Homocysteine-induced acute excitotoxicity in cerebellar granule cells in vitro is accompanied by PP2A-mediated dephosphorylation of tau

Our results demonstrate that acute exposure of primary rat cerebellar granule cell cultures to homocysteine at millimolar concentrations induces a glutamate receptor-mediated decrease in tau protein phosphorylation, which is accompanied by excitotoxic neuronal damage. This contrasts with the previously reported hyperphosphorylation of tau in homocysteine-treated neurons, and with the assumption that hyperhomocysteinemia is one of the risk factors in Alzheimer disease, in which abnormal hyperphosphorylation of tau protein leads to neurofibrillary degeneration. The mechanisms of homocysteine neurotoxicity have been explained mainly either by disturbances in methylation processes, that may also lead to the accumulation of phosphorylated tau due to reduced activity of tau-selective protein phosphatase 2A, or by excitotoxicity. Since the relationships between homocysteine excitotoxicity and tau phosphorylation are unclear, the aim of this study was to characterize these processes in neurons acutely treated with homocysteine at neurotoxic concentrations, and to link them to the activities of glutamate receptors and protein phosphatase 2A. Within 24 h following a 30 min exposure of neuronal cultures to 20 mM d,l-homocysteine, significant neurotoxicity was induced. This could be reduced by treatment with an uncompetitive NMDA receptor antagonist, MK-801 (0.5 μM), or by mGlu1 and mGlu5 receptor antagonists, LY367385 and MPEP, respectively (both at 25 μM). Western blot analysis showed a rapid decrease in immunostaining of phospho-tau, 2 h after incubation of cell cultures with 15 mM d,l-homocysteine, which persisted for 6 h after the insult. Application of MK-801, LY367385 or okadaic acid (100 nM), an inhibitor of protein phosphatases 1 and 2A, significantly prevented dephosphorylation of tau, implying a role for the activation of glutamate receptors and protein phosphatase 2A. The phosphorylation of tau may be increased or reduced by treatment with homocysteine, and the nature of the cellular response to this sulfur-containing amino acid depends on the neuronal phenotype.