Folate deficiency is associated with Alzheimer's disease (AD) and has been suggested to contribute to the pathogenesis of AD. Hyperphosphorylation of tau and deposition of beta-amyloid derived from amyloid precursor protein (APP) are characteristic features of the neurodegenerative pathology of AD. To investigate the molecular mechanisms underlying the association between folate deficiency and AD pathogenesis, we treated rat primary neuron cultures with methotrexate (MTX), a folate antagonist. After MTX treatment, levels of phosphorylated tau, APP, and P-secretase were increased, as shown by Western-blot and immunocytochernistry analyses, and the neuronal viability was reduced, as assessed by the NITS assay, indicating that folate deficiency increases characteristic AD pathologies. Interestingly, levels of methylated protein phosphatase-2A (PP2A), which is the active form of the putative tau phosphatase PP2A, were reduced. These novel findings indicate that folate deficiency increases the characteristic AD pathology including tau phosphorylation presumably by PP2A inactivation. (C) 2007 Elsevier Inc. All rights reserved.