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Initial characterization of a Syap1 knock-out mouse and distribution of Syap1 in mouse brain and cultured motoneurons

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ABSTRACT

Synapse-associated protein 1 (Syap1/BSTA) is the mammalian homologue of Sap47 (synapse-associated protein of 47 kDa) in Drosophila. Sap47 mutant larvae show reduced short-term synaptic plasticity and a defect in associative behavioral plasticity. In cultured adipocytes, Syap1 functions as part of a complex that phosphorylates protein kinase Bα/Akt1 (Akt1) at Ser473 and promotes differentiation. The role of Syap1 in the vertebrate nervous system is unknown. Here, we generated a Syap1 knock-out mouse and show that lack of Syap1 is compatible with viability and fertility. Adult knock-out mice show no overt defects in brain morphology. In wild-type brain, Syap1 is found widely distributed in synaptic neuropil, notably in regions rich in glutamatergic synapses, but also in perinuclear structures associated with the Golgi apparatus of specific groups of neuronal cell bodies. In cultured motoneurons, Syap1 is located in axons and growth cones and is enriched in a perinuclear region partially overlapping with Golgi markers. We studied in detail the influence of Syap1 knockdown and knockout on structure and development of these cells. Importantly, Syap1 knockout does not affect motoneuron survival or axon growth. Unexpectedly, neither knockdown nor knockout of Syap1 in cultured motoneurons is associated with reduced Ser473 or Thr308 phosphorylation of Akt. Our findings demonstrate a widespread expression of Syap1 in the mouse central nervous system with regionally specific distribution patterns as illustrated in particular for olfactory bulb, hippocampus, and cerebellum.

Electronic supplementary material: The online version of this article (doi:10.1007/s00418-016-1457-0) contains supplementary material, which is available to authorized users.

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Schematic of the targeted mutation 1a allele (Syap1tm1a(EUCOMM)Hmgu) of the mouse Syap1 gene (a, details in text), amino acid sequence of mouse Syap1 protein (b), and fusion protein (red amino acids) expressed in knock-out mouse (b, c). Syap1 exons 1–3 encoding the red sequences in b are spliced to sequences encoding a fragment of mouse engrailed-2 protein shown in c. Conserved amino acids of the BSD domain (underlined in b) are marked in blue
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Fig1: Schematic of the targeted mutation 1a allele (Syap1tm1a(EUCOMM)Hmgu) of the mouse Syap1 gene (a, details in text), amino acid sequence of mouse Syap1 protein (b), and fusion protein (red amino acids) expressed in knock-out mouse (b, c). Syap1 exons 1–3 encoding the red sequences in b are spliced to sequences encoding a fragment of mouse engrailed-2 protein shown in c. Conserved amino acids of the BSD domain (underlined in b) are marked in blue

Mentions: After expansion of the clone according to the EUCOMM recommendations, 10–15 ES cells were injected into BALB/c host blastocysts. Injected embryos were cultured for 3–4 h to recover and then transferred into the right uterus horn of 2.5 dpc pseudopregnant CD1 surrogate mothers. The offspring were selected based on their chimeric coat color. High-percentage male chimeras were bred with C57BL/6N females, and the F1 offspring were genotyped by PCR for the neomycin transferase selection marker (Neo) and by Southern analysis. Due to the X-chromosomal location of Syap1, all mutants of the F1 generation were heterozygous females which were mated to C57BL/6J males. Mutants of the F2 generation were intercrossed in order to produce homozygous mutant females. Due to the engrailed-2 splice acceptor site (SA) and a coding region of the mouse engrailed-2 protein (En-2) upstream of lacZ, a bi-cistronic transcript is generated producing a fusion protein consisting of a short Syap1 fragment translated from exon-1 to exon-3 of Syap1 and an En-2 fragment (Fig. 1). The Syap1 fragment of this protein is predicted to be non-functional as it lacks the conserved BSD domain (Doerks et al. 2002) which has been shown to be a requirement for Syap1 function (Yao et al. 2013). Therefore, unless there is residual splicing from exon-3 to exon-4, mice homo- or hemizygous for the Syap1tm1a allele will be Syap1 mutants. Due to an IRES sequence upstream of lacZ, β-galactosidase is translated from the bi-cistronic mRNA and can provide information of Syap1 expression in wild-type mice. The targeting construct Syap1tm1a also allows for reconstitution of gene function and the generation of a Syap1tm1c conditional knock-out allele following exposure to site-specific Flp recombination.Fig. 1


Initial characterization of a Syap1 knock-out mouse and distribution of Syap1 in mouse brain and cultured motoneurons
Schematic of the targeted mutation 1a allele (Syap1tm1a(EUCOMM)Hmgu) of the mouse Syap1 gene (a, details in text), amino acid sequence of mouse Syap1 protein (b), and fusion protein (red amino acids) expressed in knock-out mouse (b, c). Syap1 exons 1–3 encoding the red sequences in b are spliced to sequences encoding a fragment of mouse engrailed-2 protein shown in c. Conserved amino acids of the BSD domain (underlined in b) are marked in blue
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5037158&req=5

Fig1: Schematic of the targeted mutation 1a allele (Syap1tm1a(EUCOMM)Hmgu) of the mouse Syap1 gene (a, details in text), amino acid sequence of mouse Syap1 protein (b), and fusion protein (red amino acids) expressed in knock-out mouse (b, c). Syap1 exons 1–3 encoding the red sequences in b are spliced to sequences encoding a fragment of mouse engrailed-2 protein shown in c. Conserved amino acids of the BSD domain (underlined in b) are marked in blue
Mentions: After expansion of the clone according to the EUCOMM recommendations, 10–15 ES cells were injected into BALB/c host blastocysts. Injected embryos were cultured for 3–4 h to recover and then transferred into the right uterus horn of 2.5 dpc pseudopregnant CD1 surrogate mothers. The offspring were selected based on their chimeric coat color. High-percentage male chimeras were bred with C57BL/6N females, and the F1 offspring were genotyped by PCR for the neomycin transferase selection marker (Neo) and by Southern analysis. Due to the X-chromosomal location of Syap1, all mutants of the F1 generation were heterozygous females which were mated to C57BL/6J males. Mutants of the F2 generation were intercrossed in order to produce homozygous mutant females. Due to the engrailed-2 splice acceptor site (SA) and a coding region of the mouse engrailed-2 protein (En-2) upstream of lacZ, a bi-cistronic transcript is generated producing a fusion protein consisting of a short Syap1 fragment translated from exon-1 to exon-3 of Syap1 and an En-2 fragment (Fig. 1). The Syap1 fragment of this protein is predicted to be non-functional as it lacks the conserved BSD domain (Doerks et al. 2002) which has been shown to be a requirement for Syap1 function (Yao et al. 2013). Therefore, unless there is residual splicing from exon-3 to exon-4, mice homo- or hemizygous for the Syap1tm1a allele will be Syap1 mutants. Due to an IRES sequence upstream of lacZ, β-galactosidase is translated from the bi-cistronic mRNA and can provide information of Syap1 expression in wild-type mice. The targeting construct Syap1tm1a also allows for reconstitution of gene function and the generation of a Syap1tm1c conditional knock-out allele following exposure to site-specific Flp recombination.Fig. 1

View Article: PubMed Central - PubMed

ABSTRACT

Synapse-associated protein 1 (Syap1/BSTA) is the mammalian homologue of Sap47 (synapse-associated protein of 47 kDa) in Drosophila. Sap47 mutant larvae show reduced short-term synaptic plasticity and a defect in associative behavioral plasticity. In cultured adipocytes, Syap1 functions as part of a complex that phosphorylates protein kinase Bα/Akt1 (Akt1) at Ser473 and promotes differentiation. The role of Syap1 in the vertebrate nervous system is unknown. Here, we generated a Syap1 knock-out mouse and show that lack of Syap1 is compatible with viability and fertility. Adult knock-out mice show no overt defects in brain morphology. In wild-type brain, Syap1 is found widely distributed in synaptic neuropil, notably in regions rich in glutamatergic synapses, but also in perinuclear structures associated with the Golgi apparatus of specific groups of neuronal cell bodies. In cultured motoneurons, Syap1 is located in axons and growth cones and is enriched in a perinuclear region partially overlapping with Golgi markers. We studied in detail the influence of Syap1 knockdown and knockout on structure and development of these cells. Importantly, Syap1 knockout does not affect motoneuron survival or axon growth. Unexpectedly, neither knockdown nor knockout of Syap1 in cultured motoneurons is associated with reduced Ser473 or Thr308 phosphorylation of Akt. Our findings demonstrate a widespread expression of Syap1 in the mouse central nervous system with regionally specific distribution patterns as illustrated in particular for olfactory bulb, hippocampus, and cerebellum.

Electronic supplementary material: The online version of this article (doi:10.1007/s00418-016-1457-0) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus