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

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.


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Sections of wild type (a, b, e, f) and Syap1 knock-out (c, d, g, h) brains stained with Syap1 antiserum (left) and DAPI (right). In coronal sections (a, c), diffuse Syap1-specific immunolabeling is found throughout all parts of the brain, preferentially localized in the gray matter neuropil. Arrowheads point to hypothalamic cells that unspecifically cross-react with the Syap1 antiserum as they are detected both in wild-type and knock-out sections but not in controls without primary antibody (see Fig. S3). The asterisk indicates a thalamic region containing numerous cells with strong perinuclear Syap1-specific immunofluorescence labeling (enlarged in Fig. 5). Sagittal sections reveal particularly intense immunofluorescence for Syap1 in the molecular layer of the cerebellar cortex (e, g). Scale bars 1 mm (upper), 200 µm (lower) panels
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Fig3: Sections of wild type (a, b, e, f) and Syap1 knock-out (c, d, g, h) brains stained with Syap1 antiserum (left) and DAPI (right). In coronal sections (a, c), diffuse Syap1-specific immunolabeling is found throughout all parts of the brain, preferentially localized in the gray matter neuropil. Arrowheads point to hypothalamic cells that unspecifically cross-react with the Syap1 antiserum as they are detected both in wild-type and knock-out sections but not in controls without primary antibody (see Fig. S3). The asterisk indicates a thalamic region containing numerous cells with strong perinuclear Syap1-specific immunofluorescence labeling (enlarged in Fig. 5). Sagittal sections reveal particularly intense immunofluorescence for Syap1 in the molecular layer of the cerebellar cortex (e, g). Scale bars 1 mm (upper), 200 µm (lower) panels

Mentions: With the availability of Syap1 knock-out animals, it now is possible to reliably determine the distribution of Syap1 in wild-type tissues at the cellular and subcellular level. Since so far we have no information about the function of Syap1 in mammalian nervous system, we here give an overview on Syap1 localization in various parts of the mouse brain and report in more detail the distribution in selected regions. Comparison of coronal sections of wild-type and Syap1 knock-out brains immuno-reacted with the Syap1 antiserum reveals intense Syap1-specific immunofluorescence throughout the brain with regionally distinct distribution patterns (Fig. 3). Fluorescent signals observed in both wild-type and knock-out sections (e.g., individual cells in the hypothalamus, arrowheads in Fig. 3a, c; distinct fiber-like structures in other brain areas, see below) are lacking in negative controls incubated without Syap1 antiserum (Fig. S3). This indicates the cross-reactivity of the antiserum with an unknown antigen and emphasizes the necessity of comparing wild-type and knock-out sections processed in parallel for specifically characterizing Syap1 distribution in the various brain areas, as done in the present study.Fig. 3


Initial characterization of a Syap1 knock-out mouse and distribution of Syap1 in mouse brain and cultured motoneurons
Sections of wild type (a, b, e, f) and Syap1 knock-out (c, d, g, h) brains stained with Syap1 antiserum (left) and DAPI (right). In coronal sections (a, c), diffuse Syap1-specific immunolabeling is found throughout all parts of the brain, preferentially localized in the gray matter neuropil. Arrowheads point to hypothalamic cells that unspecifically cross-react with the Syap1 antiserum as they are detected both in wild-type and knock-out sections but not in controls without primary antibody (see Fig. S3). The asterisk indicates a thalamic region containing numerous cells with strong perinuclear Syap1-specific immunofluorescence labeling (enlarged in Fig. 5). Sagittal sections reveal particularly intense immunofluorescence for Syap1 in the molecular layer of the cerebellar cortex (e, g). Scale bars 1 mm (upper), 200 µm (lower) panels
© Copyright Policy - OpenAccess
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Fig3: Sections of wild type (a, b, e, f) and Syap1 knock-out (c, d, g, h) brains stained with Syap1 antiserum (left) and DAPI (right). In coronal sections (a, c), diffuse Syap1-specific immunolabeling is found throughout all parts of the brain, preferentially localized in the gray matter neuropil. Arrowheads point to hypothalamic cells that unspecifically cross-react with the Syap1 antiserum as they are detected both in wild-type and knock-out sections but not in controls without primary antibody (see Fig. S3). The asterisk indicates a thalamic region containing numerous cells with strong perinuclear Syap1-specific immunofluorescence labeling (enlarged in Fig. 5). Sagittal sections reveal particularly intense immunofluorescence for Syap1 in the molecular layer of the cerebellar cortex (e, g). Scale bars 1 mm (upper), 200 µm (lower) panels
Mentions: With the availability of Syap1 knock-out animals, it now is possible to reliably determine the distribution of Syap1 in wild-type tissues at the cellular and subcellular level. Since so far we have no information about the function of Syap1 in mammalian nervous system, we here give an overview on Syap1 localization in various parts of the mouse brain and report in more detail the distribution in selected regions. Comparison of coronal sections of wild-type and Syap1 knock-out brains immuno-reacted with the Syap1 antiserum reveals intense Syap1-specific immunofluorescence throughout the brain with regionally distinct distribution patterns (Fig. 3). Fluorescent signals observed in both wild-type and knock-out sections (e.g., individual cells in the hypothalamus, arrowheads in Fig. 3a, c; distinct fiber-like structures in other brain areas, see below) are lacking in negative controls incubated without Syap1 antiserum (Fig. S3). This indicates the cross-reactivity of the antiserum with an unknown antigen and emphasizes the necessity of comparing wild-type and knock-out sections processed in parallel for specifically characterizing Syap1 distribution in the various brain areas, as done in the present study.Fig. 3

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