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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.


Related in: MedlinePlus

Syap1 knockdown or knockout does not significantly influence total Akt phosphorylation at Thr308 and Ser473 in primary motoneurons. a Western blot of serum-starved cells stimulated with BDNF (20 ng/ml) in a time series ranging from 2 s to 30 min. Maximum Akt Thr308 (left blot) and Ser473 (right blot) phosphorylation is achieved after 2–5 min of neurotrophin stimulation. b Western blots of motoneurons from wild-type and Syap1 knock-out embryos stimulated for 5 min with BDNF did not reveal a reduction in Akt phosphorylation at Thr308 (left blot) or Ser473 (right blot) due to Syap1 knockout. Calnexin and pan-Akt served as loading controls while GFP levels indicate a positive infection of the cells. c, dBlots of Syap1 shRNA-infected motoneurons and uninfected and mock-infected controls stimulated for two (c) or five (d) minutes with BDNF. No differences in Akt phosphorylation at Thr308 (left blots) and Ser473 (right blots) were observed after Syap1 knockdown compared to controls. The detection of Syap1 (right blots) demonstrates the strong reduction in Syap1 protein levels by the knockdown. Quantification of the signals of these and similar blots is shown in Fig. S8
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Fig13: Syap1 knockdown or knockout does not significantly influence total Akt phosphorylation at Thr308 and Ser473 in primary motoneurons. a Western blot of serum-starved cells stimulated with BDNF (20 ng/ml) in a time series ranging from 2 s to 30 min. Maximum Akt Thr308 (left blot) and Ser473 (right blot) phosphorylation is achieved after 2–5 min of neurotrophin stimulation. b Western blots of motoneurons from wild-type and Syap1 knock-out embryos stimulated for 5 min with BDNF did not reveal a reduction in Akt phosphorylation at Thr308 (left blot) or Ser473 (right blot) due to Syap1 knockout. Calnexin and pan-Akt served as loading controls while GFP levels indicate a positive infection of the cells. c, dBlots of Syap1 shRNA-infected motoneurons and uninfected and mock-infected controls stimulated for two (c) or five (d) minutes with BDNF. No differences in Akt phosphorylation at Thr308 (left blots) and Ser473 (right blots) were observed after Syap1 knockdown compared to controls. The detection of Syap1 (right blots) demonstrates the strong reduction in Syap1 protein levels by the knockdown. Quantification of the signals of these and similar blots is shown in Fig. S8

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
Syap1 knockdown or knockout does not significantly influence total Akt phosphorylation at Thr308 and Ser473 in primary motoneurons. a Western blot of serum-starved cells stimulated with BDNF (20 ng/ml) in a time series ranging from 2 s to 30 min. Maximum Akt Thr308 (left blot) and Ser473 (right blot) phosphorylation is achieved after 2–5 min of neurotrophin stimulation. b Western blots of motoneurons from wild-type and Syap1 knock-out embryos stimulated for 5 min with BDNF did not reveal a reduction in Akt phosphorylation at Thr308 (left blot) or Ser473 (right blot) due to Syap1 knockout. Calnexin and pan-Akt served as loading controls while GFP levels indicate a positive infection of the cells. c, dBlots of Syap1 shRNA-infected motoneurons and uninfected and mock-infected controls stimulated for two (c) or five (d) minutes with BDNF. No differences in Akt phosphorylation at Thr308 (left blots) and Ser473 (right blots) were observed after Syap1 knockdown compared to controls. The detection of Syap1 (right blots) demonstrates the strong reduction in Syap1 protein levels by the knockdown. Quantification of the signals of these and similar blots is shown in Fig. S8
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Fig13: Syap1 knockdown or knockout does not significantly influence total Akt phosphorylation at Thr308 and Ser473 in primary motoneurons. a Western blot of serum-starved cells stimulated with BDNF (20 ng/ml) in a time series ranging from 2 s to 30 min. Maximum Akt Thr308 (left blot) and Ser473 (right blot) phosphorylation is achieved after 2–5 min of neurotrophin stimulation. b Western blots of motoneurons from wild-type and Syap1 knock-out embryos stimulated for 5 min with BDNF did not reveal a reduction in Akt phosphorylation at Thr308 (left blot) or Ser473 (right blot) due to Syap1 knockout. Calnexin and pan-Akt served as loading controls while GFP levels indicate a positive infection of the cells. c, dBlots of Syap1 shRNA-infected motoneurons and uninfected and mock-infected controls stimulated for two (c) or five (d) minutes with BDNF. No differences in Akt phosphorylation at Thr308 (left blots) and Ser473 (right blots) were observed after Syap1 knockdown compared to controls. The detection of Syap1 (right blots) demonstrates the strong reduction in Syap1 protein levels by the knockdown. Quantification of the signals of these and similar blots is shown in Fig. S8
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.


Related in: MedlinePlus