Methotrexate decreases PP2A methylation and increases tau phosphorylation in neuron

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Abstract

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 β-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 β-secretase were increased, as shown by Western-blot and immunocytochemistry analyses, and the neuronal viability was reduced, as assessed by the MTS 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.

Section snippets

Materials and methods

Neuron culture. Primary cultures of rat cortical neurons were prepared from the brains of embryonic-day-16 pups as previously described [28]. Briefly, the cerebral cortices were dissected in calcium- and magnesium-free Hank’s balanced salt solution and incubated with a 0.125% trypsin solution for 10 min at 37 oC. Trypsin was inactivated with Dulbecco’s modified Eagle’s medium (DMEM) containing 20% fetal bovine serum, and the cortical tissue was further dissociated by serial trituration using a

Results

To evaluate the effects of MTX on tau phosphorylation levels, we analyzed the neuronal extract by Western-blot analysis using antibodies that specifically recognize phosphorylated or dephosphorylated tau. Levels of phosphorylated tau that was recognized by PHF-1 and pS396 antibodies increased in a time-dependent manner, and densitometric analysis indicated that the PHF-1 level increased by about 150% after 2 days of okadaic acid (OA) treatment (Fig. 2A). Consistently, dephosphorylated tau

Discussion

Here, we showed that MTX can increase tau phosphorylation, APP accumulation, and BACE upregulation, all of which are associated with PP2A inactivation. The cellular mechanisms by which tau is abnormally phosphorylated may contribute to the pathogenesis of AD, and thus identification of the enzymes responsible for the regulation of tau phosphorylation in neurons is important to understand its role in neurodegeneration.

There is increasing evidence to show that PP2A is downregulated in AD brain

Acknowledgment

This study was supported by a grant from the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea (A0400042).

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