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Cell surface heparan sulfate proteoglycan syndecan-2 induces the maturation of dendritic spines in rat hippocampal neurons.

Ethell IM, Yamaguchi Y - J. Cell Biol. (1999)

Bottom Line: We demonstrate that the cell surface heparan sulfate proteoglycan syndecan-2 plays a critical role in spine development.Deletion of the COOH-terminal EFYA motif of syndecan-2, the binding site for PDZ domain proteins, abrogates the spine-promoting activity of syndecan-2.Our results indicate that syndecan-2 plays a direct role in the development of postsynaptic specialization through its interactions with PDZ domain proteins.

View Article: PubMed Central - PubMed

Affiliation: The Burnham Institute, La Jolla, California 92037, USA.

ABSTRACT
Dendritic spines are small protrusions that receive synapses, and changes in spine morphology are thought to be the structural basis for learning and memory. We demonstrate that the cell surface heparan sulfate proteoglycan syndecan-2 plays a critical role in spine development. Syndecan-2 is concentrated at the synapses, specifically on the dendritic spines of cultured hippocampal neurons, and its accumulation occurs concomitant with the morphological maturation of spines from long thin protrusions to stubby and headed shapes. Early introduction of syndecan-2 cDNA into immature hippocampal neurons, by transient transfection, accelerates spine formation from dendritic protrusions. Deletion of the COOH-terminal EFYA motif of syndecan-2, the binding site for PDZ domain proteins, abrogates the spine-promoting activity of syndecan-2. Syndecan-2 clustering on dendritic protrusions does not require the PDZ domain-binding motif, but another portion of the cytoplasmic domain which includes a protein kinase C phosphorylation site. Our results indicate that syndecan-2 plays a direct role in the development of postsynaptic specialization through its interactions with PDZ domain proteins.

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Cytoplasmic domain  of syndecan-2, but not COOH-terminal PDZ binding motif, is  required for the targeting of  syndecan-2 to dendritic spines.  Hippocampal neurons were  transfected with full-length syndecan-2 (A), with the syndecan-2 ΔEFYA deletion mutant  (B), or with the syndecan-2  Δcyto mutant (C). Neurons  were cotransfected with GFP as  in Fig. 6. Cells were analyzed 7 d  after transfection by confocal  microscopy after immunostaining with anti–syndecan-2 antibodies which recognize the  extracellular domain of syndecan-2 (red). Both full-length  syndecan-2 (A) and the syndecan-2 ΔEFYA deletion mutant  (B) are sorted and expressed on  dendritic spines/protrusions. In  contrast, the syndecan-2 Δcyto  deletion mutant does not show  any specific sorting, being distributed diffusely on entire neuronal surfaces (C). Note: correct  cell surface folding of syndecan-2  Δcyto deletion mutant was confirmed (see Results). Bars, 20 μm.
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Figure 7: Cytoplasmic domain of syndecan-2, but not COOH-terminal PDZ binding motif, is required for the targeting of syndecan-2 to dendritic spines. Hippocampal neurons were transfected with full-length syndecan-2 (A), with the syndecan-2 ΔEFYA deletion mutant (B), or with the syndecan-2 Δcyto mutant (C). Neurons were cotransfected with GFP as in Fig. 6. Cells were analyzed 7 d after transfection by confocal microscopy after immunostaining with anti–syndecan-2 antibodies which recognize the extracellular domain of syndecan-2 (red). Both full-length syndecan-2 (A) and the syndecan-2 ΔEFYA deletion mutant (B) are sorted and expressed on dendritic spines/protrusions. In contrast, the syndecan-2 Δcyto deletion mutant does not show any specific sorting, being distributed diffusely on entire neuronal surfaces (C). Note: correct cell surface folding of syndecan-2 Δcyto deletion mutant was confirmed (see Results). Bars, 20 μm.

Mentions: Interestingly, although deletion of the PDZ domain binding motif abrogated the ability of syndecan-2 to induce spine maturation, it did not affect the targeting of syndecan-2 into dendritic protrusions. Syndecan-2 that lacked the EFYA motif was still sorted to dendrites and showed a punctate distribution (Fig. 7 B), similar to that of full-length syndecan-2 (Fig. 7 A). High-power views revealed the clustering of truncated syndecan-2 on dendritic protrusions that had immature morphologies (Fig. 6 B). In some instances, clusters of truncated syndecan-2 were found at the tips of the protrusions. Quantitative analysis showed that the numbers of syndecan-2 clusters in dendrites were essentially the same between full-length syndecan-2 and syndecan-2 ΔEFYA transfected neurons (Table II). However, clustering of syndecan-2 in dendritic protrusions was completely abolished in neurons transfected with another syndecan-2 mutant that lacked most of the cytoplasmic domain, except three juxtamembrane amino acids (syndecan-2 Δcyto). This three amino acid tail was left to ensure correct folding of the deletion mutant. A similar strategy was used by Carey et al. (1996) with a syndecan-1 deletion mutant that shares 100% homology with other syndecans in transmembrane and juxtamembrane cytoplasmic domains. Cell surface anchoring of our mutant was also confirmed by confocal microscopy in Z-plane (data not shown). Syndecan-2 Δcyto was diffusely distributed on the surface of neurons without any specific sorting pattern (Fig. 7 C). Together, these results demonstrate that the clustering of syndecan-2 on the dendritic protrusions is mediated, at least in part, by the cytoplasmic domain, but is independent of interactions with PDZ domain proteins, suggesting that a part of the cytoplasmic domain other than the EFYA motif is involved in the targeting of syndecan-2 to dendritic protrusions.


Cell surface heparan sulfate proteoglycan syndecan-2 induces the maturation of dendritic spines in rat hippocampal neurons.

Ethell IM, Yamaguchi Y - J. Cell Biol. (1999)

Cytoplasmic domain  of syndecan-2, but not COOH-terminal PDZ binding motif, is  required for the targeting of  syndecan-2 to dendritic spines.  Hippocampal neurons were  transfected with full-length syndecan-2 (A), with the syndecan-2 ΔEFYA deletion mutant  (B), or with the syndecan-2  Δcyto mutant (C). Neurons  were cotransfected with GFP as  in Fig. 6. Cells were analyzed 7 d  after transfection by confocal  microscopy after immunostaining with anti–syndecan-2 antibodies which recognize the  extracellular domain of syndecan-2 (red). Both full-length  syndecan-2 (A) and the syndecan-2 ΔEFYA deletion mutant  (B) are sorted and expressed on  dendritic spines/protrusions. In  contrast, the syndecan-2 Δcyto  deletion mutant does not show  any specific sorting, being distributed diffusely on entire neuronal surfaces (C). Note: correct  cell surface folding of syndecan-2  Δcyto deletion mutant was confirmed (see Results). Bars, 20 μm.
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Related In: Results  -  Collection

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Figure 7: Cytoplasmic domain of syndecan-2, but not COOH-terminal PDZ binding motif, is required for the targeting of syndecan-2 to dendritic spines. Hippocampal neurons were transfected with full-length syndecan-2 (A), with the syndecan-2 ΔEFYA deletion mutant (B), or with the syndecan-2 Δcyto mutant (C). Neurons were cotransfected with GFP as in Fig. 6. Cells were analyzed 7 d after transfection by confocal microscopy after immunostaining with anti–syndecan-2 antibodies which recognize the extracellular domain of syndecan-2 (red). Both full-length syndecan-2 (A) and the syndecan-2 ΔEFYA deletion mutant (B) are sorted and expressed on dendritic spines/protrusions. In contrast, the syndecan-2 Δcyto deletion mutant does not show any specific sorting, being distributed diffusely on entire neuronal surfaces (C). Note: correct cell surface folding of syndecan-2 Δcyto deletion mutant was confirmed (see Results). Bars, 20 μm.
Mentions: Interestingly, although deletion of the PDZ domain binding motif abrogated the ability of syndecan-2 to induce spine maturation, it did not affect the targeting of syndecan-2 into dendritic protrusions. Syndecan-2 that lacked the EFYA motif was still sorted to dendrites and showed a punctate distribution (Fig. 7 B), similar to that of full-length syndecan-2 (Fig. 7 A). High-power views revealed the clustering of truncated syndecan-2 on dendritic protrusions that had immature morphologies (Fig. 6 B). In some instances, clusters of truncated syndecan-2 were found at the tips of the protrusions. Quantitative analysis showed that the numbers of syndecan-2 clusters in dendrites were essentially the same between full-length syndecan-2 and syndecan-2 ΔEFYA transfected neurons (Table II). However, clustering of syndecan-2 in dendritic protrusions was completely abolished in neurons transfected with another syndecan-2 mutant that lacked most of the cytoplasmic domain, except three juxtamembrane amino acids (syndecan-2 Δcyto). This three amino acid tail was left to ensure correct folding of the deletion mutant. A similar strategy was used by Carey et al. (1996) with a syndecan-1 deletion mutant that shares 100% homology with other syndecans in transmembrane and juxtamembrane cytoplasmic domains. Cell surface anchoring of our mutant was also confirmed by confocal microscopy in Z-plane (data not shown). Syndecan-2 Δcyto was diffusely distributed on the surface of neurons without any specific sorting pattern (Fig. 7 C). Together, these results demonstrate that the clustering of syndecan-2 on the dendritic protrusions is mediated, at least in part, by the cytoplasmic domain, but is independent of interactions with PDZ domain proteins, suggesting that a part of the cytoplasmic domain other than the EFYA motif is involved in the targeting of syndecan-2 to dendritic protrusions.

Bottom Line: We demonstrate that the cell surface heparan sulfate proteoglycan syndecan-2 plays a critical role in spine development.Deletion of the COOH-terminal EFYA motif of syndecan-2, the binding site for PDZ domain proteins, abrogates the spine-promoting activity of syndecan-2.Our results indicate that syndecan-2 plays a direct role in the development of postsynaptic specialization through its interactions with PDZ domain proteins.

View Article: PubMed Central - PubMed

Affiliation: The Burnham Institute, La Jolla, California 92037, USA.

ABSTRACT
Dendritic spines are small protrusions that receive synapses, and changes in spine morphology are thought to be the structural basis for learning and memory. We demonstrate that the cell surface heparan sulfate proteoglycan syndecan-2 plays a critical role in spine development. Syndecan-2 is concentrated at the synapses, specifically on the dendritic spines of cultured hippocampal neurons, and its accumulation occurs concomitant with the morphological maturation of spines from long thin protrusions to stubby and headed shapes. Early introduction of syndecan-2 cDNA into immature hippocampal neurons, by transient transfection, accelerates spine formation from dendritic protrusions. Deletion of the COOH-terminal EFYA motif of syndecan-2, the binding site for PDZ domain proteins, abrogates the spine-promoting activity of syndecan-2. Syndecan-2 clustering on dendritic protrusions does not require the PDZ domain-binding motif, but another portion of the cytoplasmic domain which includes a protein kinase C phosphorylation site. Our results indicate that syndecan-2 plays a direct role in the development of postsynaptic specialization through its interactions with PDZ domain proteins.

Show MeSH
Related in: MedlinePlus