2 Department of Physiology, Yamagata University School of Medicine, Yamagata 990-9585, Japan; 3 Department of Biomembrane and Biofunctional Chemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan; and 4 Department of Membrane Biochemistry, Tokyo Metropolitan Institute for Gerontology, Sacaecho, Itabashi-ku, Tokyo 173-0015, Japan
Received on December 3, 2001; revised on January 23, 2002; accepted on January 28, 2002.
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Abstract |
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Key words: ATP/ecto-protein kinase/GM1 and GQ1b/hippocampus/long-term potentiation
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Introduction |
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The reversible phosphorylation of intracellular proteins is a key regulatory mechanism in activity-dependent LTP induction in these cells (Bliss and Collingridge, 1993) and involves the transfer by the enzyme protein kinase of the gamma-phosphate of ATP to form a covalent bond with specific proteins. Another line of investigation has provided evidence for the existence of ecto-protein kinases on the surface of neuronal cells and shown how the regulatory effects of protein phosphorylation systems in neuronal signal transduction can extend to the extracellular environment (Ehrlich et al., 1986
, 1988; Chen et al., 1996
). We have previously shown that ATP, released by HFS, can act as an orthophosphate donor for the extracellular phosphorylation of synaptic membrane proteins during LTP induction (Fujii et al., 1995b
, 2000).
In the central nervous systems, gangliosides (sialic acidcontaining glycosphingolipids) are involved in many physiological functions, such as neurogenesis, proliferation, synaptogenesis, and synaptic transmission. In hippocampal CA1 neurons, activity-dependent LTP is enhanced by addition of the monosialoganglioside GM1 or by an increase in endogenous GM1 levels as a result of enzymatic treatment (Wieraszko and Seifert, 1985). Preincubation of hippocampal slices for 1 h with GM1 enhances HFS-induced LTP in medium containing normal (2.5 mM), low (1.0 mM), or high (5.0 mM) concentrations of Ca2+ (Hwang et al., 1992
). Furthermore, LTP induction is enhanced in normal (2.5 mM) Ca2+ medium by application of either GM1 or the tetrasialoganglioside GQ1b during HFS, or in low (1.01.1 mM) Ca2+ medium during HFS by incubation of slices with either GM1 or GQ1b, whereas the blocking effect of an NMDA receptor antagonist on LTP induction in normal Ca2+ medium is reversed by preincubation of slices with GQ1b (Furuse et al., 1998
). These results suggest that in hippocampal CA1 neurons, gangliosides participate in activity-dependent LTP through the modulation of NMDA receptors/Ca2+ channels activity.
Extracellularly applied ATP causes LTP (ATP-induced LTP) in hippocampal CA1 neurons (Wieraszko and Seyfried, 1989); this can involve ATP receptors (Edwards et al., 1992
), ATP hydrolysis products (Fujii et al., 1999b
), and/or ecto-protein kinase (Fujii et al., 1995a
), but the exact mechanism is still unknown. In the presence of ATP, gangliosides stimulate ecto-protein kinase activity in cultured cells, thus increasing extracellular phosphorylation (Tsuji et al., 1988
), and extracellular ATP plays a role in ATP-induced LTP as an orthophosphate donor for the extracellular phosphorylation of membrane proteins (Fujii et al., 1995a
, 1999a). However, no cooperative effect between extracellular ATP and gangliosides on ATP-induced LTP has been reported in hippocampal CA1 neurons. The aim of the present study was to determine whether the previously observed effects of GQ1b on ATP-induced LTP involved the modulation of NMDA receptors/Ca2+ channels by extracellular phosphorylation.
In cultured cortical neurons, a ceramide analog, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propranol (D-PDMP) or its L-enantiomer (L-PDMP) respectively inhibits or stimulates the biosynthesis of gangliosides, including GQ1b (Mizutani et al., 1996; Inokuchi et al., 1995
, 1997; Usuki et al., 1996
). L-PDMP also stimulates ganglioside biosynthesis in vivo in the rat brain when injected IP twice a day for 6 days (Yamagishi et al., 1999
). In the present study, we used both PDMPs in an in vivo approach to test the effects of gangliosides on ATP-induced LTP in hippocampal CA1 neuron slices. In addition, we studied the in vitro effect of GQ1b or GM1 on ATP-induced LTP in CA1 neurons by incubating naive hippocampal slices with GQ1b or GM1 for at least 2 h before electrophysiological testing.
K-252b is a derivative of K-252a, a potent protein kinase inhibitor effective on several types of kinase (Kase et al., 1987). Nagashima et al. (1991)
reported that, at concentrations up to 300 nM, K-252b does not permeate through the neuronal cell membrane and does not show any general toxicity for PC 12 cells at concentrations up to 10 µM, whereas K-252a, at a concentration of 300 nM, rapidly accumulates in the cytoplasm and completely inhibits the formation of neurites by nerve growth factortreated PC 12 cells. On the basis of this result, they suggested that K-252b is a potent ecto-protein kinase inhibitor. In the cultured cortical neurons, K-252b not only blocks the synapse formation but also inhibits the phosphorylation of some cell surface proteins, one of which may be the extracellular domains of microtuble-associated protein 1B (Kuroda et al., 1992
; Muramoto et al., 1993
).
In preliminary studies, we have shown that K-252b suppresses ATP-induced LTP in CA1 neurons of hippocampal slice preparations (Fujii et al., 1995a, 1999a). Tsuji et al. (1983)
observed the highly specific neurogenic effect of GQ1b on human neuroblastoma cell lines and demonstrated that the stimulatory effect of GQ1b on neurogenesis was closely associated with the phosphorylation of cell surface proteins (Tsuji et al., 1988
). Because K-252b inhibits the GQ1b-dependent neuritogenesis as well as the GQ1b-stimulated protein phosphorylation, they suggest the existence of a ganglioside-stimulated extracellular protein phosphorylation system in neurons (Tsuji et al., 1992
). In the present study, to elucidate the involvement of this system in ATP-induced LTP, we examined whether pretreatment of slice preparations with GQ1b interfered with K-252b-induced LTP suppression at hippocampal CA1 synapses.
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Results |
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In the same cells prepared from guinea pigs treated with L-PDMP, a robust LTP was induced by an identical perfusion (Figure 1), the S-EPSP 3540 min after the end of perfusion being 153.7 ± 13.3% (N = 8) of control levels, significantly greater than that measured 3540 min after application of 5 µM ATP to control slices (P < 0.05, Figure 1B). This shows that ATP-induced LTP in CA1 neurons is enhanced by in vivo treatment with L-PDMP, which up-regulates ganglioside biosynthesis.
To confirm the effects of up-regulation of ganglioside biosynthesis on ATP-induced LTP, slices from 12 naive guinea pigs were incubated with GQ1b or GM1 (both 4 µg/ml) for 28 h before being placed in the recording chamber. In GQ1b-treated slices, a robust LTP was induced by application of 5 µM ATP for 10 min (Figures 2A and B), the S-EPSP measured 3540 min after the end of drug perfusion being 139.0 ± 4.0% (N = 6) of the control level, significantly greater than that measured 3540 min after application of 5 µM ATP to control slices (P < 0.01, Figure 2C). In contrast, in GM1-treated slices, perfusion with 5 µM ATP for 10 min caused a transient depression of the S-EPSP, which, on removal of ATP, then increased gradually to a small potentiated plateau (Figures 2A and B), the S-EPSP measured 3540 min after the end of drug application being 109.0 ± 2.0% (N = 6) of the control level, not significantly different from that measured 3540 min after application of 5 µM ATP to control slices (Figure 2C).
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However, in slices preincubated for more than 2 h with GQ1b, coapplication of 40 nM K-252b and 10 µM ATP induced LTP, the S-EPSP measured 3540 min after the end of the perfusion being 120.4 ± 4.3% (N = 5), significantly (P < 0.05) greater than that seen in nonpretreated slices perfused with ATP and K-252b (Figure 4B). This indicates that pretreatment with GQ1b interferes with K-252b-induced LTP suppression. We therefore conclude that in hippocampal CA1 neurons, the enhancing effect of GQ1b on ATP-induced LTP is mediated by extracellular phosphorylation of membrane proteins at hippocampal CA1 synapses.
The LTP blockade induced by 5 µM AP5 was not enhanced by coapplication of 40 nM K-252b and ATP. As shown in Figure 4B, coapplication of 10 µM ATP, 40 nM K-252b, and 5 µM AP5 for 10 min suppressed LTP induction in naive slices, the S-EPSP measured 3540 min after the end of the drug perfusion being 111.2 ± 3.5% (N = 6) of the control level, showing that the degree of LTP blockade induced by AP5 was not affected by the presence of K-252b. As the blockade of ATP-induced LTP was not saturated by application of 5 µM AP5 (Figures 3B and 4A) and the LTP blockade induced by 5 µM AP5 was not enhanced by coapplication of 40 nM K-252b during ATP perfusion (Figure 4B), this indicates that the mechanism of LTP blockade by AP5 was occluded by K-252b, implying that AP5 and K-252b have common sites of action in the signaling pathways involved in ATP-induced LTP in hippocampal CA1 neurons. Thus, we conclude that the enhancing effects of GQ1b on ATP-induced LTP involve the phosphorylation of extracellular domains of synaptic membrane proteins, one of which could be the NMDA receptor/Ca2+ channel.
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Discussion |
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One explanation for the different effects of GQ1b and GM1 on ATP-induced LTP might be the manner in which Ca2+ is bound by these two molecules, because GM1 contains only one negatively charged sialic acid group, whereas GQ1b contains four, each of which can bind to positively charged molecules outside the cell, such as Ca2+ (Ando, 1983). In our previous study (Furuse et al., 1998
), we measured activity-dependent LTP of the population spikes in CA1 neurons when slices were incubated in low Ca2+ (1.01.1 mM) medium containing either GM1 or GQ1b; we found that the LTP in these treated slices was significantly increased compared to in controls and that this was especially apparent in slices treated with GQ1b. Because, in hippocampal CA1 neurons, Ca2+ influx through NMDA receptors/Ca2+ channels plays a key role in the formation of both ATP-induced LTP (Fujii et al., 1999a
) and activity-dependent LTP (Bliss and Collingridge, 1993
; Lisman, 1994
), it is possible that GQ1b-Ca2+ complexes, acting as Ca2+ donors, are involved in both LTPs and may provide more Ca2+ release and/or Ca2+ influx through NMDA receptors than GM1-Ca2+ complexes.
Another possible explanation for the different effects of GQ1b and GM1 on ATP-induced LTP might be properties specific to GQ1b, which, in the presence of ATP, stimulates ecto-protein kinase, thus enhancing extracellular phosphorylation (Tsuji et al., 1988), triggering the biological process resulting in LTP (Fujii et al., 1995a
, 1999a). The results of the present study indicate that the enhancing effects of GQ1b on ATP-induced LTP involve the modulation of NMDA receptors/Ca2+ channels (Figures 3 and 4A) and the phosphorylation of extracellular domains of synaptic membrane proteins (Figure 4B), one of which could be the NMDA receptor/Ca2+ channel. Thus it is possible that GQ1b is involved in the formation of ATP-induced LTP through the modulation of NMDA receptors/Ca2+ channels.
One specific effect of GQ1b on synaptic activity has been demonstrated using cultured rat cerebral cortical neurons (Mizutani et al., 1996). In this study, the synchronous oscillatory activity between neurons, monitored using fura-2 calcium imaging, was suppressed after total ganglioside depletion of neurons using an inhibitor of glucosylceramide synthesis and, when a series of gangliosides (GM3, GM1, GD3, GD1b, and GQ1b) were added back to the ganglioside-depleted cells, only GQ1b was able to normalize the reduced synaptic activity. These results are further evidence that GQ1b is essential for synaptic activity.
Prolonged treatment of rats with L-PDMP starting 24 h after transient ischemia ameliorates deficits in previously acquired spatial memory (Inokuchi et al., 1997). Moreover, under similar treatment conditions, L-PDMP stimulates brain biosynthesis of gangliosides, including GQ1b (Yamagishi et al., 1999
). Because LTP is considered to be the cellular basis of learning and memory (Bliss and Collingridge, 1993
), the present results suggest a functional role of gangliosides in synaptic plasticities and in memory formation in the brain and that up-regulation of de novo synthesis of gangliosides may have potential in the treatment of memory deficits.
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Materials and methods |
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The field EPSP in CA1 neurons was then recorded, a test stimulation of 0.05 Hz being delivered to the Schaffer collaterals/commisural afferents. The effects of bath application of 5 or 10 µM ATP (Sigma, St. Louis, MO) were evaluated by the change in the S-EPSP recorded before perfusion and 3540 min after the end of perfusion. The mean amplitude of the S-EPSP in the 10-min period before drug application was defined as 100%, and changes in responses after drug application expressed as the mean ± SEM (%) of this control level. Based on the percentage change after ATP application, responses were classified as LTP or failure of LTP, the dividing line being taken as a response 120% of the control level. The results were analyzed for statistical significance (P < 0.05 or P < 0.01) using Students t-test (two-tailed).
Two methods were used to evaluate the effects of gangliosides on ATP-induced LTP in hippocampal CA1 neurons. In the first, ATP-induced LTP was studied using slices from animals in which ganglioside biosynthesis was inhibited or stimulated in vivo by prolonged D- or L-PDMP treatment. Three guinea pigs were injected IP twice a day for 6 days with D-PDMP or L-PDMP (40 mg/kg body weight per injection; 20 mg/ml in saline containing 5% Tween 80; Seikagaku-Kogyo), then slices were prepared from the hippocampi as described. In a second approach, ATP-induced LTP was induced by addition of 5 or 10 µM ATP to naive slices preincubated in vitro for 28 h with either GQ1b or GM1 (both 4 µg/ml; provided by the Tokyo Metropolitan Institute for Gerontology). In addition, naive and GQ1b-pretreated (4 µg/ml) slices were treated for 10 min with either 10 µM ATP alone or in combination with 5 or 50 µM AP5 (Tocris Neuramine, Bristol, UK, an NMDA receptor antagonist) and/or 40 nM K-252b (Calbiochem, La Jolla, CA).
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Abbreviations |
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Footnotes |
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References |
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