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

Abstract

Calsyntenins/alcadeins are type I transmembrane proteins with two extracellular cadherin domains highly expressed in mammalian brain. They form a tripartite complex with X11/X11L and APP (amyloid precursor protein) and are proteolytically processed in a similar fashion to APP. Although a genetic association of calsyntenin-2 with human memory performance has recently been reported, physiological roles and molecular functions of the protein in the nervous system are poorly understood. Here, we show that CASY-1, the Caenorhabditis elegans ortholog of calsyntenins/alcadeins, is essential for multiple types of learning. Through a genetic screen, we found that casy-1 mutants show defects in salt chemotaxis learning. casy-1 mutants also show defects in temperature learning, olfactory adaptation, and integration of two sensory signals. casy-1 is widely expressed in the nervous system. Expression of casy-1 in a single sensory neuron and at the postdevelopmental stage is sufficient for its function in salt chemotaxis learning. The fluorescent protein-tagged ectodomain of CASY-1 is released from neurons. Moreover, functional domain analyses revealed that both cytoplasmic and transmembrane domains of this protein are dispensable, whereas the ectodomain, which contains the LG/LNS-like domain, is critically required for learning. These results suggest that learning is modulated by the released ectodomain of CASY-1.

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.

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).

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).

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.

Fig. 5.

A model for CASY-1 function in salt chemotaxis learning. CASY-1 expressed in the ASER sensory neuron is constitutively cleaved, and the ectodomain is released. Released ectodomain acts on either the neuron itself or other nearby targets and modulates salt chemotaxis learning. It is also endocytosed by the scavenger cell, coelomocyte. Localization of CASY-1 was mainly observed in the cell body and is likely to be released from the cell body, but a small amount might be also transported to the synaptic sites and released there.