Limits...
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.


Syap1 accumulates close to the Golgi organelle. (a–d) Perinuclear Syap1 immunoreactivity partially co-stains with the cis/medial-Golgi marker GM130 (a, c) and with γ-adaptin, a component of the adapter protein complex 1 (b, d) (maximum intensity projections). Blue nuclear DAPI staining. (e–h) Super-resolution imaging by SIM of Syap1 and GM130 or γ-adaptin. Typical single optical sections are shown. (e, g) Syap1 and GM130 distribution, (f, h) Syap1 and γ-adaptin distribution. Orange arrowheads point to co-occurrence of both labels, and white arrowheads indicate Syap1-positive, GM130- or γ-adaptin-negative signals. Scale barsa, b, e, f 10 µm; c, d 5 µm; g, h 2.5 µm
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5037158&req=5

Fig11: Syap1 accumulates close to the Golgi organelle. (a–d) Perinuclear Syap1 immunoreactivity partially co-stains with the cis/medial-Golgi marker GM130 (a, c) and with γ-adaptin, a component of the adapter protein complex 1 (b, d) (maximum intensity projections). Blue nuclear DAPI staining. (e–h) Super-resolution imaging by SIM of Syap1 and GM130 or γ-adaptin. Typical single optical sections are shown. (e, g) Syap1 and GM130 distribution, (f, h) Syap1 and γ-adaptin distribution. Orange arrowheads point to co-occurrence of both labels, and white arrowheads indicate Syap1-positive, GM130- or γ-adaptin-negative signals. Scale barsa, b, e, f 10 µm; c, d 5 µm; g, h 2.5 µm

Mentions: In order to further define the subcellular localization of Syap1 in the cultured motoneurons, co-staining with antibodies against the cis/medial-Golgi apparatus marker GM130, and against γ-adaptin, a component of the adaptor protein complex 1 (AP1) particularly accumulated in the trans-Golgi network (Boehm and Bonifacino 2001), was performed (Fig. 11). Syap1 staining was found concentrated next to the nucleus in close proximity to the cis/medial-Golgi label (Fig. 11c, maximum intensity projections; orange arrowheads point at co-occurrence of both signals, while white arrowheads mark Syap1-positive, GM130-negative spots). Analyses of confocal optical sections revealed an average Pearson’s correlation coefficient after Costes’ randomization (PCC) of 0.66 ± 0.01 (pooled data of N = 50 cells, 4 independent cultures), indicating a non-random correlation between both immunoreactive signals. Syap1 and γ-adaptin IR (Fig. 11d) showed a PCC of 0.72 ± 0.01 (pooled data of N = 35 cells, 5 independent cultures). Confocal microscopy thus indicates a pronounced localization of Syap1 to the perinuclear Golgi apparatus, confirming observations in the brain sections. To examine the localization of Syap1 at even higher resolution, structured illumination microscopy (SIM) was performed. SIM is a super-resolution imaging approach using standard fluorescent probes as immunocytochemical tags and offers here an x–y-resolution in the range of 120 nm (see Schermelleh et al. 2010). SIM microscopy again showed a fine-structured, punctate distribution of Syap1 with a strong accumulation in the perinuclear Golgi region and a punctate staining throughout the cytoplasm. SIM optical sections reveal a close proximity to GM130 in the perinuclear area, but only a rare co-occurrence of both labels. Syap1 immunoreactivity did not follow the typical cisternae-like label of the cis/medial-Golgi marker GM130 (Fig. 11e, g; orange arrowheads point to co-occurrence of both signals, while white arrowheads point to Syap1-positive, GM130-negative labels). Similarly, SIM images reveal an even less frequent overlap of Syap1- and γ-adaptin IR (Fig. 11f, h; yellow arrowheads point to co-occurrence of both signals, while white arrowheads point to Syap1-positive, γ-adaptin-negative labels).Fig. 11


Initial characterization of a Syap1 knock-out mouse and distribution of Syap1 in mouse brain and cultured motoneurons
Syap1 accumulates close to the Golgi organelle. (a–d) Perinuclear Syap1 immunoreactivity partially co-stains with the cis/medial-Golgi marker GM130 (a, c) and with γ-adaptin, a component of the adapter protein complex 1 (b, d) (maximum intensity projections). Blue nuclear DAPI staining. (e–h) Super-resolution imaging by SIM of Syap1 and GM130 or γ-adaptin. Typical single optical sections are shown. (e, g) Syap1 and GM130 distribution, (f, h) Syap1 and γ-adaptin distribution. Orange arrowheads point to co-occurrence of both labels, and white arrowheads indicate Syap1-positive, GM130- or γ-adaptin-negative signals. Scale barsa, b, e, f 10 µm; c, d 5 µm; g, h 2.5 µm
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5037158&req=5

Fig11: Syap1 accumulates close to the Golgi organelle. (a–d) Perinuclear Syap1 immunoreactivity partially co-stains with the cis/medial-Golgi marker GM130 (a, c) and with γ-adaptin, a component of the adapter protein complex 1 (b, d) (maximum intensity projections). Blue nuclear DAPI staining. (e–h) Super-resolution imaging by SIM of Syap1 and GM130 or γ-adaptin. Typical single optical sections are shown. (e, g) Syap1 and GM130 distribution, (f, h) Syap1 and γ-adaptin distribution. Orange arrowheads point to co-occurrence of both labels, and white arrowheads indicate Syap1-positive, GM130- or γ-adaptin-negative signals. Scale barsa, b, e, f 10 µm; c, d 5 µm; g, h 2.5 µm
Mentions: In order to further define the subcellular localization of Syap1 in the cultured motoneurons, co-staining with antibodies against the cis/medial-Golgi apparatus marker GM130, and against γ-adaptin, a component of the adaptor protein complex 1 (AP1) particularly accumulated in the trans-Golgi network (Boehm and Bonifacino 2001), was performed (Fig. 11). Syap1 staining was found concentrated next to the nucleus in close proximity to the cis/medial-Golgi label (Fig. 11c, maximum intensity projections; orange arrowheads point at co-occurrence of both signals, while white arrowheads mark Syap1-positive, GM130-negative spots). Analyses of confocal optical sections revealed an average Pearson’s correlation coefficient after Costes’ randomization (PCC) of 0.66 ± 0.01 (pooled data of N = 50 cells, 4 independent cultures), indicating a non-random correlation between both immunoreactive signals. Syap1 and γ-adaptin IR (Fig. 11d) showed a PCC of 0.72 ± 0.01 (pooled data of N = 35 cells, 5 independent cultures). Confocal microscopy thus indicates a pronounced localization of Syap1 to the perinuclear Golgi apparatus, confirming observations in the brain sections. To examine the localization of Syap1 at even higher resolution, structured illumination microscopy (SIM) was performed. SIM is a super-resolution imaging approach using standard fluorescent probes as immunocytochemical tags and offers here an x–y-resolution in the range of 120 nm (see Schermelleh et al. 2010). SIM microscopy again showed a fine-structured, punctate distribution of Syap1 with a strong accumulation in the perinuclear Golgi region and a punctate staining throughout the cytoplasm. SIM optical sections reveal a close proximity to GM130 in the perinuclear area, but only a rare co-occurrence of both labels. Syap1 immunoreactivity did not follow the typical cisternae-like label of the cis/medial-Golgi marker GM130 (Fig. 11e, g; orange arrowheads point to co-occurrence of both signals, while white arrowheads point to Syap1-positive, GM130-negative labels). Similarly, SIM images reveal an even less frequent overlap of Syap1- and γ-adaptin IR (Fig. 11f, h; yellow arrowheads point to co-occurrence of both signals, while white arrowheads point to Syap1-positive, γ-adaptin-negative labels).Fig. 11

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.