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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.

No MeSH data available.


pAktSer473/Akt ratios are not altered in different compartments of stimulated motoneurons. After 5 DIV in the presence of CNTF (5 ng/ml), the cells were serum as well as neurotrophic factor deprived for 6–7 h and stimulated for 5 min with BDNF (20 ng/ml), followed by fixation and staining against pAktSer473, Akt, and GFP. Compartment-specific quantification of the pAktSer473/Akt ratios is shown in Fig. S9. pAktSer473/Akt ratios strongly increase upon BDNF stimulation, but comparable pAktSer473/Akt values were achieved after Syap1 knockdown or mock treatment in all the tested compartments of the motoneurons. Scale bars Soma and axon: 10 µm; growth cone: 5 µm
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Fig14: pAktSer473/Akt ratios are not altered in different compartments of stimulated motoneurons. After 5 DIV in the presence of CNTF (5 ng/ml), the cells were serum as well as neurotrophic factor deprived for 6–7 h and stimulated for 5 min with BDNF (20 ng/ml), followed by fixation and staining against pAktSer473, Akt, and GFP. Compartment-specific quantification of the pAktSer473/Akt ratios is shown in Fig. S9. pAktSer473/Akt ratios strongly increase upon BDNF stimulation, but comparable pAktSer473/Akt values were achieved after Syap1 knockdown or mock treatment in all the tested compartments of the motoneurons. Scale bars Soma and axon: 10 µm; growth cone: 5 µm

Mentions: In adipocyte differentiation, Syap1 regulates growth factor-induced Akt1 phosphorylation at Ser473, and Syap1 knockdown by shRNA strongly reduces this phosphorylation (Yao et al. 2013). To investigate whether knockdown or knockout of Syap1 in motoneurons also causes reduced Akt phosphorylation, we stimulated primary motoneurons after 5 DIV in the presence of CNTF (5 ng/ml) and serum, followed by neurotrophic factor deprivation for variable times (see Methods), with BDNF (20 ng/ml) and tested the levels of Akt phosphorylation at Thr308 and Ser473 using phospho-specific antisera. In order to determine Akt phosphorylation kinetics at Thr308 and Ser473, we first stimulated motoneurons for increasing durations ranging from 2 s to 30 min. Maximal Akt phosphorylation was achieved after 2–5 min of stimulation for both sites (Thr308 and Ser473) (Fig. 13a, n = 3). Since localization data were identical for knock-out and knock-down motoneurons (Fig. 10), and Western blots confirmed almost complete Syap1 deficiency in knock-down neurons (Fig. 13c, d right panels), the effect of Syap1 deficiency on Akt phosphorylation was determined with knock-out neurons after 5 min of BDNF stimulation (Fig. 13b), whereas experiments after 2- and 5-min stimulation (Figs. 13c, d, S8) and tests for subcompartimental differences in stimulation effects (Figs. 14, S9) were carried out on knock-down neurons which could be generated in comparatively large quantities from motoneurons pooled from wild-type embryos. The Western blots of Fig. 13 revealed no reduction in Akt phosphorylation at Thr308 or Ser473 upon Syap1 knockout or knockdown compared to the controls. For the quantitative evaluation, densitometric signal values were normalized to the stimulated uninfected controls. The results depicted in Fig. S8 show no significant difference in Akt phosphorylation after Syap1 knockout (Fig. S8a, 5-min stimulation, values normalized to stimulated wild type; Thr308(left): unstimulated, wild type 0.13 ± 0.06; unstimulated, knockout: 0.15 ± 0.06; stimulated, knockout: 1.58 ± 0.06 (n = 3), Ser473 (right) unstimulated, wild type 0.23 ± 0.06; unstimulated, knockout: 0.24 ± 0.08; stimulated, knockout: 1.48 ± 0.29 (n = 3)) or knockdown (Fig. S8b, values normalized to uninfected stimulated pAkt/Akt ratio, 2-min stimulation; Thr308(left): unstimulated, uninfected: 0.12 ± 0.06; stimulated, sh-Syap1-infected: 1.58 ± 0.16; stimulated, mock-infected: 1.27 ± 0.26 (n = 3), Ser473 (right) unstimulated, uninfected: 0.39 ± 0.05; stimulated, sh-Syap1-infected: 1.27 ± 0.13, stimulated, mock-infected: 1.08 ± 0.04 (n = 3)); Fig. S8c, 5-min stimulation; Thr308 (left): unstimulated, uninfected: 0.06 ± 0.03; stimulated, sh-Syap1-infected: 1.31 ± 0.15; stimulated, mock-infected: 0.92 ± 0.05 (n = 3); Ser473 (right) unstimulated, uninfected: 0.23 ± 0.07; stimulated, sh-Syap1-infected: 1.72 ± 0.17; stimulated, mock-infected: 1.54 ± 0.24 (n = 3)).Fig. 13


Initial characterization of a Syap1 knock-out mouse and distribution of Syap1 in mouse brain and cultured motoneurons
pAktSer473/Akt ratios are not altered in different compartments of stimulated motoneurons. After 5 DIV in the presence of CNTF (5 ng/ml), the cells were serum as well as neurotrophic factor deprived for 6–7 h and stimulated for 5 min with BDNF (20 ng/ml), followed by fixation and staining against pAktSer473, Akt, and GFP. Compartment-specific quantification of the pAktSer473/Akt ratios is shown in Fig. S9. pAktSer473/Akt ratios strongly increase upon BDNF stimulation, but comparable pAktSer473/Akt values were achieved after Syap1 knockdown or mock treatment in all the tested compartments of the motoneurons. Scale bars Soma and axon: 10 µm; growth cone: 5 µm
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Fig14: pAktSer473/Akt ratios are not altered in different compartments of stimulated motoneurons. After 5 DIV in the presence of CNTF (5 ng/ml), the cells were serum as well as neurotrophic factor deprived for 6–7 h and stimulated for 5 min with BDNF (20 ng/ml), followed by fixation and staining against pAktSer473, Akt, and GFP. Compartment-specific quantification of the pAktSer473/Akt ratios is shown in Fig. S9. pAktSer473/Akt ratios strongly increase upon BDNF stimulation, but comparable pAktSer473/Akt values were achieved after Syap1 knockdown or mock treatment in all the tested compartments of the motoneurons. Scale bars Soma and axon: 10 µm; growth cone: 5 µm
Mentions: In adipocyte differentiation, Syap1 regulates growth factor-induced Akt1 phosphorylation at Ser473, and Syap1 knockdown by shRNA strongly reduces this phosphorylation (Yao et al. 2013). To investigate whether knockdown or knockout of Syap1 in motoneurons also causes reduced Akt phosphorylation, we stimulated primary motoneurons after 5 DIV in the presence of CNTF (5 ng/ml) and serum, followed by neurotrophic factor deprivation for variable times (see Methods), with BDNF (20 ng/ml) and tested the levels of Akt phosphorylation at Thr308 and Ser473 using phospho-specific antisera. In order to determine Akt phosphorylation kinetics at Thr308 and Ser473, we first stimulated motoneurons for increasing durations ranging from 2 s to 30 min. Maximal Akt phosphorylation was achieved after 2–5 min of stimulation for both sites (Thr308 and Ser473) (Fig. 13a, n = 3). Since localization data were identical for knock-out and knock-down motoneurons (Fig. 10), and Western blots confirmed almost complete Syap1 deficiency in knock-down neurons (Fig. 13c, d right panels), the effect of Syap1 deficiency on Akt phosphorylation was determined with knock-out neurons after 5 min of BDNF stimulation (Fig. 13b), whereas experiments after 2- and 5-min stimulation (Figs. 13c, d, S8) and tests for subcompartimental differences in stimulation effects (Figs. 14, S9) were carried out on knock-down neurons which could be generated in comparatively large quantities from motoneurons pooled from wild-type embryos. The Western blots of Fig. 13 revealed no reduction in Akt phosphorylation at Thr308 or Ser473 upon Syap1 knockout or knockdown compared to the controls. For the quantitative evaluation, densitometric signal values were normalized to the stimulated uninfected controls. The results depicted in Fig. S8 show no significant difference in Akt phosphorylation after Syap1 knockout (Fig. S8a, 5-min stimulation, values normalized to stimulated wild type; Thr308(left): unstimulated, wild type 0.13 ± 0.06; unstimulated, knockout: 0.15 ± 0.06; stimulated, knockout: 1.58 ± 0.06 (n = 3), Ser473 (right) unstimulated, wild type 0.23 ± 0.06; unstimulated, knockout: 0.24 ± 0.08; stimulated, knockout: 1.48 ± 0.29 (n = 3)) or knockdown (Fig. S8b, values normalized to uninfected stimulated pAkt/Akt ratio, 2-min stimulation; Thr308(left): unstimulated, uninfected: 0.12 ± 0.06; stimulated, sh-Syap1-infected: 1.58 ± 0.16; stimulated, mock-infected: 1.27 ± 0.26 (n = 3), Ser473 (right) unstimulated, uninfected: 0.39 ± 0.05; stimulated, sh-Syap1-infected: 1.27 ± 0.13, stimulated, mock-infected: 1.08 ± 0.04 (n = 3)); Fig. S8c, 5-min stimulation; Thr308 (left): unstimulated, uninfected: 0.06 ± 0.03; stimulated, sh-Syap1-infected: 1.31 ± 0.15; stimulated, mock-infected: 0.92 ± 0.05 (n = 3); Ser473 (right) unstimulated, uninfected: 0.23 ± 0.07; stimulated, sh-Syap1-infected: 1.72 ± 0.17; stimulated, mock-infected: 1.54 ± 0.24 (n = 3)).Fig. 13

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.