Reg1ulatory role and molecular interactions of a cell-surface heparan sulfate proteoglycan (N-syndecan) in hippocampal long-term potentiation

Abstract

The cellular mechanisms responsible for synaptic plasticity involve interactions between neurons and the extracellular matrix. Heparan sulfates (HSs) constitute a group of glycosaminoglycans that accumulate in the beta-amyloid deposits in Alzheimer's disease and influence the development of neuron-target contacts by interacting with other cell surface and matrix molecules. However, the contribution of HSs to brain function is unknown. We found that HSs play a crucial role in long-term potentiation (LTP), a finding that is consistent with the idea that converging molecular mechanisms are used in the development of neuron-target contacts and in activity-induced synaptic plasticity in adults. Enzymatic cleavage of HS by heparitinase as well as addition of soluble heparin-type carbohydrates prevented expression of LTP in response to 100 Hz/1 sec stimulation of Schaffer collaterals in rat hippocampal slices. A prominent carrier protein for the type of glycans implicated in LTP regulation in the adult hippocampus was identified as N-syndecan (syndecan-3), a transmembrane proteoglycan that was expressed at the processes of the CA1 pyramidal neurons in an activity-dependent manner. Addition of soluble N-syndecan into the CA1 dendritic area prevented tetanus-induced LTP. A major substrate of src-type kinases, cortactin (p80/85), and the tyrosine kinase fyn copurified with N-syndecan from hippocampus. Moreover, association of both cortactin and fyn to N-syndecan was rapidly increased after induction of LTP. N-syndecan may thus act as an important regulator in the activity-dependent modulation of neuronal connectivity by transmitting signals between extracellular heparin-binding factors and the fyn signaling pathway.

Fig. 1.

Enzymatic cleavage of heparan sulfate prevents LTP but has no effect on single pulse-evoked synaptic responses in the area CA1 of hippocampal slices. A, Effect of HFS on the fEPSP slope in rat hippocampal slices (300 μm) preincubated with heparitinase–0.2% BSA (20 U/ml; volume, 500 μl; 3 hr; +24°C) (•) or 0.2% BSA only (○) (average ± SEM;n = 7 on both groups; p < 0.01; Student’s t test). fEPSP traces before and 30 min after the HFS are shown superimposed on the right.B, Slopes of fEPSPs plotted as a function of presynaptic fiber volley (psfv) amplitude show the lack of effect of heparitinase on baseline synaptic responses. Data were obtained from five heparitinase-treated (•) and five control (○) slices (average ± SEM shown).

Fig. 2.

Heparin prevents LTP in a manner dependent on the sulfation pattern of the glycan, but has no effect on pharmacologically isolated NMDA receptor-mediated responses. A, Effect of heparin (n = 8) and chondroitin sulfate (CS) (n = 7) on LTP. Both glycans were bath-applied at the concentration of 100 ng/ml as shown by thebar. Top traces show sample fEPSPs before (a) and after (b) application of the glycan, and 60 min after HFS (c). B, Pooled data showing the effect of selectively 2-O-desulfated (2-O-ds) and 6-O-desulfated (6-O-ds) heparins on LTP. Because the desulfated glycans were available in low amounts, these experiments were performed in static perfusion (volume, 1 ml). The values represent fEPSP slope (% from control ± SEM;n = 4) 30 min after the HFS (*p< 0.01; one-way ANOVA with Tukey post hoc comparison).C, Averaged amplitude of NMDA receptor-mediated responses recorded under current clamp from CA1 pyramidal neurones in the presence of 10 μm NBQX and 100 μm PiTX. Application of heparin (0.5 μg/ml) (shown by the bar) did not affect the amplitude of these responses (n= 3) at the resting membrane potential. The lack of effect of heparin on the voltage dependence of NMDA responses is shown inD.

Fig. 3.

N-syndecan is isolated as the major HB-GAM-binding HSPG from adult hippocampus. A, Adult rat hippocampi (10 gm wet tissue) were solubilized in octyl glucoside and fractionated by salt gradient elution on HB-GAM–Sepharose. Alcian blue–silver staining was used to detect both proteins and proteoglycans, and it revealed a proteoglycan-type smear in fractions eluting at 0.4–0.6 m NaCl (fractions 9–13). The figure shows a Western blot of fractions deglycosylated by nitrous acid. The immunoblot was stained with affinity-purified antibodies againstN-syndecan. B, Western blot of the hippocampal fractions with anti-N-syndecan antibodies showing that heparitinase digestion reduces the 200 kDa proteoglycan (lane 1) to a 120 kDa core protein (lane 2).

Fig. 4.

Effect of soluble N-syndecan on LTP. After a stable baseline recording, N-syndecan (20 μg/ml) (•) was pressure-injected into the CA1 dendritic area close to the recording site (arrow). Control injections were performed with saline (○). LTP was induced by HFS 10 min after the injection. The data represents the average ± SEM of six experiments (p < 0.05; Student’st test). Sample responses before (a) and after (b) injection, and 30 min after high-frequency stimulation (c) are shown.

Fig. 5.

Expression of N-syndecan in adult rat hippocampus. In situ hybridization with antisense (A) and sense (B)N-syndecan RNA probes in hippocampal sections. Light microscopic (C) and electron microscopic (D) visualization of N-syndecan immunoreactivity in CA3 stratum radiatum. In (D), the arrows point to anti-N-syndecan immunoperoxidase labeling at the surface of neuronal processes (7500× magnification). Parallel control stainings with nonimmune rabbit IgG showed no reactivity (data not shown).

Fig. 6.

Changes in the expression ofN-syndecan after LTP induction. In vivorecording showing the effect of 100 Hz/sec Schaffer collateral stimulation on the fEPSP slope. Sample fEPSPs before and 1 hr after the HFS are shown on the right (A).In situ hybridization with antisenseN-syndecan probes in hippocampus of a control animal (B) and after induction of LTP (C). Note that these sections are not comparable to Figure 5 because of different duration of color development. Light microscopic pictures from the area CA1 showing a low level ofN-syndecan immunoreactivity in control animals (D) and the enhanced staining of neuronal fibers after HFS (E). Electron microscopic micrograph showing N-syndecan immunostaining at the periphery of presynaptic and postsynaptic structures (arrows) in the CA1 dendritic area of a stimulated hippocampus (F) (15,000× magnification).

Fig. 7.

Hippocampal N-syndecan copurifies with cortactin and the tyrosine kinase fyn.A, Western blot of the HB-GAM affinity-purified hippocampal extracts showing copurification ofN-syndecan, cortactin, and an src family kinase to fractions 9–13. The polyclonal antibody against src family (SRC-2) recognizes fyn, c-src, and yes-kinases. B, Immunoblotting of the fractions with monoclonal antibodies detects fyn but no other src family kinases in the N-syndecan containing hippocampal fractions.

Fig. 8.

Association between N-syndecan and cortactin/fyn is increased by an LTP-inducing HFS. A, Western blot from HB-GAM–Sepharose-precipitated extracts from the area CA1 of hippocampal slices, which have been maintained under control conditions or in which LTP has been induced by HFS.N-syndecan and components associated to it were displaced from HB-GAM–Sepharose by heparin (10 μg/ml). The amount of cortactin was markedly increased after 20 min but already increased 10 min after HFS. An increase in the amount ofN-syndecan-associated fyn was also detected.B, Quantification of N-syndecan, cortactin, and fyn from immunoblots of four independent experiments.N-syndecan was detected similarly in the samples from control and stimulated slices (*p < 0.05; Student’s t test).

Fig. 9.

A schematic picture depicting interactions ofN-syndecan with intracellular and extracellular molecules in the regulation of neuronal plasticity.