Role of group I metabotropic glutamate receptors and NMDA receptors in homocysteine-evoked acute neurodegeneration of cultured cerebellar granule neurones

Hyperhomocysteinemia is a risk factor in neurodegeneration. It has been suggested that apart from disturbances in methylation processes, the mechanisms of this effect may include excitotoxicity mediated by the N-methyl-D-aspartate (NMDA) receptors. In this study we demonstrate that apart from NMDA receptors, also group I metabotropic glutamate receptors participate in acute homocysteine (Hcy)-induced neurotoxicity in cultured rat cerebellar granule neurones. Primary neuronal cultures were incubated for 30 min in the Mg2+-free ionic medium containing homocysteine and other ligands, and neurodegenerative changes were assessed 24 h later using propidium iodide staining. D,L-Homocysteine given alone appeared to be a weak neurotoxin, with EC50 of 17.4 mM, whereas EC50 for L-glutamate was 0.17 mM. Addition of 50 muM glycine enhanced homocysteine neurotoxicity, and only that portion of neurotoxicity was abolished by 0.5 muM MK-801, an uncompetitive NMDA receptor antagonist. The net stimulation of Ca-45 uptake by granule cells incubated in the presence of 25 MM D,L-homocysteine with 50 muM glycine was only 3% of the net uptake evoked by 1 mM glutamate. Application of an antagonist of group I metabotropic glutamate receptors (mGluRs) LY367385 at 25 and 250 muM concentrations, induced a dose-dependent partial neuroprotection, whereas given together with MK-801 completely prevented neurotoxicity. In the absence of glycine, LY367385 and MK-801 given alone failed to induce neuroprotection, while applied together completely prevented homocysteine neurotoxicity. Agonist of group I mGluRs, 10 trans-azetidine-2,3-dicarboxylic acid (t-ADA) induced significant neurotoxicity. This study shows for the first time that acute homocysteine-induced neurotoxicity is mediated both by group I mGluRs and NMDA receptors, and is not accompanied by massive influx of extracellular Ca2+ to neurones. (C) 2003 Elsevier Science Ltd. All rights reserved.