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

Abstract

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 30min in the Mg2+-free ionic medium containing homocysteine and other ligands, and neurodegenerative changes were assessed 24h later using propidium iodide staining. d,l-Homocysteine given alone appeared to be a weak neurotoxin, with EC50 of 17.4mM, whereas EC50 for l-glutamate was 0.17mM. Addition of 50μM glycine enhanced homocysteine neurotoxicity, and only that portion of neurotoxicity was abolished by 0.5μM MK-801, an uncompetitive NMDA receptor antagonist. The net stimulation of 45Ca uptake by granule cells incubated in the presence of 25mM d,l-homocysteine with 50μM glycine was only 3% of the net uptake evoked by 1mM glutamate. Application of an antagonist of group I metabotropic glutamate receptors (mGluRs) LY367385 at 25 and 250μM 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.