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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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The expression of heparan  sulfate on dendritic spines of cultured  hippocampal neurons. (A–E) Double  immunostaining of hippocampal neurons at 30 DIV with anti-MAP2 (red)  and the 10E4 anti–heparan sulfate  (green) antibodies. Cell surface heparan sulfate immunoreactivity is detected on the cell bodies and along  dendrites of the pyramidal neurons. Interneurons occasionally present in  these cultures are not stained for heparan sulfate (arrows). (D and E) High-power confocal image shows cell surface heparan sulfate immunoreactivity  (yellow) as a puncta on the surface of  MAP2-positive dendrites (red), in patterns and shapes reminiscent of dendritic spines. Bar, 20 μm in A and 10  μm in D and E. (F–H) Double immunostaining of 30 DIV hippocampal  neurons with anti-NCAM (red) and  anti–heparan sulfate 10E4 (green) antibodies. Heparan sulfate immunoreactivity is detected on dendritic spines,  which appear as small protrusions on  dendritic shafts. Bar, 10 μm.
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Figure 3: The expression of heparan sulfate on dendritic spines of cultured hippocampal neurons. (A–E) Double immunostaining of hippocampal neurons at 30 DIV with anti-MAP2 (red) and the 10E4 anti–heparan sulfate (green) antibodies. Cell surface heparan sulfate immunoreactivity is detected on the cell bodies and along dendrites of the pyramidal neurons. Interneurons occasionally present in these cultures are not stained for heparan sulfate (arrows). (D and E) High-power confocal image shows cell surface heparan sulfate immunoreactivity (yellow) as a puncta on the surface of MAP2-positive dendrites (red), in patterns and shapes reminiscent of dendritic spines. Bar, 20 μm in A and 10 μm in D and E. (F–H) Double immunostaining of 30 DIV hippocampal neurons with anti-NCAM (red) and anti–heparan sulfate 10E4 (green) antibodies. Heparan sulfate immunoreactivity is detected on dendritic spines, which appear as small protrusions on dendritic shafts. Bar, 10 μm.

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)

The expression of heparan  sulfate on dendritic spines of cultured  hippocampal neurons. (A–E) Double  immunostaining of hippocampal neurons at 30 DIV with anti-MAP2 (red)  and the 10E4 anti–heparan sulfate  (green) antibodies. Cell surface heparan sulfate immunoreactivity is detected on the cell bodies and along  dendrites of the pyramidal neurons. Interneurons occasionally present in  these cultures are not stained for heparan sulfate (arrows). (D and E) High-power confocal image shows cell surface heparan sulfate immunoreactivity  (yellow) as a puncta on the surface of  MAP2-positive dendrites (red), in patterns and shapes reminiscent of dendritic spines. Bar, 20 μm in A and 10  μm in D and E. (F–H) Double immunostaining of 30 DIV hippocampal  neurons with anti-NCAM (red) and  anti–heparan sulfate 10E4 (green) antibodies. Heparan sulfate immunoreactivity is detected on dendritic spines,  which appear as small protrusions on  dendritic shafts. Bar, 10 μm.
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Figure 3: The expression of heparan sulfate on dendritic spines of cultured hippocampal neurons. (A–E) Double immunostaining of hippocampal neurons at 30 DIV with anti-MAP2 (red) and the 10E4 anti–heparan sulfate (green) antibodies. Cell surface heparan sulfate immunoreactivity is detected on the cell bodies and along dendrites of the pyramidal neurons. Interneurons occasionally present in these cultures are not stained for heparan sulfate (arrows). (D and E) High-power confocal image shows cell surface heparan sulfate immunoreactivity (yellow) as a puncta on the surface of MAP2-positive dendrites (red), in patterns and shapes reminiscent of dendritic spines. Bar, 20 μm in A and 10 μm in D and E. (F–H) Double immunostaining of 30 DIV hippocampal neurons with anti-NCAM (red) and anti–heparan sulfate 10E4 (green) antibodies. Heparan sulfate immunoreactivity is detected on dendritic spines, which appear as small protrusions on dendritic shafts. Bar, 10 μm.
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