Neuroprotection produced by the NAALADase inhibitor 2-PMPA in rat cerebellar neurons

doi:10.1016/S0014-2999(00)00519-7

European Journal of Pharmacology

Volume 402, Issues 1–2, 18 August 2000, Pages 31-37

https://doi.org/10.1016/S0014-2999(00)00519-7 Get rights and content

Abstract

The present study examined the neuroprotective actions of the N-acetylated-α-linked-acidic dipeptidase (NAALADase) inhibitor 2-(phosphonomethyl)pentanedioic acid (2-PMPA) in four in vitro models of neurotoxicity. Using neuron-enriched primary cultures derived from rat embryo (E15) cerebellum, 2-PMPA afforded 100% neuroprotection from injuries induced by hypoxia (EC₅₀=8.4 μM). In contrast, against glutamate or N-methyl-d-aspartate (NMDA) injury, 2-PMPA was less potent and its efficacy limited to a maximum of 46% and 16%, respectively. 2-PMPA was not effective against veratridine-induced injury. Also, the less potent analog of 2-PMPA, 2-[phosphonomethyl]succinic acid (2-PMSA), was ineffective. Unlike 2-PMPA, the endogenous NAALADase substrate and mGlu₃ receptor agonist N-acetyl-aspartyl-glutamate (NAAG) was neuroprotective against all four injury mechanisms and compared to 2-PMPA, exhibited a different “phosphate effect” on neuroprotection. These results confirm the superior efficacy of 2-PMPA to protect against injury caused by cellular anoxia, and are discussed relative to upstream modulation of hyperglutamatergic activity vs. downstream modulation of metabotropic receptors as possible targets for ischemia/stroke therapy.

Introduction

Glutamate is the major excitatory neurotransmitter in mammalian brain. Excessive activation of cellular glutamatergic processes (i.e. receptors, ion channels, transcription proteins, etc.) is believed to represent the seminal cellular mechanism promoting neurodegeneration leading to neuron dysfunction and cell death. Regardless of etiology, or subsequent clinical event (i.e. stroke, brain trauma, progressive neurodegenerative diseases, chemical/biological terrorism, etc.), the arrest of hyperglutamatergic activity and subsequent attenuation of the excitotoxic cascade remains a dynamic challenge to drug development and experimental therapeutics, and one of significant clinical potential.

Much research has focused on the two primary classes of glutamate receptors (ionotropic and metabotropic) and the role of intracellular calcium and other downstream mechanisms representing the excitotoxic “secondary signaling cascade”. Alternatively, upstream modulation of glutamate by interfering with its neuropeptide precursor N-acetyl-aspartyl-glutamate (NAAG) (Coyle, 1997), via inhibition of the hydrolyzing enzyme N-acetylated-α-linked-acidic dipeptidase (NAALADase), has recently received attention as a novel strategy to suppress excitotoxic glutamate neurotransmission pathogenic to several brain disorders (Slusher et al., 1999).

The purpose of this study was to examine the neuroprotective properties of an inhibitor of NAALADase, 2-(phosphonomethyl)pentanedioic acid (2-PMPA) (Jackson et al., 1996), in different in vitro neuronal culture models of cell death. Using cerebellar neurons, neuroprotection was studied following hypoxia/hypoglycemia, N-methyl-d-aspartate (NMDA), glutamate, or veratridine-induced neuronal injury.

Section snippets

Cerebellum cultures

Neuronal cultures were prepared from prenatal day 15 Sprague–Dawley rat embryo cerebellum (whole cerebellum). The harvested neurons were plated in 48-well culture plates (5×10⁵ cells/well) pre-coated with poly-l-lysine. The cultures were maintained in a medium containing equal parts of Eagle's basal media (without glutamine) and Ham's F12K media supplemented with 10% heat-inactivated horse serum, 10% fetal bovine serum, glucose (600 μg/ml), glutamine (100 μg/ml), penicillin (50 units/ml), and

Neurotoxicity

Exposure of normal cerebellar neurons (Fig. 1A) to hypoxia/hypoglycemia (Fig. 1B), glutamate (Fig. 1C), NMDA (Fig. 1D), or veratridine (Fig. 1E), was highly neurotoxic. Following hypoxia/hypoglycemia, neuronal cell death in untreated neurons approached 70% (65±4%). Neuronal cell death following glutamate, NMDA or veratridine exposures were 75±5%, 70±5% and 60±4%, respectively.

Effect of 2-PMPA

Co-treatment with 2-PMPA was neuroprotective (Fig. 1, F–I), and the degree of neuroprotection dependent upon the

Discussion

Consistent with our recent report describing neuroprotective effects of 2-PMPA in experimental models of anoxia/ischemia mediated neuronal death (Slusher et al., 1999), the results of the present study confirmed that 2-PMPA protects neurons from injury induced by cellular anoxia (i.e. hypoxia/hypoglycemia deprivation) and that delayed treatment with 2-PMPA is also neuroprotective, at least when the injury is caused by anoxia (i.e. hypoxia/hypoglycemia model). Also, it has been demonstrated here

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Neuroprotection produced by the NAALADase inhibitor 2-PMPA in rat cerebellar neurons

Abstract

Introduction

Section snippets

Cerebellum cultures

Neurotoxicity

Effect of 2-PMPA

Discussion

Int. Rev. Neurobiol.

Neurobiol. Dis.

Neurosci. Lett.

Neuropharmacology

J. Biol. Chem.

Binding of the NAALADase inhibitor 2-PMPA to brain membranes exhibiting two-site kinetics

Soc. Neurosci. Abstr.

Dodecylglycerol provides partial protection against glutamate toxicity in neuronal cultures derived from different regions of embryonic rat brain

Mol. Chem. Neuropathol.

Activity of N-acetylaspartylglutamate at human recombinant glutamate receptors

Soc. Neurosci. Abstr.