Tau pathology involves protein phosphatase 2A in parkinsonism-dementia of Guam

Abstract

Parkinsonism-dementia (PD) of Guam is a neurodegenerative disease with parkinsonism and early-onset Alzheimer-like dementia associated with neurofibrillary tangles composed of hyperphosphorylated microtubule-associated protein, tau. β-N-methylamino-l-alanine (BMAA) has been suspected of being involved in the etiology of PD, but the mechanism by which BMAA leads to tau hyperphosphorylation is not known. We found a decrease in protein phosphatase 2A (PP2A) activity associated with an increase in inhibitory phosphorylation of its catalytic subunit PP2Ac at Tyr(307) and abnormal hyperphosphorylation of tau in brains of patients who had Guam PD. To test the possible involvement of BMAA in the etiopathogenesis of PD, we studied the effect of this environmental neurotoxin on PP2A activity and tau hyperphosphorylation in mouse primary neuronal cultures and metabolically active rat brain slices. BMAA treatment significantly decreased PP2A activity, with a concomitant increase in tau kinase activity resulting in elevated tau hyperphosphorylation at PP2A favorable sites. Moreover, we found an increase in the phosphorylation of PP2Ac at Tyr(307) in BMAA-treated rat brains. Pretreatment with metabotropic glutamate receptor 5 (mGluR5) and Src antagonists blocked the BMAA-induced inhibition of PP2A and the abnormal hyperphosphorylation of tau, indicating the involvement of an Src-dependent PP2A pathway. Coimmunoprecipitation experiments showed that BMAA treatment dissociated PP2Ac from mGluR5, making it available for phosphorylation at Tyr(307). These findings suggest a scenario in which BMAA can lead to tau pathology by inhibiting PP2A through the activation of mGluR5, the consequent release of PP2Ac from the mGluR5-PP2A complex, and its phosphorylation at Tyr(307) by Src.

Keywords: Alzheimer's disease; amyotrophic lateral sclerosis; cycad; tau phosphorylation; tauopathies.

Fig. 1.

PP2A activity is decreased because of the increase in phosphorylation of PP2Ac at Tyr³⁰⁷, and tau is hyperphosphorylated in Guam PD brains. (A) PP2A activity assayed by phosphatase ELISA in Guam PD and Guam nonneurological control frontal cortices. (B) Western blots of PP2A and its inhibitors. (C) Quantitative analysis of blots in B. (D) Western blots of tau phosphorylated at different sites. (E) Quantitative analysis of blots in D. Data are expressed as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 2.

BMAA inhibits PP2A activity and increases tau hyperphosphorylation in mouse hippocampal primary neurons. (A and B) PP2A activity (A) and PP2Ac protein level (B) were measured in mixed mouse primary hippocampal neurons at 0, 1, 3, 6, and 24 h after treatment with BMAA (1 mM). (C) Representative Western blots showing hyperphosphorylation of tau. (D) Quantification of hyperphosphorylation of tau at different sites and R134d (total tau) shown after normalization with GAPDH. (E and F) Representative photomicrographs (E) and the corresponding quantitation of phospho-tau (12E8) staining (F) in control and BMAA-treated cultured primary neurons. (Scale bar, 100 µm.) (Magnification: 40×.) (G) LDH release was measured 24 h after treatment with BMAA (1 mM) in cultured neurons; 2 mM glutamate was used as a positive control. Data are expressed as mean ± SEM for three separate experiments. *P < 0.05; **P < 0.01.

Fig. 3.

BMAA induces an increase in pTyr³⁰⁷ PP2Ac, inhibits the phosphatase activity, and activates several tau protein kinases in rat brain slices. Rat hippocampal brain slices were treated with BMAA (1 mM) for 2 h, and the tissue homogenate was used for Western blots and to measure PP2A activity. (A) PP2A activity. (B) Western blots. (C) The quantitative analysis of pTyr³⁰⁷-PP2Ac, DML³⁰⁹-PP2Ac, I₁^PP2A, and I₂^PP2A of Western blots in B after normalization with total PP2Ac. (D) Western blots of brain slices treated with PP2, an Src kinase inhibitor (10 µM), for 30 min before treatment with BMAA. (E) Quantitative analysis of blots in D. (F and G) The Western blot pattern of total and phosphorylated kinases (F) and quantitation of phosphorylated kinases normalized with the level of corresponding kinase (G). Data are expressed as mean ± SEM for three separate experiments. *P < 0.05; **P < 0.01.

Fig. 4.

The i.c.v. infusion of BMAA induces an increase in pTyr³⁰⁷-PP2Ac, inhibition of PP2A activity, and hyperphosphorylation of tau in vivo in rat pups. Rat postnatal day 2 hippocampus was collected at the indicated time points after i.c.v. injection of BMAA (1.3 μmol) and was analyzed by Western blots to detect tau phosphorylation at individual sites and PP2A phosphorylation at Tyr³⁰⁷. (A) Representative Western blots. (B) pTyr³⁰⁷-PP2A level normalized with total PP2A level. (C and D) PP2A activity (C) and quantification of hyperphosphorylation of tau normalized with total tau (92e) (D). Data are expressed as mean ± SEM for two separate experiments (n = 4–5). pPP2Ac, pY³⁰⁷-PP2Ac. *P < 0.05; **P < 0.01.

Fig. 5.

Up-regulation of PP2A by DES or PP2A (DsRed) reverses BMAA-induced tau hyperphosphorylation in rat brain slices and mouse hippocampal primary neurons. (A) Western blots of brain slices treated with BMAA (1 mM) for 2 h with or without pretreatment with the PP2A activator DES (15 nM). (B) Quantitation of the blots in A. (C) The primary hippocampal neurons were transfected with DsRed-vector or DsRed-PP2Ac at 6 days in vitro (DIV) and then were treated with BMAA (1 mM) for 3 h at 8 DIV. The neurons were fixed and costained with DsRed (red) and 12E8 (green). (Scale bar, 50 µm.) (Magnification: 40×). (D) Quantitation of 12E8 staining normalized with DsRed. (E) In vitro PP2A activity was measured using purified PP2A holoenzyme in the presence or absence of BMAA. Data are expressed as mean ± SEM for three separate experiments. *P < 0.05; **P < 0.01.

Fig. 6.

BMAA induces tau hyperphosphorylation by activating mGluR5. Rat Hippocampal brain slices were incubated with BMAA (1 mM) for 2 h with or without the 30-min pretreatment with MPEP (50 µM), MK-801 (10 µM), or CNQX (20 µM). (A) Representative Western blots. (B) Quantitation of blots from A. (C) PP2A activity. (D) PP2Ac immunoprecipitates blotted for coimmunoprecipitating mGluR5 after BMAA treatment in primary neuronal cultures. (E) Quantitation of data from D. Data are expressed as mean ± SEM for three separate experiments. *P < 0.05; **P < 0.01.

Fig. 7.

A proposed schematic representation of the possible mechanism leading to neurofibrillary degeneration in PD. BMAA activates the mGluR5 receptor and dissociates PP2Ac from the receptor. Src kinase then acts on free and available PP2Ac and phosphorylates at Tyr³⁰⁷, inhibiting its activity and shifting the balance toward tau kinases, thus leading to the hyperphosphorylation of tau as evident in Guam PD brain.