Chapter 22 Neurotrophic and Neuroprotective Actions of an Enhancer of Ganglioside Biosynthesis

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To address the role of brain gangliosides in synaptic plasticity, the synthetic ceramide analog, 1‐phenyl‐2‐decanoylamino‐3‐morpholino‐1‐propanol (PDMP) was used to manipulate the biosynthesis of gangliosides in cultured cortical neurons. Spontaneous synchronized oscillatory activity of intracellular Ca2+ between the neurons, which represents synapse formation, was suppressed by the depletion of endogenous gangliosides by dthreo‐PDMP, an inhibitor of glucosylceramide synthase. On the other hand, the enantiomer of inhibitor, lthreo‐PDMP, could elevate cellular levels of gangliosides by upregulating several glycosyltransferases responsible for ganglioside biosynthesis. This review presents our findings on the neurotrophic actions of lthreo‐PDMP in vitro and in vivo. We found that l‐PDMP could upregulate neurite outgrowth, and functional synapse formation through activating GM3, GD3, and GQ1b synthases. Simultaneously, the activity of p42 mitogen‐activated protein kinase was also facilitated by l‐PDMP. To evaluate the efficacy of this drug on long term memory, rats were trained for 2 weeks using an 8‐arm radial maze task, and then forebrain ischemia was induced by four‐vessel occlusion. Repeated treatment of l‐PDMP starting 24 h after the ischemia, improved the deficit of the well‐learned spatial memory and prevented the ischemia‐induced apoptosis in hippocampus, demonstrating the potential therapeutic use of the ceramide analog for treatment of neurodegenerative disorders.

Introduction

Gangliosides, a family of sialic acid‐containing glycosphingolipids (GSLs), are abundant in CNS (Fig. 1). It has often been reported that exogenous gangliosides can elicit neurite outgrowth and neural repair in vitro and in vivo (Pepeu et al., 1994, Tsuji et al., 1992, Wu et al., 1991). In particular, GM1 and GQ1b have been found not only to enhance nerve growth factor (NGF)‐induced neurite outgrowth, but also to display NGF‐like activities themselves (Ferrari et al., 1993, Maysinger et al., 1993, Mutoh et al., 1998, Tsuji et al., 1983). The development of GM2/GD2 synthase knockout mice revealed that a lack of complex gangliosides induces abnormal conduction velocity in somatosensory nerves (Takamiya et al., 1996). The mice also exhibited decreased central myelination and axonal degeneration (Sheikh et al., 1999). Moreover, GM1 reduced glutamate, aspartate, γ‐amino‐n‐butyric acid (GABA), and glycine efflux from the cerebral cortex after transient cerebral ischemia in rats (Phillis and O'Regan, 1995), and has been developed clinically for the treatment of neuronal dysfunction.

Various studies have been conducted on excitatory amino acids and their receptors to explain the neuronal cell death (necrosis) after cerebral ischemia. The mechanism of ischemia‐induced neuronal damage and the efficacies of antagonists were reviewed by Hara et al. (1994). Recently, using caspase‐1 knockout or transgenic mice (Friedlander et al., 1997) and Bcl‐2 transgenic mice (Martinou et al., 1994), it was shown that the neuronal cell death induced by cerebral ischemia includes apoptosis.

GM1 prevented apoptotic cell death by enhancing TrkA dimerization and consequent autophosphorylation in PC12 cells (Ferrari and Greene, 1998) and decreased the severity of ischemic brain lesions in experimental models (Frontczak‐Baniewicz et al., 2000, Hicks et al., 1998). Also in neuron‐rich cortical cultures, GM1 and other gangliosides attenuated serum deprivation‐induced neuronal apoptosis (Ryu et al., 1999).

We demonstrated that the glucosylceramide synthase inhibitor, dthreo‐PDMP (d‐PDMP; Inokuchi and Radin, 1987, Inokuchi et al., 1989, Inokuchi et al., 1990), inhibited functional synapse formation (Mizutani et al., 1996), neurite outgrowth, autophosphorylation of Trk, and Trk‐initiated intracellular protein kinase cascades (Mutoh et al., 1998). These suppressive effects were specifically reversed by the addition of GQ1b or GM1. Conversely, it was found that the enantiomeric form of d‐PDMP, lthreo‐PDMP (l‐PDMP), increased the cellular content and biosynthesis of gangliosides in B16 melanoma cells (Inokuchi et al., 1989, Inokuchi et al., 1995) and stimulated neurite outgrowth (Usuki et al., 1996) and functional synapse formation with a concomitant increase of ganglioside biosynthesis in primary cultured rat embryonic cortical neurons (Inokuchi et al., 1997). Thus, l‐PDMP or its analogs might be valuable for clinical use in the postischemic treatment of cerebro‐vascular diseases.

In this review, I summarize our approaches to explore a new therapeutic intervention for the treatment of neurological disorders by enhancing the ganglioside biosynthesis.

Section snippets

Development of a Ceramide Analog PDMP

1‐Phenyl‐2‐decanoylamino‐3‐morpholino‐1‐propanol (PDMP) is a synthetic analog of ceramide and possesses two chiral centers at the Cl and C2 positions, to which hydroxyl and aminoacyl groups are attached. It thus forms four isomers (Fig. 2). The stereospecific action of PDMP isomers on UDP‐glucose: N‐acylsphingosine glucosyltransferase [glucosylceramide (GIcCer) synthase] has been clearly demonstrated, as only d‐PDMP was able to inhibit GlcCer synthase and the other isomers, including the lthreo

Effects of L‐ and D‐PDMP on Neurite Extension

When explants from embryonic neocortical neurons were cultured with l‐PDMP for 2 days in serum‐free medium, marked increases in dense neurite outgrowth and the numbers of neurite branches were observed (Usuki et al., 1996). On the other hand, treatment with d‐PDMP resulted in an inhibition of neurite outgrowth. A typical microphotograph is presented in Fig. 3. d‐PDMP showed a dose‐dependent inhibitory effect from 5 to 20 μM. On the other hand, l‐PDMP enhanced the neurite outgrowth over the same

Facilitation of Functional Synapse Formation and Ganglioside Synthesis by l‐PDMP

l‐PDMP at 20 and 40 μM in primary culture of cortical neurons increased the frequency of spontaneous synchronous oscillations between the neurons on day 9 of in vitro culture (DIV), a 50% increase being achieved compared to controls with no l‐PDMP added (Fig. 4A). d‐PDMP, which blocks GSL biosynthesis, showed the opposite effect as reported by Mizutani et al. (1996). The decreased functional synapse formation was normalized by supplementation of GQ1b but not by the other gangliosides, suggesting

Upregulation of p42 MAPK Activity

Activation of p42 mitogen‐activated protein kinase (MAPK) was found to occur in response to glutamate agonist stimulation (Bading and Greenberg, 1991, English and Sweatt, 1996) and was correlated with the spontaneous synaptic activity in cortical neurons (Fiore et al., 1993). Since the cortical cell culture forms glutamatergic synapses (Robinson et al., 1993), we measured the content and activity of p42 MAPK in the cortical cells treated with or without 20 μM l‐PDMP (Inokuchi et al., 1997). As

Improvement of the Spatial Memory Deficit and the Apoptotic Neuronal Death in Ischemic Rats

In the course of trials to evaluate the efficacy of l‐PDMP on memory in vivo, we have tested the effect of the two PDMP isomers on the deficit of previously acquired special memory after transient forebrain ischemia in rats (Inokuchi et al., 1997, Yamagishi et al., 2003). The experimental protocol is shown in Fig. 7A. Vehicle‐treated repeated cerebral ischemia rats showed a significant deficit in spatial cognition in the 8‐arm radial maze task on the 7th day after reperfusion. On the other

Effect of l‐PDMP on Biosynthesis of Cortical Gangliosides after Repeated Cerebral Ischemia

The successful results of l‐PDMP treatment in ischemic rats encouraged us to investigate whether l‐PDMP is able to stimulate ganglioside biosynthesis in vivo under a similar drug administration schedule (Yamagishi et al., 2003). As a result of metabolic radiolabeling of gangliosides in the ischemic brain, we found that l‐PDMP stimulated the biosynthesis of major gangliosides involving GM3 at the top of the biosynthetic pathway (Fig. 9). In particular, the biosynthesis of b‐series gangliosides

Discussion

It has been shown that the biosynthesis of polysialogangliosides and their expression are correlated with differentiation and synaptogenesis of neuronal cells (Ledeen, 1985, Rosner, 1992). We demonstrated that a synthetic ceramide analog, l‐PDMP, upregulates the biosynthesis of b‐series gangliosides in a long‐term primary culture of cortical neurons by activating GM3, GD3, and GQ1b synthases (Inokuchi et al., 1997). Under the same culture conditions, l‐PDMP facilitates neurite outgrowth (Usuki

Acknowledgments

The author thanks Dr Norman Radin (Emeritus Professor University of Michigan) for continuous encouragement and many colleagues cited this review, whose contributions are indispensable for this study.

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