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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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Localization of Syap1 protein in cultured primary motoneurons. a Representative images of primary motoneurons cultured for 5 days and stained against Syap1 and acetylated tubulin or co-stained with phalloidin, as indicated. Syap1 knock-out cells (b, d, f) and shRNA–Syap1-treated cells (h, j, l) show a robust reduction in Syap1 immunoreactivity compared to wild-type (a, c, e) or uninfected (g, i, k) and sh-mismatch RNA (“mock”)-infected (not shown) controls. Syap1 signals are observed in the dendrites, soma, axon, and distal part of the axon. The magnified images of the somata (c, i) reveal punctate Syap1 staining in the cytoplasm and dendrites with a prominent perinuclear accumulation. Syap1 signals are also present in growth cones (e, k). Blue nuclear DAPI staining. Scale barsa, b, g, h: 25 µm; c–f, i–l: 10 µm
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Fig10: Localization of Syap1 protein in cultured primary motoneurons. a Representative images of primary motoneurons cultured for 5 days and stained against Syap1 and acetylated tubulin or co-stained with phalloidin, as indicated. Syap1 knock-out cells (b, d, f) and shRNA–Syap1-treated cells (h, j, l) show a robust reduction in Syap1 immunoreactivity compared to wild-type (a, c, e) or uninfected (g, i, k) and sh-mismatch RNA (“mock”)-infected (not shown) controls. Syap1 signals are observed in the dendrites, soma, axon, and distal part of the axon. The magnified images of the somata (c, i) reveal punctate Syap1 staining in the cytoplasm and dendrites with a prominent perinuclear accumulation. Syap1 signals are also present in growth cones (e, k). Blue nuclear DAPI staining. Scale barsa, b, g, h: 25 µm; c–f, i–l: 10 µm

Mentions: Motoneurons were isolated and enriched from E13.5 mouse embryos as described in “Methods” section. Cells from wild-type and knock-out embryos were cultured for 5 days, PFA-fixed and stained against Syap1 and acetylated tubulin, and counterstained with DAPI. In addition, wild-type cells infected either with the Syap1 knock-down virus or with the mock virus were compared. Strong signals obtained by confocal immunofluorescence microscopy were seen with the Syap1 antiserum in wild-type cells (Fig. 10a, c, e, g, i, k), but no specific staining was obtained in Syap1-/- knock-out cells (Fig. 10b, d, f). Residual fluorescence represents cross-reactivity of the primary antiserum as determined by omission of first antibody (not shown). A robust depletion of Syap1 signal after lentiviral Syap1 knockdown was likewise apparent (Fig. 10h, j, l) by comparison with mock-treated (not shown) or uninfected control cells (Fig. 10g, i, k). Syap1 immunofluorescence revealed a punctate staining pattern which could be observed in somata, dendrites, axons, and growth cones of the motoneurons and was strongly reduced in knock-out or knock-down cells. The nucleus showed no distinct Syap1 signal. At higher magnification of the cell bodies, a strong perinuclear Syap1 signal was detected (Fig. 10c, i) which was clearly lacking in knock-out and knock-down neurons (Fig. 10d, j). In axonal growth cones, punctate Syap1 signals appeared densest in regions containing high levels of F-actin as identified by Phalloidin staining (Cooper 1987; Fig. 10e, k). Low levels of fluorescent signals throughout terminal axons in knock-out and knock-down neurons (Fig. 10f, l) confirmed the specificity of strong Syap1 signals in actin-rich growth cone regions.Fig. 10


Initial characterization of a Syap1 knock-out mouse and distribution of Syap1 in mouse brain and cultured motoneurons
Localization of Syap1 protein in cultured primary motoneurons. a Representative images of primary motoneurons cultured for 5 days and stained against Syap1 and acetylated tubulin or co-stained with phalloidin, as indicated. Syap1 knock-out cells (b, d, f) and shRNA–Syap1-treated cells (h, j, l) show a robust reduction in Syap1 immunoreactivity compared to wild-type (a, c, e) or uninfected (g, i, k) and sh-mismatch RNA (“mock”)-infected (not shown) controls. Syap1 signals are observed in the dendrites, soma, axon, and distal part of the axon. The magnified images of the somata (c, i) reveal punctate Syap1 staining in the cytoplasm and dendrites with a prominent perinuclear accumulation. Syap1 signals are also present in growth cones (e, k). Blue nuclear DAPI staining. Scale barsa, b, g, h: 25 µm; c–f, i–l: 10 µm
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Fig10: Localization of Syap1 protein in cultured primary motoneurons. a Representative images of primary motoneurons cultured for 5 days and stained against Syap1 and acetylated tubulin or co-stained with phalloidin, as indicated. Syap1 knock-out cells (b, d, f) and shRNA–Syap1-treated cells (h, j, l) show a robust reduction in Syap1 immunoreactivity compared to wild-type (a, c, e) or uninfected (g, i, k) and sh-mismatch RNA (“mock”)-infected (not shown) controls. Syap1 signals are observed in the dendrites, soma, axon, and distal part of the axon. The magnified images of the somata (c, i) reveal punctate Syap1 staining in the cytoplasm and dendrites with a prominent perinuclear accumulation. Syap1 signals are also present in growth cones (e, k). Blue nuclear DAPI staining. Scale barsa, b, g, h: 25 µm; c–f, i–l: 10 µm
Mentions: Motoneurons were isolated and enriched from E13.5 mouse embryos as described in “Methods” section. Cells from wild-type and knock-out embryos were cultured for 5 days, PFA-fixed and stained against Syap1 and acetylated tubulin, and counterstained with DAPI. In addition, wild-type cells infected either with the Syap1 knock-down virus or with the mock virus were compared. Strong signals obtained by confocal immunofluorescence microscopy were seen with the Syap1 antiserum in wild-type cells (Fig. 10a, c, e, g, i, k), but no specific staining was obtained in Syap1-/- knock-out cells (Fig. 10b, d, f). Residual fluorescence represents cross-reactivity of the primary antiserum as determined by omission of first antibody (not shown). A robust depletion of Syap1 signal after lentiviral Syap1 knockdown was likewise apparent (Fig. 10h, j, l) by comparison with mock-treated (not shown) or uninfected control cells (Fig. 10g, i, k). Syap1 immunofluorescence revealed a punctate staining pattern which could be observed in somata, dendrites, axons, and growth cones of the motoneurons and was strongly reduced in knock-out or knock-down cells. The nucleus showed no distinct Syap1 signal. At higher magnification of the cell bodies, a strong perinuclear Syap1 signal was detected (Fig. 10c, i) which was clearly lacking in knock-out and knock-down neurons (Fig. 10d, j). In axonal growth cones, punctate Syap1 signals appeared densest in regions containing high levels of F-actin as identified by Phalloidin staining (Cooper 1987; Fig. 10e, k). Low levels of fluorescent signals throughout terminal axons in knock-out and knock-down neurons (Fig. 10f, l) confirmed the specificity of strong Syap1 signals in actin-rich growth cone regions.Fig. 10

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