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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.

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Verification of Syap1 knockout and demonstration of Syap1 protein expression in different tissues. a qRT-PCR with mRNA from mouse cortex and primers connecting Syap1 exon-3 and exon-4 demonstrates that intact transcript levels are reduced in the Syap1tm1a mutant (curves3, 4) by a factor of ~30 compared to wild type (curves1, 2). Curve5 indicates background (no reverse transcriptase). b Lysates of the indicated tissues analyzed by Western blots show comparable levels of Syap1 protein expression in all tested brain regions of wild-type mice. However, the protein is also detected in non-neural tissues such as liver (left half of blot). No Syap1 signals are obtained for tissues from Syaptm1a mutant mice (right half of blot). c No trace of the Syap1 protein is detected by Western blots in lysates of hippocampus from Syaptm1a mutant (lane 2) even after increased protein loading and extended exposure. The two weak upper bands are unspecific, and the weak lower bands presumably represent Syap1 degradation products. Top parts of blots in b and c: Calnexin signals as loading controls
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Fig2: Verification of Syap1 knockout and demonstration of Syap1 protein expression in different tissues. a qRT-PCR with mRNA from mouse cortex and primers connecting Syap1 exon-3 and exon-4 demonstrates that intact transcript levels are reduced in the Syap1tm1a mutant (curves3, 4) by a factor of ~30 compared to wild type (curves1, 2). Curve5 indicates background (no reverse transcriptase). b Lysates of the indicated tissues analyzed by Western blots show comparable levels of Syap1 protein expression in all tested brain regions of wild-type mice. However, the protein is also detected in non-neural tissues such as liver (left half of blot). No Syap1 signals are obtained for tissues from Syaptm1a mutant mice (right half of blot). c No trace of the Syap1 protein is detected by Western blots in lysates of hippocampus from Syaptm1a mutant (lane 2) even after increased protein loading and extended exposure. The two weak upper bands are unspecific, and the weak lower bands presumably represent Syap1 degradation products. Top parts of blots in b and c: Calnexin signals as loading controls

Mentions: Heterozygous X-chromosomal Syap1 gene-trap mice (Syap1+/tm1a) in C57BL/6N background were crossed to C57BL/6J mice in order to produce mutant males and females and to reduce non-C57BL/6J genetic background. Hemizygous Syap1tm1a F2 males were crossed to Syap1+/tm1a heterozygote females to obtain homozygous Syap1tm1a/tm1a (Syap1-/-) female animals. Frequencies of offspring genotypes so far were compatible with an assumption of reduced viability of females homozygous for the Syap1tm1a allele (data Table 1). No obvious differences in size or morphology between homozygous, hemizygous, heterozygous mutant, and wild-type littermates of the same sex were noted. The weights of mutant and wild-type males were not significantly different (weight ratio mutant/wild-type 0.996 ± 0.025, 4 age groups, 12 mutant and 12 wild-type animals, P = 0.56). Since the tma1 insertion allele does not delete Syap1 coding sequences (Fig. 1), we determined whether any residual intact Syap1 transcript or protein was generated by exon-3 to exon-4 splicing (ignoring the closer En-2 splice acceptor site (SA) of the tm1a insertion). qRT-PCR of cDNA isolated from wild-type and Syap1-/- mutant brain with primers connecting exon-3 to exon-4 showed a reduction in the mutant of transcripts expressing these exons to 5.68 ± 0.54 % of wild-type levels (n = 6 independent experiments) (Fig. 2a). Transcripts containing exon-8 and exon-9 were reduced in the mutant to 14.8 ± 2.9 % (n = 3). Using an antiserum against Syap1 (characterized in Online Resource 1), we observed in Western blots of tissue lysates from wild type a clear Syap1 signal for all brain areas, for spinal cord, for sciatic nerve, but also for non-neuronal tissues such as diaphragm (Fig. S2a) and liver at the expected apparent relative molecular weight of 56 kDa (Fig. 2b). Corresponding tissue lysates from Syap1-/- mutant mice showed no Syap1 signal. When more protein was loaded and exposure was extended, background signals and Syap1 degradation products showed up in the wild-type lane, but no Syap1 signal was detected in tissues from mutant mice (Fig. 2c). Under these conditions, the wild-type signal of hippocampal homogenate was still detectable when a 1:100 dilution of the lysates was loaded (Fig. S2b). These results demonstrate that Syap1 is abundant in nervous tissue, but it is not a nervous system-specific protein. In view of the observation that in the Syap1 mutants intact Syap1 protein is absent (or reduced to less than 1 % of its normal level) and that in the Syap1–En2 fusion protein more than 80 % of the Syap1 primary structure is missing [including the functionally relevant BSD domain (Doerks et al. 2002; Yao et al. 2013)] (Fig. 1), we conclude that the Syap1tm1a allele may be regarded as a allele on a functional level.Table 1


Initial characterization of a Syap1 knock-out mouse and distribution of Syap1 in mouse brain and cultured motoneurons
Verification of Syap1 knockout and demonstration of Syap1 protein expression in different tissues. a qRT-PCR with mRNA from mouse cortex and primers connecting Syap1 exon-3 and exon-4 demonstrates that intact transcript levels are reduced in the Syap1tm1a mutant (curves3, 4) by a factor of ~30 compared to wild type (curves1, 2). Curve5 indicates background (no reverse transcriptase). b Lysates of the indicated tissues analyzed by Western blots show comparable levels of Syap1 protein expression in all tested brain regions of wild-type mice. However, the protein is also detected in non-neural tissues such as liver (left half of blot). No Syap1 signals are obtained for tissues from Syaptm1a mutant mice (right half of blot). c No trace of the Syap1 protein is detected by Western blots in lysates of hippocampus from Syaptm1a mutant (lane 2) even after increased protein loading and extended exposure. The two weak upper bands are unspecific, and the weak lower bands presumably represent Syap1 degradation products. Top parts of blots in b and c: Calnexin signals as loading controls
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Fig2: Verification of Syap1 knockout and demonstration of Syap1 protein expression in different tissues. a qRT-PCR with mRNA from mouse cortex and primers connecting Syap1 exon-3 and exon-4 demonstrates that intact transcript levels are reduced in the Syap1tm1a mutant (curves3, 4) by a factor of ~30 compared to wild type (curves1, 2). Curve5 indicates background (no reverse transcriptase). b Lysates of the indicated tissues analyzed by Western blots show comparable levels of Syap1 protein expression in all tested brain regions of wild-type mice. However, the protein is also detected in non-neural tissues such as liver (left half of blot). No Syap1 signals are obtained for tissues from Syaptm1a mutant mice (right half of blot). c No trace of the Syap1 protein is detected by Western blots in lysates of hippocampus from Syaptm1a mutant (lane 2) even after increased protein loading and extended exposure. The two weak upper bands are unspecific, and the weak lower bands presumably represent Syap1 degradation products. Top parts of blots in b and c: Calnexin signals as loading controls
Mentions: Heterozygous X-chromosomal Syap1 gene-trap mice (Syap1+/tm1a) in C57BL/6N background were crossed to C57BL/6J mice in order to produce mutant males and females and to reduce non-C57BL/6J genetic background. Hemizygous Syap1tm1a F2 males were crossed to Syap1+/tm1a heterozygote females to obtain homozygous Syap1tm1a/tm1a (Syap1-/-) female animals. Frequencies of offspring genotypes so far were compatible with an assumption of reduced viability of females homozygous for the Syap1tm1a allele (data Table 1). No obvious differences in size or morphology between homozygous, hemizygous, heterozygous mutant, and wild-type littermates of the same sex were noted. The weights of mutant and wild-type males were not significantly different (weight ratio mutant/wild-type 0.996 ± 0.025, 4 age groups, 12 mutant and 12 wild-type animals, P = 0.56). Since the tma1 insertion allele does not delete Syap1 coding sequences (Fig. 1), we determined whether any residual intact Syap1 transcript or protein was generated by exon-3 to exon-4 splicing (ignoring the closer En-2 splice acceptor site (SA) of the tm1a insertion). qRT-PCR of cDNA isolated from wild-type and Syap1-/- mutant brain with primers connecting exon-3 to exon-4 showed a reduction in the mutant of transcripts expressing these exons to 5.68 ± 0.54 % of wild-type levels (n = 6 independent experiments) (Fig. 2a). Transcripts containing exon-8 and exon-9 were reduced in the mutant to 14.8 ± 2.9 % (n = 3). Using an antiserum against Syap1 (characterized in Online Resource 1), we observed in Western blots of tissue lysates from wild type a clear Syap1 signal for all brain areas, for spinal cord, for sciatic nerve, but also for non-neuronal tissues such as diaphragm (Fig. S2a) and liver at the expected apparent relative molecular weight of 56 kDa (Fig. 2b). Corresponding tissue lysates from Syap1-/- mutant mice showed no Syap1 signal. When more protein was loaded and exposure was extended, background signals and Syap1 degradation products showed up in the wild-type lane, but no Syap1 signal was detected in tissues from mutant mice (Fig. 2c). Under these conditions, the wild-type signal of hippocampal homogenate was still detectable when a 1:100 dilution of the lysates was loaded (Fig. S2b). These results demonstrate that Syap1 is abundant in nervous tissue, but it is not a nervous system-specific protein. In view of the observation that in the Syap1 mutants intact Syap1 protein is absent (or reduced to less than 1 % of its normal level) and that in the Syap1–En2 fusion protein more than 80 % of the Syap1 primary structure is missing [including the functionally relevant BSD domain (Doerks et al. 2002; Yao et al. 2013)] (Fig. 1), we conclude that the Syap1tm1a allele may be regarded as a allele on a functional level.Table 1

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