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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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Syap1 distribution in the hippocampus. a In wild-type sections, Syap1-specific immunofluorescence is present in general neuropil, high levels of Syap1-labeling are observed in CA2 perikarya (large arrowheads), and Syap1 immunoreactivity is also detectable to a weaker extent in CA1 or CA3 perikarya (small arrowheads), compared with knock-out sections (b, d). The infra- and suprapyramidal mossy fiber pathways arising from dentate gyrus granule cells show intense Syap1-specific immunoreactivity (arrows). Syap1 is also found in the stratum lucidum. (c, d) The enlargement of the CA2 region double stained with anti-Syap1 and GM130 demonstrates the association of perinuclear Syap1 with the Golgi apparatus of these cells. Hi, hilus; SG, stratum granulosum; SL, stratum lucidum; SLM, stratum lacunosum-moleculare; SM, stratum moleculare; SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum. Scale bars 200 µm (a, b), 50 µm (c, d), 10 µm (inset in c)
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Fig6: Syap1 distribution in the hippocampus. a In wild-type sections, Syap1-specific immunofluorescence is present in general neuropil, high levels of Syap1-labeling are observed in CA2 perikarya (large arrowheads), and Syap1 immunoreactivity is also detectable to a weaker extent in CA1 or CA3 perikarya (small arrowheads), compared with knock-out sections (b, d). The infra- and suprapyramidal mossy fiber pathways arising from dentate gyrus granule cells show intense Syap1-specific immunoreactivity (arrows). Syap1 is also found in the stratum lucidum. (c, d) The enlargement of the CA2 region double stained with anti-Syap1 and GM130 demonstrates the association of perinuclear Syap1 with the Golgi apparatus of these cells. Hi, hilus; SG, stratum granulosum; SL, stratum lucidum; SLM, stratum lacunosum-moleculare; SM, stratum moleculare; SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum. Scale bars 200 µm (a, b), 50 µm (c, d), 10 µm (inset in c)

Mentions: In the thalamus, a region of enhanced Syap1 immunolabeling is observed (marked by an asterisk in Fig. 3a, compared with knockout in b). At higher magnification (Fig. 5a, c), it becomes apparent that this is caused by perinuclear accumulations of Syap1-immunoreactive material in numerous neurons (identified by their typical nuclei in DAPI staining, Fig. 5a′, c′, or by double staining with anti-NeuN antibodies, a neuron-specific marker (Fig. S4b), in close association with immunofluorescence for GM130, a marker for the cis/medial-Golgi apparatus (Nakamura et al. 1995) in dual immunolabelings (Fig. 5a, a′). In astrocytes and presumably other non-neuronal cells displaying comparatively small and heterochromatin-rich nuclei in DAPI stainings, accumulation of Syap1-immunoreactivity near the Golgi apparatus is lacking (arrow in Figs. 5a′, S4a, b). Also, the fact that apparently not all neuronal nuclei are surrounded by Syap1-immunoreactivity (Fig. 5c′) indicates that Syap1 expression is under cell-(sub)type-specific control. This becomes even more obvious in the hippocampus. Here, we find low levels of Syap1-specific immunofluorescence in the pyramidal layer of CA1 and CA3 (small arrowheads in Fig. 6a), but high concentrations in practically all perikarya of the CA2 pyramidal layer (large arrowheads in Fig. 6a, at higher magnification in c). Again, association with GM130-immunoreactivity is notable in CA2 neuronal perikarya (Fig. 6c, inset). This observation induced us to ask whether the structure of the Golgi apparatus was noticeably altered when Syap1 protein was missing. We therefore compared GM130 staining in wild-type and Syap1 knock-out brain sections at higher magnification. No qualitative effect of Syap1 knockout on Golgi structure as revealed by light microscopical GM130 staining was noted (Fig. S6). Medium intense, diffuse Syap1-immunoreactivity is observed in the dentate gyrus stratum moleculare and in the CA stratum lacunosum-moleculare. In CA1, the strata oriens and radiatum display medium high and low immunoreactivity levels, respectively. While dentate gyrus granule cells display scarce perinuclear labeling, the hilus contains strong punctate Syap1-immunoreactivity (Figs. 6a, S5a) which extends into the CA3 region, forming two bands of particularly intense fluorescence adjacent to CA3 somata (arrows in Figs. 6a, S5b) corresponding to the supra- and infrapyramidal mossy fiber pathways, the suprapyramidal band extending throughout the stratum lucidum of CA3 into CA2 (Fig. 6a). In dual immunolabelings, partial co-localization of Syap1 with the vesicular glutamate transporter vGlut1 is observed in the mossy fiber pathway (Fig. S5a, b), indicating the presence of the protein in the particularly large glutamatergic mossy fiber boutons but also in additional non-vGlut1 immunoreactive elements.Fig. 5


Initial characterization of a Syap1 knock-out mouse and distribution of Syap1 in mouse brain and cultured motoneurons
Syap1 distribution in the hippocampus. a In wild-type sections, Syap1-specific immunofluorescence is present in general neuropil, high levels of Syap1-labeling are observed in CA2 perikarya (large arrowheads), and Syap1 immunoreactivity is also detectable to a weaker extent in CA1 or CA3 perikarya (small arrowheads), compared with knock-out sections (b, d). The infra- and suprapyramidal mossy fiber pathways arising from dentate gyrus granule cells show intense Syap1-specific immunoreactivity (arrows). Syap1 is also found in the stratum lucidum. (c, d) The enlargement of the CA2 region double stained with anti-Syap1 and GM130 demonstrates the association of perinuclear Syap1 with the Golgi apparatus of these cells. Hi, hilus; SG, stratum granulosum; SL, stratum lucidum; SLM, stratum lacunosum-moleculare; SM, stratum moleculare; SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum. Scale bars 200 µm (a, b), 50 µm (c, d), 10 µm (inset in c)
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Fig6: Syap1 distribution in the hippocampus. a In wild-type sections, Syap1-specific immunofluorescence is present in general neuropil, high levels of Syap1-labeling are observed in CA2 perikarya (large arrowheads), and Syap1 immunoreactivity is also detectable to a weaker extent in CA1 or CA3 perikarya (small arrowheads), compared with knock-out sections (b, d). The infra- and suprapyramidal mossy fiber pathways arising from dentate gyrus granule cells show intense Syap1-specific immunoreactivity (arrows). Syap1 is also found in the stratum lucidum. (c, d) The enlargement of the CA2 region double stained with anti-Syap1 and GM130 demonstrates the association of perinuclear Syap1 with the Golgi apparatus of these cells. Hi, hilus; SG, stratum granulosum; SL, stratum lucidum; SLM, stratum lacunosum-moleculare; SM, stratum moleculare; SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum. Scale bars 200 µm (a, b), 50 µm (c, d), 10 µm (inset in c)
Mentions: In the thalamus, a region of enhanced Syap1 immunolabeling is observed (marked by an asterisk in Fig. 3a, compared with knockout in b). At higher magnification (Fig. 5a, c), it becomes apparent that this is caused by perinuclear accumulations of Syap1-immunoreactive material in numerous neurons (identified by their typical nuclei in DAPI staining, Fig. 5a′, c′, or by double staining with anti-NeuN antibodies, a neuron-specific marker (Fig. S4b), in close association with immunofluorescence for GM130, a marker for the cis/medial-Golgi apparatus (Nakamura et al. 1995) in dual immunolabelings (Fig. 5a, a′). In astrocytes and presumably other non-neuronal cells displaying comparatively small and heterochromatin-rich nuclei in DAPI stainings, accumulation of Syap1-immunoreactivity near the Golgi apparatus is lacking (arrow in Figs. 5a′, S4a, b). Also, the fact that apparently not all neuronal nuclei are surrounded by Syap1-immunoreactivity (Fig. 5c′) indicates that Syap1 expression is under cell-(sub)type-specific control. This becomes even more obvious in the hippocampus. Here, we find low levels of Syap1-specific immunofluorescence in the pyramidal layer of CA1 and CA3 (small arrowheads in Fig. 6a), but high concentrations in practically all perikarya of the CA2 pyramidal layer (large arrowheads in Fig. 6a, at higher magnification in c). Again, association with GM130-immunoreactivity is notable in CA2 neuronal perikarya (Fig. 6c, inset). This observation induced us to ask whether the structure of the Golgi apparatus was noticeably altered when Syap1 protein was missing. We therefore compared GM130 staining in wild-type and Syap1 knock-out brain sections at higher magnification. No qualitative effect of Syap1 knockout on Golgi structure as revealed by light microscopical GM130 staining was noted (Fig. S6). Medium intense, diffuse Syap1-immunoreactivity is observed in the dentate gyrus stratum moleculare and in the CA stratum lacunosum-moleculare. In CA1, the strata oriens and radiatum display medium high and low immunoreactivity levels, respectively. While dentate gyrus granule cells display scarce perinuclear labeling, the hilus contains strong punctate Syap1-immunoreactivity (Figs. 6a, S5a) which extends into the CA3 region, forming two bands of particularly intense fluorescence adjacent to CA3 somata (arrows in Figs. 6a, S5b) corresponding to the supra- and infrapyramidal mossy fiber pathways, the suprapyramidal band extending throughout the stratum lucidum of CA3 into CA2 (Fig. 6a). In dual immunolabelings, partial co-localization of Syap1 with the vesicular glutamate transporter vGlut1 is observed in the mossy fiber pathway (Fig. S5a, b), indicating the presence of the protein in the particularly large glutamatergic mossy fiber boutons but also in additional non-vGlut1 immunoreactive elements.Fig. 5

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