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A conserved function of C. elegans CASY-1 calsyntenin in associative learning.

Hoerndli FJ, Walser M, Fröhli Hoier E, de Quervain D, Papassotiropoulos A, Hajnal A - PLoS ONE (2009)

Bottom Line: Whole-genome association studies in humans have enabled the unbiased discovery of new genes associated with human memory performance.However, such studies do not allow for a functional or causal testing of newly identified candidate genes.Our experiments demonstrate a remarkable conservation of the molecular function of Calsyntenins between nematodes and humans and point at a role of C. elegans casy-1 in regulating a glutamate receptor signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: Institute of Zoology, University of Zürich, Zürich, Switzerland.

ABSTRACT

Background: Whole-genome association studies in humans have enabled the unbiased discovery of new genes associated with human memory performance. However, such studies do not allow for a functional or causal testing of newly identified candidate genes. Since polymorphisms in Calsyntenin 2 (CLSTN2) showed a significant association with episodic memory performance in humans, we tested the C. elegans CLSTN2 ortholog CASY-1 for possible functions in the associative behavior of C. elegans.

Methodology/principal findings: Using three different associative learning paradigms and functional rescue experiments, we show that CASY-1 plays an important role during associative learning in C. elegans. Furthermore, neuronal expression of human CLSTN2 in C. elegans rescues the learning defects of casy-1 mutants. Finally, genetic interaction studies and neuron-specific expression experiments suggest that CASY-1 may regulate AMPA-like GLR-1 glutamate receptor signaling.

Conclusion/significance: Our experiments demonstrate a remarkable conservation of the molecular function of Calsyntenins between nematodes and humans and point at a role of C. elegans casy-1 in regulating a glutamate receptor signaling pathway.

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Related in: MedlinePlus

Olfactory associative learning defects in casy-1(tm718) mutants.(A) Rooted tree diagram showing the sequence similarities between the invertebrate and the three classes of vertebrate calsyntenins. The protein sequences of CLSTN1, CLSTN2 and CLSTN3 from Homo sapiens (Hs), Mus musculus (Mm), Danio rerio (Dr) and the single calsyntenins from Drosophila melanogaster (Dm), Apis mellifera (Am) and Caenorhabditis elegans (Ce) were aligned using the ClustalX program, and a rooted tree was drawn using PHYLIP. Note that the invertebrate calsyntenins and the vertebrate CLSTN2 proteins originate from a common branch. (B) Chemotaxis of wild-type and casy-1(tm718) worms towards 10−2, 10−3 and 10−4 fold dilution of Diacetyl in 100% EtOH(V/v) assay in the absence of conditioning. The assays were repeated on three different days using one plate for each condition and were quantified using the chemotaxis Index CI (CI = (worms in DA - worms at EtOH)/ total number of worms, see methods). Error bars indicate the standard error of mean. White bars: wild-type N2, Black bars: casy-1(tm718). (C) Swimming assay of casy-1(tm718), wild-type, nicotinic Acetylcholine-receptor acr-16 knock out (ok789) and levamisole acetylcholine-receptor unc-29 subunit knock-out (x29). Number of body bends per minute counted manually, and blinded to the respective genotypes (N = 20). (D) Chemotaxis of starvation conditioned wild-type and casy-1(tm718) animals. The experiment was repeated on three separate days with six replicates per assay. The results of a Student t-test are indicated as * = p<0.05 and ** = p<0.01. (E) Food sensing assay. Locomotion rate of wild-type and casy-1(tm718) worms in body bends/20 seconds of worms transferred from a food plate to another food plate (FED), or worms allowed to starve on an empty agar plate for 1 hr (STARVED). White bars: wild-type, Black bars: casy-1(tm718). (F) Adaptation assay. Comparison of the chemotaxis Index CI of wild-type and casy-1(tm718) to 0.1% DA after starving for 1 hour without DA (White bars), with 100% DA (Black Bars) and on food for 2 hours with 100% DA (Grey bars). Assays were repeated on two different days using 3 replicates per condition. For the complete dataset of the behavioral assays, see Table S1.
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pone-0004880-g001: Olfactory associative learning defects in casy-1(tm718) mutants.(A) Rooted tree diagram showing the sequence similarities between the invertebrate and the three classes of vertebrate calsyntenins. The protein sequences of CLSTN1, CLSTN2 and CLSTN3 from Homo sapiens (Hs), Mus musculus (Mm), Danio rerio (Dr) and the single calsyntenins from Drosophila melanogaster (Dm), Apis mellifera (Am) and Caenorhabditis elegans (Ce) were aligned using the ClustalX program, and a rooted tree was drawn using PHYLIP. Note that the invertebrate calsyntenins and the vertebrate CLSTN2 proteins originate from a common branch. (B) Chemotaxis of wild-type and casy-1(tm718) worms towards 10−2, 10−3 and 10−4 fold dilution of Diacetyl in 100% EtOH(V/v) assay in the absence of conditioning. The assays were repeated on three different days using one plate for each condition and were quantified using the chemotaxis Index CI (CI = (worms in DA - worms at EtOH)/ total number of worms, see methods). Error bars indicate the standard error of mean. White bars: wild-type N2, Black bars: casy-1(tm718). (C) Swimming assay of casy-1(tm718), wild-type, nicotinic Acetylcholine-receptor acr-16 knock out (ok789) and levamisole acetylcholine-receptor unc-29 subunit knock-out (x29). Number of body bends per minute counted manually, and blinded to the respective genotypes (N = 20). (D) Chemotaxis of starvation conditioned wild-type and casy-1(tm718) animals. The experiment was repeated on three separate days with six replicates per assay. The results of a Student t-test are indicated as * = p<0.05 and ** = p<0.01. (E) Food sensing assay. Locomotion rate of wild-type and casy-1(tm718) worms in body bends/20 seconds of worms transferred from a food plate to another food plate (FED), or worms allowed to starve on an empty agar plate for 1 hr (STARVED). White bars: wild-type, Black bars: casy-1(tm718). (F) Adaptation assay. Comparison of the chemotaxis Index CI of wild-type and casy-1(tm718) to 0.1% DA after starving for 1 hour without DA (White bars), with 100% DA (Black Bars) and on food for 2 hours with 100% DA (Grey bars). Assays were repeated on two different days using 3 replicates per condition. For the complete dataset of the behavioral assays, see Table S1.

Mentions: To test a causal relationship between CLSTN2 function and learning and memory, we searched the genomes of invertebrate model organisms for CLSTN2 orthologs. While vertebrate genomes typically encode three Calsyntenin family members, the genomes of invertebrates like Drosophila melanogaster and C. elegans contain only a single Calsyntenin gene (Fig. 1A). Protein sequence alignment of the three vertebrate Calsyntenin family members with the invertebrate Calsyntenins indicates that the single C. elegans homolog CASY-1 as well as Drosophila Calsyntenin are most similar to vertebrate CLSTN2 (Fig. 1A).


A conserved function of C. elegans CASY-1 calsyntenin in associative learning.

Hoerndli FJ, Walser M, Fröhli Hoier E, de Quervain D, Papassotiropoulos A, Hajnal A - PLoS ONE (2009)

Olfactory associative learning defects in casy-1(tm718) mutants.(A) Rooted tree diagram showing the sequence similarities between the invertebrate and the three classes of vertebrate calsyntenins. The protein sequences of CLSTN1, CLSTN2 and CLSTN3 from Homo sapiens (Hs), Mus musculus (Mm), Danio rerio (Dr) and the single calsyntenins from Drosophila melanogaster (Dm), Apis mellifera (Am) and Caenorhabditis elegans (Ce) were aligned using the ClustalX program, and a rooted tree was drawn using PHYLIP. Note that the invertebrate calsyntenins and the vertebrate CLSTN2 proteins originate from a common branch. (B) Chemotaxis of wild-type and casy-1(tm718) worms towards 10−2, 10−3 and 10−4 fold dilution of Diacetyl in 100% EtOH(V/v) assay in the absence of conditioning. The assays were repeated on three different days using one plate for each condition and were quantified using the chemotaxis Index CI (CI = (worms in DA - worms at EtOH)/ total number of worms, see methods). Error bars indicate the standard error of mean. White bars: wild-type N2, Black bars: casy-1(tm718). (C) Swimming assay of casy-1(tm718), wild-type, nicotinic Acetylcholine-receptor acr-16 knock out (ok789) and levamisole acetylcholine-receptor unc-29 subunit knock-out (x29). Number of body bends per minute counted manually, and blinded to the respective genotypes (N = 20). (D) Chemotaxis of starvation conditioned wild-type and casy-1(tm718) animals. The experiment was repeated on three separate days with six replicates per assay. The results of a Student t-test are indicated as * = p<0.05 and ** = p<0.01. (E) Food sensing assay. Locomotion rate of wild-type and casy-1(tm718) worms in body bends/20 seconds of worms transferred from a food plate to another food plate (FED), or worms allowed to starve on an empty agar plate for 1 hr (STARVED). White bars: wild-type, Black bars: casy-1(tm718). (F) Adaptation assay. Comparison of the chemotaxis Index CI of wild-type and casy-1(tm718) to 0.1% DA after starving for 1 hour without DA (White bars), with 100% DA (Black Bars) and on food for 2 hours with 100% DA (Grey bars). Assays were repeated on two different days using 3 replicates per condition. For the complete dataset of the behavioral assays, see Table S1.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2654071&req=5

pone-0004880-g001: Olfactory associative learning defects in casy-1(tm718) mutants.(A) Rooted tree diagram showing the sequence similarities between the invertebrate and the three classes of vertebrate calsyntenins. The protein sequences of CLSTN1, CLSTN2 and CLSTN3 from Homo sapiens (Hs), Mus musculus (Mm), Danio rerio (Dr) and the single calsyntenins from Drosophila melanogaster (Dm), Apis mellifera (Am) and Caenorhabditis elegans (Ce) were aligned using the ClustalX program, and a rooted tree was drawn using PHYLIP. Note that the invertebrate calsyntenins and the vertebrate CLSTN2 proteins originate from a common branch. (B) Chemotaxis of wild-type and casy-1(tm718) worms towards 10−2, 10−3 and 10−4 fold dilution of Diacetyl in 100% EtOH(V/v) assay in the absence of conditioning. The assays were repeated on three different days using one plate for each condition and were quantified using the chemotaxis Index CI (CI = (worms in DA - worms at EtOH)/ total number of worms, see methods). Error bars indicate the standard error of mean. White bars: wild-type N2, Black bars: casy-1(tm718). (C) Swimming assay of casy-1(tm718), wild-type, nicotinic Acetylcholine-receptor acr-16 knock out (ok789) and levamisole acetylcholine-receptor unc-29 subunit knock-out (x29). Number of body bends per minute counted manually, and blinded to the respective genotypes (N = 20). (D) Chemotaxis of starvation conditioned wild-type and casy-1(tm718) animals. The experiment was repeated on three separate days with six replicates per assay. The results of a Student t-test are indicated as * = p<0.05 and ** = p<0.01. (E) Food sensing assay. Locomotion rate of wild-type and casy-1(tm718) worms in body bends/20 seconds of worms transferred from a food plate to another food plate (FED), or worms allowed to starve on an empty agar plate for 1 hr (STARVED). White bars: wild-type, Black bars: casy-1(tm718). (F) Adaptation assay. Comparison of the chemotaxis Index CI of wild-type and casy-1(tm718) to 0.1% DA after starving for 1 hour without DA (White bars), with 100% DA (Black Bars) and on food for 2 hours with 100% DA (Grey bars). Assays were repeated on two different days using 3 replicates per condition. For the complete dataset of the behavioral assays, see Table S1.
Mentions: To test a causal relationship between CLSTN2 function and learning and memory, we searched the genomes of invertebrate model organisms for CLSTN2 orthologs. While vertebrate genomes typically encode three Calsyntenin family members, the genomes of invertebrates like Drosophila melanogaster and C. elegans contain only a single Calsyntenin gene (Fig. 1A). Protein sequence alignment of the three vertebrate Calsyntenin family members with the invertebrate Calsyntenins indicates that the single C. elegans homolog CASY-1 as well as Drosophila Calsyntenin are most similar to vertebrate CLSTN2 (Fig. 1A).

Bottom Line: Whole-genome association studies in humans have enabled the unbiased discovery of new genes associated with human memory performance.However, such studies do not allow for a functional or causal testing of newly identified candidate genes.Our experiments demonstrate a remarkable conservation of the molecular function of Calsyntenins between nematodes and humans and point at a role of C. elegans casy-1 in regulating a glutamate receptor signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: Institute of Zoology, University of Zürich, Zürich, Switzerland.

ABSTRACT

Background: Whole-genome association studies in humans have enabled the unbiased discovery of new genes associated with human memory performance. However, such studies do not allow for a functional or causal testing of newly identified candidate genes. Since polymorphisms in Calsyntenin 2 (CLSTN2) showed a significant association with episodic memory performance in humans, we tested the C. elegans CLSTN2 ortholog CASY-1 for possible functions in the associative behavior of C. elegans.

Methodology/principal findings: Using three different associative learning paradigms and functional rescue experiments, we show that CASY-1 plays an important role during associative learning in C. elegans. Furthermore, neuronal expression of human CLSTN2 in C. elegans rescues the learning defects of casy-1 mutants. Finally, genetic interaction studies and neuron-specific expression experiments suggest that CASY-1 may regulate AMPA-like GLR-1 glutamate receptor signaling.

Conclusion/significance: Our experiments demonstrate a remarkable conservation of the molecular function of Calsyntenins between nematodes and humans and point at a role of C. elegans casy-1 in regulating a glutamate receptor signaling pathway.

Show MeSH
Related in: MedlinePlus