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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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Postsynaptic localization of heparan sulfate in primary  cultures of rat hippocampal neurons. (A–E) Confocal images of  double immunolabeling of hippocampal neurons at 30 DIV for  cell surface heparan sulfate 10E4 (D, green) and presynaptic  marker, synapsin I (E, red). Cell surface heparan sulfate immunoreactivity (green) shows a punctate pattern on the surface of  dendrites similar to synapsin I–positive staining. (B and C) High-  power view reveals close apposition with only partial overlap  (yellow) of immunoreactivities for synapsin I (red) and cell surface heparan sulfate (green), suggestive of synaptic localization  of heparan sulfate. Bars, 20 μm in A and 10 μm in B and C. (F–I)  Double immunolabeling with anti–heparan sulfate (green) and  anti–PSD-95 (red) antibodies in methanol-permeabilized 30 DIV  rat hippocampal neurons. Punctate immunoreactivity for heparan sulfate (green) was colocalized with PSD-95 (red) mostly on  dendrites and partially cell bodies (yellow in F). (G) High-power  view shows colocalization of heparan sulfate (green) and PSD-95  (red) in yellow. Some nonoverlapping immunostaining for heparan sulfate is seen in perikaryon and proximal dendrites due to  intracellular heparan sulfate immunoreactivity, and was not seen  in the case of alive cell surface immunolabeling (D, see Materials  and Methods). Bars, 20 μm in F and 10 μm in G.
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Figure 2: Postsynaptic localization of heparan sulfate in primary cultures of rat hippocampal neurons. (A–E) Confocal images of double immunolabeling of hippocampal neurons at 30 DIV for cell surface heparan sulfate 10E4 (D, green) and presynaptic marker, synapsin I (E, red). Cell surface heparan sulfate immunoreactivity (green) shows a punctate pattern on the surface of dendrites similar to synapsin I–positive staining. (B and C) High- power view reveals close apposition with only partial overlap (yellow) of immunoreactivities for synapsin I (red) and cell surface heparan sulfate (green), suggestive of synaptic localization of heparan sulfate. Bars, 20 μm in A and 10 μm in B and C. (F–I) Double immunolabeling with anti–heparan sulfate (green) and anti–PSD-95 (red) antibodies in methanol-permeabilized 30 DIV rat hippocampal neurons. Punctate immunoreactivity for heparan sulfate (green) was colocalized with PSD-95 (red) mostly on dendrites and partially cell bodies (yellow in F). (G) High-power view shows colocalization of heparan sulfate (green) and PSD-95 (red) in yellow. Some nonoverlapping immunostaining for heparan sulfate is seen in perikaryon and proximal dendrites due to intracellular heparan sulfate immunoreactivity, and was not seen in the case of alive cell surface immunolabeling (D, see Materials and Methods). Bars, 20 μm in F and 10 μm in G.

Mentions: To localize cell surface heparan sulfate immunoreactivity on hippocampal neurons at 30 DIV, cells were first stained alive for heparan sulfate, with subsequent fixation and double immunostaining for synapsin I, a specific marker of presynaptic boutons. Confocal microscopy revealed close apposition of cell surface heparan sulfate and synapsin I immunoreactivities (Fig. 2, A–E). Frequently they showed partial, but not complete, overlap (Fig. 2, B and C). This staining pattern suggests that cell surface heparan sulfate is associated with the synaptic junctions of cultured hippocampal neurons. Double labeling of methanol-fixed 30 DIV hippocampal neurons with antibodies to heparan sulfate and PSD-95 further confirmed the synaptic localization of heparan sulfate. Punctate immunoreactivities of heparan sulfate and PSD-95 colocalized well on dendrites (Fig. 2, F–I). There was some nonoverlapping immunostaining for heparan sulfate in perikaryon and proximal dendrites. This staining was not seen in the case of immunolabeling of live cells (Fig. 2 D), suggesting that some heparan sulfates are associated with intracellular compartments. Double staining for heparan sulfate and either MAP2, a dendritic marker (Fig. 3, A–E), or NCAM (Fig. 3, F–H) further demonstrated a punctate distribution of cell surface heparan sulfate along the dendrites of hippocampal neurons. High-power confocal imaging revealed the localization of heparan sulfate on small protrusions on the shafts of dendrites (Fig. 3, D, E, and H). These results strongly suggest that heparan sulfate is present in dendritic spines. Interestingly, while pyramidal neurons, which comprise the majority of cells in these cultures, had these heparan sulfate-immunoreactive protrusions, we occasionally found neurons that were positive for MAP2 but negative for heparan sulfate (arrows in Fig. 3, A–C). These neurons had smaller cell bodies and more satellite shapes than pyramidal neurons, a morphology consistent with that of local interneurons, that were present in these cultures as a minor population. It has been shown that local interneurons tend to lack dendritic spines (Harris and Kater, 1994). Taken together, these results demonstrate localization of cell surface heparan sulfate to the dendritic spines of hippocampal pyramidal neurons.


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)

Postsynaptic localization of heparan sulfate in primary  cultures of rat hippocampal neurons. (A–E) Confocal images of  double immunolabeling of hippocampal neurons at 30 DIV for  cell surface heparan sulfate 10E4 (D, green) and presynaptic  marker, synapsin I (E, red). Cell surface heparan sulfate immunoreactivity (green) shows a punctate pattern on the surface of  dendrites similar to synapsin I–positive staining. (B and C) High-  power view reveals close apposition with only partial overlap  (yellow) of immunoreactivities for synapsin I (red) and cell surface heparan sulfate (green), suggestive of synaptic localization  of heparan sulfate. Bars, 20 μm in A and 10 μm in B and C. (F–I)  Double immunolabeling with anti–heparan sulfate (green) and  anti–PSD-95 (red) antibodies in methanol-permeabilized 30 DIV  rat hippocampal neurons. Punctate immunoreactivity for heparan sulfate (green) was colocalized with PSD-95 (red) mostly on  dendrites and partially cell bodies (yellow in F). (G) High-power  view shows colocalization of heparan sulfate (green) and PSD-95  (red) in yellow. Some nonoverlapping immunostaining for heparan sulfate is seen in perikaryon and proximal dendrites due to  intracellular heparan sulfate immunoreactivity, and was not seen  in the case of alive cell surface immunolabeling (D, see Materials  and Methods). Bars, 20 μm in F and 10 μm in G.
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Figure 2: Postsynaptic localization of heparan sulfate in primary cultures of rat hippocampal neurons. (A–E) Confocal images of double immunolabeling of hippocampal neurons at 30 DIV for cell surface heparan sulfate 10E4 (D, green) and presynaptic marker, synapsin I (E, red). Cell surface heparan sulfate immunoreactivity (green) shows a punctate pattern on the surface of dendrites similar to synapsin I–positive staining. (B and C) High- power view reveals close apposition with only partial overlap (yellow) of immunoreactivities for synapsin I (red) and cell surface heparan sulfate (green), suggestive of synaptic localization of heparan sulfate. Bars, 20 μm in A and 10 μm in B and C. (F–I) Double immunolabeling with anti–heparan sulfate (green) and anti–PSD-95 (red) antibodies in methanol-permeabilized 30 DIV rat hippocampal neurons. Punctate immunoreactivity for heparan sulfate (green) was colocalized with PSD-95 (red) mostly on dendrites and partially cell bodies (yellow in F). (G) High-power view shows colocalization of heparan sulfate (green) and PSD-95 (red) in yellow. Some nonoverlapping immunostaining for heparan sulfate is seen in perikaryon and proximal dendrites due to intracellular heparan sulfate immunoreactivity, and was not seen in the case of alive cell surface immunolabeling (D, see Materials and Methods). Bars, 20 μm in F and 10 μm in G.
Mentions: To localize cell surface heparan sulfate immunoreactivity on hippocampal neurons at 30 DIV, cells were first stained alive for heparan sulfate, with subsequent fixation and double immunostaining for synapsin I, a specific marker of presynaptic boutons. Confocal microscopy revealed close apposition of cell surface heparan sulfate and synapsin I immunoreactivities (Fig. 2, A–E). Frequently they showed partial, but not complete, overlap (Fig. 2, B and C). This staining pattern suggests that cell surface heparan sulfate is associated with the synaptic junctions of cultured hippocampal neurons. Double labeling of methanol-fixed 30 DIV hippocampal neurons with antibodies to heparan sulfate and PSD-95 further confirmed the synaptic localization of heparan sulfate. Punctate immunoreactivities of heparan sulfate and PSD-95 colocalized well on dendrites (Fig. 2, F–I). There was some nonoverlapping immunostaining for heparan sulfate in perikaryon and proximal dendrites. This staining was not seen in the case of immunolabeling of live cells (Fig. 2 D), suggesting that some heparan sulfates are associated with intracellular compartments. Double staining for heparan sulfate and either MAP2, a dendritic marker (Fig. 3, A–E), or NCAM (Fig. 3, F–H) further demonstrated a punctate distribution of cell surface heparan sulfate along the dendrites of hippocampal neurons. High-power confocal imaging revealed the localization of heparan sulfate on small protrusions on the shafts of dendrites (Fig. 3, D, E, and H). These results strongly suggest that heparan sulfate is present in dendritic spines. Interestingly, while pyramidal neurons, which comprise the majority of cells in these cultures, had these heparan sulfate-immunoreactive protrusions, we occasionally found neurons that were positive for MAP2 but negative for heparan sulfate (arrows in Fig. 3, A–C). These neurons had smaller cell bodies and more satellite shapes than pyramidal neurons, a morphology consistent with that of local interneurons, that were present in these cultures as a minor population. It has been shown that local interneurons tend to lack dendritic spines (Harris and Kater, 1994). Taken together, these results demonstrate localization of cell surface heparan sulfate to the dendritic spines of hippocampal pyramidal neurons.

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