CASY-1, an ortholog of calsyntenins/alcadeins, is essential for learning in Caenorhabditis elegans

Identification of casy-1, a Calsyntenin/Alcadein Homolog, as a Learning-Related Gene.

We previously reported that C. elegans learns to avoid NaCl after being exposed to NaCl under starvation conditions (salt chemotaxis learning) (4, 8). To identify unknown molecules involved in salt chemotaxis learning, we performed an EMS mutagenesis screen (18). We screened for mutants that failed to avoid NaCl after conditioning in a buffer containing NaCl and obtained several candidate strains. Of these, we further characterized the JN401 mutant, which showed defective learning when conditioned in NaCl-containing buffer [Fig. 1A, casy-1(pe401), see below]. We obtained similar results in the plate-conditioning method [supporting information (SI) Fig. S1]. Chemotaxis of the mutants to various concentrations of NaCl was similar to that of wild-type animals when unconditioned (Fig. 1B). Through genetic mapping and DNA sequencing, we found that JN401 carries a missense mutation, pe401, in the gene B0034.3 (Fig. 1C). This gene had been called cdh-11. We renamed this gene casy-1 because of its orthology to calsyntenins/alcadeins. The learning defects of pe401 mutants were restored by introduction of a cosmid (B0034) or a PCR fragment corresponding to the genomic region of casy-1 (Fig. 1A), indicating that the impairment of casy-1 function causes the learning defects. casy-1 encodes the sole C. elegans ortholog of mammalian calsyntenins/alcadeins (13, 15). Calsyntenins/alcadeins are type I transmembrane proteins highly conserved in metazoa (Fig. S2) (19). They have two tandem cadherin domains and an LG/LNS domain in the ectodomain, i.e., the extracellular region. To characterize the molecular function of this gene further, deletion alleles were obtained and incorporated in subsequent analyses (Fig. 1C). Among them, tm718 and hd41 are putative null alleles because both carry a deletion in the N-terminal region of casy-1, which results in a frame shift. All these mutants exhibited strong learning defects similar to those in pe401 mutants (Fig. 1D).

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Fig. 1.

casy-1 mutants show defects in salt chemotaxis learning. (A) Chemotaxis to 50 mM NaCl after NaCl conditioning or mock conditioning is shown for wild-type N2, casy-1(pe401) mutants, and transgenic rescue lines. PCR-amplified 17-kb genomic fragment included 4.8 kb upstream and 0.8 kb downstream of the casy-1 genome region. (B) casy-1(pe401) mutants show normal sensitivity to NaCl. Naïve animals were tested for chemotaxis to indicated concentrations of NaCl. (C) Structure of casy-1 and an illustration of the predicted gene product. (D) Salt chemotaxis learning in various casy-1 mutants. Asterisks represent significant differences from control animals (−) carrying only the transformation marker in A and wild type in D (Dunnett's test; **, P < 0.001; *, P < 0.01). Two-way ANOVA did not reveal significant effect of strains in B.

Multiple Types of Learning Defects in casy-1 Mutants.

Some mutants defective in salt chemotaxis learning also exhibit defects in other types of learning or sensory processing. To investigate the possible roles of casy-1 in these types of learning, the mutants were tested for olfactory adaptation and temperature learning. In olfactory adaptation, worms were soaked in a buffer containing the odorant benzaldehyde, and then they were tested for chemotaxis to benzaldehyde (9). After preexposure to benzaldehyde, wild-type animals displayed aversive responses to benzaldehyde, whereas casy-1 mutants continued to show a strong chemotaxis to benzaldehyde (Fig. 2A). In temperature learning, worms were cultured at 20°C without food for 0–4 h, and temperature preference was assayed on a thermal gradient plate (10). Whereas wild-type animals gradually lost the preference for 20°C, casy-1 mutants retained the temperature preference after conditioning without food for 4 h (Fig. 2B). These results suggest that casy-1 is required for multiple forms of learning. On the other hand, if casy-1 mutants were not pretreated under starvation, they did not show large defects in their response to benzaldehyde (Fig. S3A), or cultivation temperature (Fig. 2B, time = 0). We also found that casy-1 is important for another type of information processing: sensory integration. In a paradigm for testing sensory integration, worms must cross an aversive barrier of Cu²⁺ to reach an attractive odorant diacetyl (12). casy-1 mutants showed defects in this type of sensory processing (Fig. 2C), whereas these mutants normally responded to diacetyl or Cu²⁺ when presented separately (Fig. S3 B and C). These results suggest that CASY-1 is not essential for primary sensory transduction, but is important for a variety of higher-order information processing.

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Fig. 2.

casy-1 mutants exhibit defects in various types of learning and sensory integration. (A) Olfactory adaptation assays. Animals were treated in a buffer with (benz-conditioned) or without (mock-conditioned) benzaldehyde for 1 h and tested for chemotaxis to 5 nl/μl benzaldehyde. (B) casy-1 mutants show defects in temperature learning. Animals grown at 20°C were transferred to bacteria-free plates at 20°C for the indicated periods and placed on thermal gradients. The fraction of animals that preferred 20°C is indicated. (C) Sensory integration assays. The fraction of animals that migrated to the attractive odorant (diacetyl) across the repulsive copper ion barrier is indicated. Two-way ANOVA revealed that there are significant effects of strains and time and their interaction in B and significant effects of strains and concentrations, but not the interaction, in C. Asterisks represent significant differences from wild type (Dunnett's test in A, and Tukey post hoc tests in B and C; **, P < 0.001; *, P < 0.01).

Temporal and Spatial Requirement of CASY-1 in Salt Chemotaxis Learning.

CASY-1 is a nonclassical cadherin-like protein. Various cadherin-like proteins are expressed and have functions in the nervous system. For example, classic neural cadherins, including N-cadherin, play a role in the developmental organization of the brain (20, 21) and synapse formation (22). A recent report showed that N-cadherin also regulates memory formation in the adult brain (23). To investigate whether CASY-1 could be required either for neural development or for postdevelopmental neural functions underlying salt chemotaxis learning, we performed rescue experiments by transient gene expression. Learning defects of the casy-1(tm718) mutants were strongly rescued when functional casy-1 was transiently expressed by a heat-inducible hsp16.2 promoter in the adult stage (Fig. 3A). This result indicates that casy-1 acts in the mature neural circuit.

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Fig. 3.

CASY-1 activity is required in a mature sensory neuron in salt chemotaxis learning. (A) Heat shock-rescue experiments were performed on two independent lines. Shown are the chemotaxis index of NaCl-conditioned casy-1(tm718) mutants that carry (Ex+) or that have lost (Ex−), the extrachromosomal array containing the hspp::casy-1 transgene. (B and C) casy-1 promoter::gfp was expressed in neurons and other tissues. The anterior part of the body (B) and neurons in the head region (C) are shown. (Scale bars, 10 μm.) VNC, ventral nerve cord. (D) Neuron-specific rescue experiments. Expression of casy-1(+) was driven by the indicated cell-specific promoters, and salt chemotaxis learning assays were performed. Neurons in which each promoter drives expression are: H20p, all neurons; gcy-5p, ASER; gcy-22p, ASER; gcy-7p, ASEL; odr-4p, amphid sensory neurons except ASE; odr-2p, AIZ, AIB and AVG etc.; ttx-3p, AIY; glr-1p, AVA, AVB, AVD, AVE and PVC etc. Asterisks represent significant differences from Ex− animals (A) and control animals carrying only the transformation marker (D) (two-way ANOVA followed by Tukey post hoc tests in A and Dunnett's test in D; **, P < 0.001; *, P < 0.01).

Mammalian calsyntenins are widely expressed in the central nervous system (13⇓–15). Given that CASY-1 is involved in behavioral plasticity, casy-1 is expected to be expressed in the nervous system. We determined the expression patterns of casy-1 using a green fluorescent protein reporter driven by the authentic casy-1 promoter. Fluorescent signals were observed throughout the nervous system and in other tissues such as intestine and gonadal sheath cells (Fig. 3B). We noticed strong GFP signals in many head neurons, including most amphid sensory neurons (Fig. 3C). The expression was observed from the embryonic stage. We also investigated the intracellular localization of CASY-1 by using fluorescent protein-tagged CASY-1 (see below). We found that CASY-1 is mainly localized to cell bodies of neurons, especially plasma membranes and intracellular membranes (Fig. S4). Localization of the protein in the neuronal processes was not evident but occasionally observed.

To identify neuron(s) in which CASY-1 acts in salt chemotaxis learning, we expressed casy-1 cDNA by several neuron-specific promoters in casy-1 mutants and examined whether each of them can rescue the learning defects of the mutants. We found that the expression of casy-1 solely in the ASER neuron is sufficient to rescue the defects (Fig. 3D). In contrast, the defects were not rescued by expression of casy-1 in other neurons. These results indicate that CASY-1 acts in the ASER neuron in salt chemotaxis learning, the same site as where the PI3K pathway acts (4).

Genetic Relationship Between casy-1 and the Insulin-Like Signaling Pathway.

Because both CASY-1 and the components of the PI3K pathway act in the ASER neuron for salt chemotaxis learning, we examined the genetic relationship between these genes. In daf-18 PTEN mutants, which have elevated level of the PI3K signaling, naïve animals show reduced chemotaxis to NaCl compared with the wild type (4). We therefore tested the phenotypes of casy-1; daf-18 double mutants and found that the casy-1 mutation suppresses the reduced chemotaxis of daf-18 (Fig. S5A). This result suggests that casy-1 acts either downstream of or in parallel to daf-18.

We next investigated the genetic interaction between casy-1 and another component of the insulin/PI3K pathway, ins-1. ins-1 acts upstream of the PI3K pathway in salt chemotaxis learning (4). We constructed a double mutant of casy-1 and ins-1 and then tested it for salt chemotaxis learning. The learning defect of casy-1; ins-1 double mutants was more severe than that of either single mutant (Fig. S5B). These results suggest that casy-1 acts in parallel to the insulin-like signaling pathway.

Release of CASY-1 Ectodomain from Neurons.

Many transmembrane proteins have been shown to undergo ectodomain shedding, the release of the extracellular domain by proteolysis (24). It has been shown that calsyntenins/alcadeins are cleaved in the extracellular region (13, 16). By analogy, we reasoned that CASY-1 might also be proteolytically cleaved. Our observation of the localization of N- and C-terminal GFP fusion proteins, NtGFP::CASY-1 and CASY-1::CtGFP, respectively, is consistent with this possibility. GFP fluorescence was observed in coelomocytes only for NtGFP::CASY-1 but not for CASY-1::CtGFP (data not shown). Coelomocytes are macrophage-like scavenger cells present in the pseudocoelomic cavity that take up various compounds from the body cavity fluid (25). Because these two constructs share the same transcriptional regulatory sequences, distinct localization patterns suggest a proteolytic cleavage of the CASY-1 protein. To confirm this hypothesis, fluorescent markers, mRFP and Venus, were simultaneously fused to the N terminus and C terminus, respectively, of CASY-1 (Fig. 4G, RYV) and were expressed in head neurons. Only the signal of mRFP, tagged to the ectodomain, was detected in coelomocytes (Fig. 4 A–C). mRFP signals in coelomocytes were observed even in normally cultured naïve animals. We made similar observations when the fusion protein was expressed in the ASER neuron alone, although the fluorescence was weaker in this case (data not shown). Therefore, we concluded that in head neurons including ASER, the ectodomain is cleaved from the full-length CASY-1 and released into the body cavity.

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Fig. 4.

The ectodomain of CASY-1 is essential for salt chemotaxis learning. (A–F) RYV (full-length casy-1 tagged with the fluorescent markers) (A–C) and RYV700 (a tagged ectodomain of casy-1) (D–F) were expressed in head neurons including the ASER neuron by the ins-1 promoter. Yellow arrowheads indicate coelomocytes, and white brackets show head neurons. Adult-stage animals are shown. (A and D) DIC image; (B and E) Venus; (C and F) mRFP. (Scale bar, 30 μm.) (G) Functional domain mapping of casy-1. Schematic depiction of deletion constructs, summary of the results of rescue experiments, and localization of mRFP signals in coelomocytes.

Functional Domain Mapping of CASY-1.

Regarding the processing of CASY-1, we attempted to determine which part of the protein is important for salt chemotaxis learning (Fig. 4G). The cytoplasmic region of mammalian calsyntenin-1 binds Ca²⁺, and it was proposed that calsyntenins may be involved in proteolysis-dependent modulation of synaptic functions through its Ca²⁺-binding capacity (13, 26). Based on this idea, the cytoplasmic region of CASY-1 may be essential for learning in C. elegans. On the other hand, our observation that the mutations of the extracellular domain lead to impairment of learning suggests the importance of the ectodomain (Fig. 1 C and D, pe401 and hd33).

We found that the fragment lacking the cytoplasmic region can rescue the learning defects of the casy-1 mutants [Fig. 4G and Fig. S6A, RYV(ΔCt)], whereas the fragments that lack the ectodomain did not rescue the defect [Fig. 4G and Fig. S6A, RYV(ΔNt) and CtV]. These results indicate that the cytoplasmic acidic region is dispensable at least for salt chemotaxis learning.

We further shortened the remaining region of the protein and tested whether the truncated proteins are functional. Unexpectedly, we found that the ectodomain alone, even without the transmembrane region, could rescue the learning defects of the casy-1 mutants (Fig. 4G and Fig. S6A, RYV800 and RYV700). In these constructs, both mRFP and Venus signals were observed in coelomocytes, suggesting that the whole protein is secreted (Fig. 4 D–F, RYV700). We also found that the ectodomain lacking the signal peptide failed to rescue [Fig. 4G, RYV700(ΔSP)], suggesting that only the secreted fragments support learning.

When shorter forms of the ectodomain were tested, they did not rescue the defect (Fig. 4G, RYV600 and RYV530). These results suggest that the central part of the extracellular region is important for learning. We expected that the LG/LNS domain is essential for learning because two mutant alleles that lead to the impairment of the extracellular region around the LNS domain cause defects in learning (Fig. 1 C and D, pe401 and hd33). LNS domains are often found in extracellular proteins and are implicated in interaction with a variety of cellular receptors or ligands (27, 28). As expected, deletion of the LNS domain abolished the functionality of CASY-1 [Fig. 4G, RYV(ΔLNS)]. Because LNS domain by itself is not enough to rescue the defects (Fig. 4G, RYV600, RYV530, and RYV331–700), we concluded that LNS domain and its flanking regions are necessary for modulating learning. Slight but nonsignificant rescue observed in the cadherin domain-deleted fragments implies that the cadherin domains may have accessory roles [Fig. 4G and Fig. S6B, RYV(ΔCads)].