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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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Knockout of Syap1 does not modify survival or axonal outgrowth of primary motoneurons. a Quantification of the survival of primary motoneurons cultured for 7 DIV in the absence or presence of BDNF. Cells were counted on day 1 and again on day 7. There is no significant difference in the survival rate of motoneurons from Syap1 knockout (ko) compared to wild-type (WT) embryos [n = 17 (WT) and 7 (ko) independent cultures]. Wild-type cells cultured without BDNF show highly reduced cell survival. b Axon length of primary motoneurons cultured for 7 DIV is not affected by Syap1 knockout (n = 288 for WT and 237 for ko)
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Fig12: Knockout of Syap1 does not modify survival or axonal outgrowth of primary motoneurons. a Quantification of the survival of primary motoneurons cultured for 7 DIV in the absence or presence of BDNF. Cells were counted on day 1 and again on day 7. There is no significant difference in the survival rate of motoneurons from Syap1 knockout (ko) compared to wild-type (WT) embryos [n = 17 (WT) and 7 (ko) independent cultures]. Wild-type cells cultured without BDNF show highly reduced cell survival. b Axon length of primary motoneurons cultured for 7 DIV is not affected by Syap1 knockout (n = 288 for WT and 237 for ko)

Mentions: Primary motoneurons in culture require neurotrophic factors for survival and growth of axons and dendrites. Thus, the role of cell-autonomous factors and their effects on signaling pathways controlling axon elongation and growth cone differentiation can be analyzed using this system (Calof and Reichardt 1984; Forscher and Smith 1988; Arakawa et al. 1990; Sanes and Lichtman 1999; Sendtner et al. 2000). To study the function of Syap1, we therefore investigated the effect of Syap1 knockout on survival and axon growth of cultured embryonic motoneurons (Fig. 12). After 7 days in vitro (DIV), there was no difference in the survival rate of Syap1 knockout in comparison with wild-type motoneurons (P > 0.3), whereas a significant reduction in viability was observed when wild-type or knock-out neurons were cultured without the neurotrophic factor BDNF (P < 0.05) (Fig. 12a; without BDNF: wild-type: 24.9 ± 1.9 % (17); Syap1 knockout: 29.4 ± 2.4 (7); with BDNF: wild-type: 64.6 ± 3.6 % (17); Syap1 knockout: 64.5 ± 5.6 % (7); (in parentheses: numbers of independent cultures). The mean values for axon length (Fig. 12b) also did not differ significantly between the genotypes (axon length wild type: 660.5 ± 16.8 µm; Syap1 knockout: 659.4 ± 19.3 µm; (n = 4 independent cultures; numbers of cells pooled per condition: wild type: N = 288, Syap1 knockout: N = 237). These results indicate that Syap1 does not play a major role in microtubule dynamics or other molecular mechanisms relevant for neurite outgrowth (Paglini et al. 1998; Sendtner et al. 2000; Rossoll et al. 2003; Jablonka et al. 2007; Selvaraj et al. 2012).Fig. 12


Initial characterization of a Syap1 knock-out mouse and distribution of Syap1 in mouse brain and cultured motoneurons
Knockout of Syap1 does not modify survival or axonal outgrowth of primary motoneurons. a Quantification of the survival of primary motoneurons cultured for 7 DIV in the absence or presence of BDNF. Cells were counted on day 1 and again on day 7. There is no significant difference in the survival rate of motoneurons from Syap1 knockout (ko) compared to wild-type (WT) embryos [n = 17 (WT) and 7 (ko) independent cultures]. Wild-type cells cultured without BDNF show highly reduced cell survival. b Axon length of primary motoneurons cultured for 7 DIV is not affected by Syap1 knockout (n = 288 for WT and 237 for ko)
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Fig12: Knockout of Syap1 does not modify survival or axonal outgrowth of primary motoneurons. a Quantification of the survival of primary motoneurons cultured for 7 DIV in the absence or presence of BDNF. Cells were counted on day 1 and again on day 7. There is no significant difference in the survival rate of motoneurons from Syap1 knockout (ko) compared to wild-type (WT) embryos [n = 17 (WT) and 7 (ko) independent cultures]. Wild-type cells cultured without BDNF show highly reduced cell survival. b Axon length of primary motoneurons cultured for 7 DIV is not affected by Syap1 knockout (n = 288 for WT and 237 for ko)
Mentions: Primary motoneurons in culture require neurotrophic factors for survival and growth of axons and dendrites. Thus, the role of cell-autonomous factors and their effects on signaling pathways controlling axon elongation and growth cone differentiation can be analyzed using this system (Calof and Reichardt 1984; Forscher and Smith 1988; Arakawa et al. 1990; Sanes and Lichtman 1999; Sendtner et al. 2000). To study the function of Syap1, we therefore investigated the effect of Syap1 knockout on survival and axon growth of cultured embryonic motoneurons (Fig. 12). After 7 days in vitro (DIV), there was no difference in the survival rate of Syap1 knockout in comparison with wild-type motoneurons (P > 0.3), whereas a significant reduction in viability was observed when wild-type or knock-out neurons were cultured without the neurotrophic factor BDNF (P < 0.05) (Fig. 12a; without BDNF: wild-type: 24.9 ± 1.9 % (17); Syap1 knockout: 29.4 ± 2.4 (7); with BDNF: wild-type: 64.6 ± 3.6 % (17); Syap1 knockout: 64.5 ± 5.6 % (7); (in parentheses: numbers of independent cultures). The mean values for axon length (Fig. 12b) also did not differ significantly between the genotypes (axon length wild type: 660.5 ± 16.8 µm; Syap1 knockout: 659.4 ± 19.3 µm; (n = 4 independent cultures; numbers of cells pooled per condition: wild type: N = 288, Syap1 knockout: N = 237). These results indicate that Syap1 does not play a major role in microtubule dynamics or other molecular mechanisms relevant for neurite outgrowth (Paglini et al. 1998; Sendtner et al. 2000; Rossoll et al. 2003; Jablonka et al. 2007; Selvaraj et al. 2012).Fig. 12

View Article: PubMed Central - PubMed

ABSTRACT

Synapse-associated protein 1 (Syap1/BSTA) is the mammalian homologue of Sap47 (synapse-associated protein of 47&nbsp;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 B&alpha;/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