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Bridging the synaptic gap: neuroligins and neurexin I in Apis mellifera.

Biswas S, Russell RJ, Jackson CJ, Vidovic M, Ganeshina O, Oakeshott JG, Claudianos C - PLoS ONE (2008)

Bottom Line: Neurexin I and neuroligin expression was found in brain tissue, with expression present throughout development, and in most cases significantly up-regulated in adults.We show neuroligins and neurexins comprise a highly conserved molecular system with likely similar functional roles in insects as vertebrates, and with scope in the honeybee to generate substantial functional diversity through alternative splicing.Our study provides important prerequisite data for using the bee as a model for vertebrate synaptic development.

View Article: PubMed Central - PubMed

Affiliation: University of Queensland, Queensland Brain Institute, Brisbane, Queensland, Australia.

ABSTRACT
Vertebrate studies show neuroligins and neurexins are binding partners in a trans-synaptic cell adhesion complex, implicated in human autism and mental retardation disorders. Here we report a genetic analysis of homologous proteins in the honey bee. As in humans, the honeybee has five large (31-246 kb, up to 12 exons each) neuroligin genes, three of which are tightly clustered. RNA analysis of the neuroligin-3 gene reveals five alternatively spliced transcripts, generated through alternative use of exons encoding the cholinesterase-like domain. Whereas vertebrates have three neurexins the bee has just one gene named neurexin I (400 kb, 28 exons). However alternative isoforms of bee neurexin I are generated by differential use of 12 splice sites, mostly located in regions encoding LNS subdomains. Some of the splice variants of bee neurexin I resemble the vertebrate alpha- and beta-neurexins, albeit in vertebrates these forms are generated by alternative promoters. Novel splicing variations in the 3' region generate transcripts encoding alternative trans-membrane and PDZ domains. Another 3' splicing variation predicts soluble neurexin I isoforms. Neurexin I and neuroligin expression was found in brain tissue, with expression present throughout development, and in most cases significantly up-regulated in adults. Transcripts of neurexin I and one neuroligin tested were abundant in mushroom bodies, a higher order processing centre in the bee brain. We show neuroligins and neurexins comprise a highly conserved molecular system with likely similar functional roles in insects as vertebrates, and with scope in the honeybee to generate substantial functional diversity through alternative splicing. Our study provides important prerequisite data for using the bee as a model for vertebrate synaptic development.

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Neuroligin and Neurexin Phylogeny.(1.1) shows the phylogenetic relationship of 53 neuroligin proteins from the honeybee (AmNLG1-5) and Drosophila fly together with other neuroligins described by Bolliger et al [105]. All sequences are represented by taxon names showing species and NCBI accession numbers. The evolutionary history was inferred using the Neighbor-Joining method [106]. An optimal tree with percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches [107]. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the JTT matrix based method [108] and are in the units of the number of amino acid substitutions per site. All positions containing alignment gaps and missing data were eliminated only in pairwise sequence comparisons (Pairwise deletion option). There were a total of 1884 positions in the final dataset. Phylogenetic analyses were conducted in MEGA3.1 [94]. Four radiations (shaded) represent relationship of vertebrate proteins NLG1-4 compared with invertebrate proteins. The phylogeny shows vertebrate and invertebrate neuroligin radiations arise from a single common ancestor found in the sea urchin (S. purpuratus XP_001192426), and displays a congruent topology with the Maximum-Likelihood tree reported by Bolliger et al. [105]. Similar analysis was performed for investigating neurexin phylogeny. (1.2) shows the evolutionary relationship of two ancestrally related clades of neurexin proteins from vertebrates and invertebrates; the neurological neurexins (NrxI) and neurexin IV (also known as neurexin). Notably, the invertebrate neurexin IV proteins form an orthologous group with CASPR. There were a total of 2125 positions in the final dataset. Also shown are the NCBI and wormbase accession numbers. Abbreviations Am: Apis mellifera, honeybee.
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pone-0003542-g001: Neuroligin and Neurexin Phylogeny.(1.1) shows the phylogenetic relationship of 53 neuroligin proteins from the honeybee (AmNLG1-5) and Drosophila fly together with other neuroligins described by Bolliger et al [105]. All sequences are represented by taxon names showing species and NCBI accession numbers. The evolutionary history was inferred using the Neighbor-Joining method [106]. An optimal tree with percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches [107]. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the JTT matrix based method [108] and are in the units of the number of amino acid substitutions per site. All positions containing alignment gaps and missing data were eliminated only in pairwise sequence comparisons (Pairwise deletion option). There were a total of 1884 positions in the final dataset. Phylogenetic analyses were conducted in MEGA3.1 [94]. Four radiations (shaded) represent relationship of vertebrate proteins NLG1-4 compared with invertebrate proteins. The phylogeny shows vertebrate and invertebrate neuroligin radiations arise from a single common ancestor found in the sea urchin (S. purpuratus XP_001192426), and displays a congruent topology with the Maximum-Likelihood tree reported by Bolliger et al. [105]. Similar analysis was performed for investigating neurexin phylogeny. (1.2) shows the evolutionary relationship of two ancestrally related clades of neurexin proteins from vertebrates and invertebrates; the neurological neurexins (NrxI) and neurexin IV (also known as neurexin). Notably, the invertebrate neurexin IV proteins form an orthologous group with CASPR. There were a total of 2125 positions in the final dataset. Also shown are the NCBI and wormbase accession numbers. Abbreviations Am: Apis mellifera, honeybee.

Mentions: We conducted in silico homology based searches of the honeybee genome [47] using predicted fruit fly (Drosophila melanogaster) and mosquito (Anopheles gambiae) neuroligin sequences [48]. The searches confirmed five Beebase Glean-3 annotations, GB18720, GB10066, GB18290, GB18836 and GB13939, as putative neuroligin sequences [49] (‘BeeBase’ http://racerx00.tamu.edu/). Specific nucleotide primers were then used to PCR amplify five neuroligin cDNAs from reverse-transcribed adult honeybee brain RNA. These are named AmNLG1-5 according to their presumptive orthologies with other invertebrate neuroligins (Figure 1.1). Sequence analysis from the cloned amplicons showed that AmNLG -1, -3, -4 and -5 were full length cDNAs, each about approximately 2.5 kb in length. Despite strong sequence conservation between predicted orthologs and various attempts using different primer and PCR conditions, we were unable to verify the 5′ coding regions of AmNLG2. This may be due in part to significant secondary structure in the mRNA transcript as a consequence of the high GC rich content at the 5′-end of the AmNLG2 gene. Verified coding sequence, including 1645 bp of the 3′ portion of AmNLG2, is given in Figure S1 of the Supplementary Data.


Bridging the synaptic gap: neuroligins and neurexin I in Apis mellifera.

Biswas S, Russell RJ, Jackson CJ, Vidovic M, Ganeshina O, Oakeshott JG, Claudianos C - PLoS ONE (2008)

Neuroligin and Neurexin Phylogeny.(1.1) shows the phylogenetic relationship of 53 neuroligin proteins from the honeybee (AmNLG1-5) and Drosophila fly together with other neuroligins described by Bolliger et al [105]. All sequences are represented by taxon names showing species and NCBI accession numbers. The evolutionary history was inferred using the Neighbor-Joining method [106]. An optimal tree with percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches [107]. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the JTT matrix based method [108] and are in the units of the number of amino acid substitutions per site. All positions containing alignment gaps and missing data were eliminated only in pairwise sequence comparisons (Pairwise deletion option). There were a total of 1884 positions in the final dataset. Phylogenetic analyses were conducted in MEGA3.1 [94]. Four radiations (shaded) represent relationship of vertebrate proteins NLG1-4 compared with invertebrate proteins. The phylogeny shows vertebrate and invertebrate neuroligin radiations arise from a single common ancestor found in the sea urchin (S. purpuratus XP_001192426), and displays a congruent topology with the Maximum-Likelihood tree reported by Bolliger et al. [105]. Similar analysis was performed for investigating neurexin phylogeny. (1.2) shows the evolutionary relationship of two ancestrally related clades of neurexin proteins from vertebrates and invertebrates; the neurological neurexins (NrxI) and neurexin IV (also known as neurexin). Notably, the invertebrate neurexin IV proteins form an orthologous group with CASPR. There were a total of 2125 positions in the final dataset. Also shown are the NCBI and wormbase accession numbers. Abbreviations Am: Apis mellifera, honeybee.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2570956&req=5

pone-0003542-g001: Neuroligin and Neurexin Phylogeny.(1.1) shows the phylogenetic relationship of 53 neuroligin proteins from the honeybee (AmNLG1-5) and Drosophila fly together with other neuroligins described by Bolliger et al [105]. All sequences are represented by taxon names showing species and NCBI accession numbers. The evolutionary history was inferred using the Neighbor-Joining method [106]. An optimal tree with percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches [107]. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the JTT matrix based method [108] and are in the units of the number of amino acid substitutions per site. All positions containing alignment gaps and missing data were eliminated only in pairwise sequence comparisons (Pairwise deletion option). There were a total of 1884 positions in the final dataset. Phylogenetic analyses were conducted in MEGA3.1 [94]. Four radiations (shaded) represent relationship of vertebrate proteins NLG1-4 compared with invertebrate proteins. The phylogeny shows vertebrate and invertebrate neuroligin radiations arise from a single common ancestor found in the sea urchin (S. purpuratus XP_001192426), and displays a congruent topology with the Maximum-Likelihood tree reported by Bolliger et al. [105]. Similar analysis was performed for investigating neurexin phylogeny. (1.2) shows the evolutionary relationship of two ancestrally related clades of neurexin proteins from vertebrates and invertebrates; the neurological neurexins (NrxI) and neurexin IV (also known as neurexin). Notably, the invertebrate neurexin IV proteins form an orthologous group with CASPR. There were a total of 2125 positions in the final dataset. Also shown are the NCBI and wormbase accession numbers. Abbreviations Am: Apis mellifera, honeybee.
Mentions: We conducted in silico homology based searches of the honeybee genome [47] using predicted fruit fly (Drosophila melanogaster) and mosquito (Anopheles gambiae) neuroligin sequences [48]. The searches confirmed five Beebase Glean-3 annotations, GB18720, GB10066, GB18290, GB18836 and GB13939, as putative neuroligin sequences [49] (‘BeeBase’ http://racerx00.tamu.edu/). Specific nucleotide primers were then used to PCR amplify five neuroligin cDNAs from reverse-transcribed adult honeybee brain RNA. These are named AmNLG1-5 according to their presumptive orthologies with other invertebrate neuroligins (Figure 1.1). Sequence analysis from the cloned amplicons showed that AmNLG -1, -3, -4 and -5 were full length cDNAs, each about approximately 2.5 kb in length. Despite strong sequence conservation between predicted orthologs and various attempts using different primer and PCR conditions, we were unable to verify the 5′ coding regions of AmNLG2. This may be due in part to significant secondary structure in the mRNA transcript as a consequence of the high GC rich content at the 5′-end of the AmNLG2 gene. Verified coding sequence, including 1645 bp of the 3′ portion of AmNLG2, is given in Figure S1 of the Supplementary Data.

Bottom Line: Neurexin I and neuroligin expression was found in brain tissue, with expression present throughout development, and in most cases significantly up-regulated in adults.We show neuroligins and neurexins comprise a highly conserved molecular system with likely similar functional roles in insects as vertebrates, and with scope in the honeybee to generate substantial functional diversity through alternative splicing.Our study provides important prerequisite data for using the bee as a model for vertebrate synaptic development.

View Article: PubMed Central - PubMed

Affiliation: University of Queensland, Queensland Brain Institute, Brisbane, Queensland, Australia.

ABSTRACT
Vertebrate studies show neuroligins and neurexins are binding partners in a trans-synaptic cell adhesion complex, implicated in human autism and mental retardation disorders. Here we report a genetic analysis of homologous proteins in the honey bee. As in humans, the honeybee has five large (31-246 kb, up to 12 exons each) neuroligin genes, three of which are tightly clustered. RNA analysis of the neuroligin-3 gene reveals five alternatively spliced transcripts, generated through alternative use of exons encoding the cholinesterase-like domain. Whereas vertebrates have three neurexins the bee has just one gene named neurexin I (400 kb, 28 exons). However alternative isoforms of bee neurexin I are generated by differential use of 12 splice sites, mostly located in regions encoding LNS subdomains. Some of the splice variants of bee neurexin I resemble the vertebrate alpha- and beta-neurexins, albeit in vertebrates these forms are generated by alternative promoters. Novel splicing variations in the 3' region generate transcripts encoding alternative trans-membrane and PDZ domains. Another 3' splicing variation predicts soluble neurexin I isoforms. Neurexin I and neuroligin expression was found in brain tissue, with expression present throughout development, and in most cases significantly up-regulated in adults. Transcripts of neurexin I and one neuroligin tested were abundant in mushroom bodies, a higher order processing centre in the bee brain. We show neuroligins and neurexins comprise a highly conserved molecular system with likely similar functional roles in insects as vertebrates, and with scope in the honeybee to generate substantial functional diversity through alternative splicing. Our study provides important prerequisite data for using the bee as a model for vertebrate synaptic development.

Show MeSH
Related in: MedlinePlus