations of neuronal proteins via HECT-type E3 ubiquitin ligases have been largely unknown, and the substrates of the other ubiquitin ligases, HECD-1, UBR-5, and Y92H12A.2, have not been reported.

In summary, this study revealed 4 findings. First, the UPS functions in the nervous system for taste avoidance learning. Second, 6 out of 9 mutants of HECT domain-containing E3 ubiquitin ligases showed several defects in salt chemotaxis. Third, these ubiquitin ligases may contribute to salt chemotaxis learning via multiple pathways after starvation conditioning. Fourth, most ubiquitin ligases function in parallel with the insulin pathway in taste avoidance learning.

Data availability

Strains are available upon request. The authors affirm that all data necessary for confirming the conclusions of the article are present within the article, figures, and tables.

Supplemental material is available at GENETICS online.

Supplementary Material

iyac025_Supplementary_Data
Click here for additional data file. (1.3MB, pdf)

Acknowledgments

The authors thank the Caenorhabditis Genetics Center, National Bioresource Project, and Tao Jiang for the experimental strains. They also thank the members of the Iino laboratory for the helpful comments and discussions.

Funding

This work was supported by Japan Society for the Promotion of Science (JSPS) Grant-in-Aid for Scientific Research (S) JP17H06113, Grants-in-Aid for Innovative Area “Artificial Intelligence and Brain Science” JP19H04980, Japan Science and Technology Agency (JST), CREST JPMJCR12W1, and University of Tokyo Center for Integrative Science of Human Behavior (CiSHuB).

Conflicts of interest

The authors declare no conflict of interest associated with this research.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

iyac025_Supplementary_Data
Click here for additional data file. (1.3MB, pdf)

Data Availability Statement

Strains are available upon request. The authors affirm that all data necessary for confirming the conclusions of the article are present within the article, figures, and tables.

Supplemental material is available at GENETICS online.

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