Protein phosphatase 2A methyltransferase links homocysteine metabolism with tau and amyloid precursor protein regulation

Abstract

Alzheimer's disease (AD) neuropathology is characterized by the accumulation of phosphorylated tau and amyloid-beta peptides derived from the amyloid precursor protein (APP). Elevated blood levels of homocysteine are a significant risk factor for many age-related diseases, including AD. Impaired homocysteine metabolism favors the formation of S-adenosylhomocysteine, leading to inhibition of methyltransferase-dependent reactions. Here, we show that incubation of neuroblastoma cells with S-adenosylhomocysteine results in reduced methylation of protein phosphatase 2A (PP2A), a major brain Ser/Thr phosphatase, most likely by inhibiting PP2A methyltransferase (PPMT). PP2A methylation levels are also decreased after ectopic expression of PP2A methylesterase in Neuro-2a (N2a) cells. Reduced PP2A methylation promotes the downregulation of B alpha-containing holoenzymes, thereby affecting PP2A substrate specificity. It is associated with the accumulation of both phosphorylated tau and APP isoforms and increased secretion of beta-secretase-cleaved APP fragments and amyloid-beta peptides. Conversely, incubation of N2a cells with S-adenosylmethionine and expression of PPMT enhance PP2A methylation. This leads to the accumulation of dephosphorylated tau and APP species and increased secretion of neuroprotective alpha-secretase-cleaved APP fragments. Remarkably, hyperhomocysteinemia induced in wild-type and cystathionine-beta-synthase +/- mice by feeding a high-methionine, low-folate diet is associated with increased brain S-adenosylhomocysteine levels, PPMT downregulation, reduced PP2A methylation levels, and tau and APP phosphorylation. We reported previously that downregulation of neuronal PPMT and PP2A methylation occur in affected brain regions from AD patients. The link between homocysteine, PPMT, PP2A methylation, and key CNS proteins involved in AD pathogenesis provides new mechanistic insights into this disorder.

Figure 1.

Incubation of N2a cells with SAM and SAH modulates PP2A methylation. A, Representative experiment showing a concomitant increase in endogenous SAM, PPMT expression, and methylated C levels in SAM-treated cells. B, Representative experiment showing a dose-dependent increase in endogenous SAH concentrations in SAH-treated cells. C, D, In these cells, the SAM/SAH ratio (C) and relative levels of methylated C subunits (D) are decreased, but PPMT expression levels (D) are not significantly affected by the short-term SAH treatment. For A–D, similar results were obtained in three separate experiments.

Figure 2.

Expression of the L309Δ C subunit mutant in N2a cells decreases endogenous PP2A methylation and inhibits the ability of PP2A to bind to and dephosphorylate tau. A, Relative expression levels of endogenous and expressed C subunits in stable N2a clones expressing HA-tagged Wt C or the L309Δ C subunit mutant. B, The expression levels of methylated C and Bα subunits are enhanced in N2a–Wt C but decreased in N2a–L309Δ cells. C, D, Cells were transiently transfected with a plasmid encoding human adult tau. C, Total cell extracts and normalized HA-tag immunoprecipitates (IP) were analyzed for the presence of tau using the phosphorylation-independent Tau-5 monoclonal antibody. D, Control and stable N2a cells were analyzed by Western blotting (WB) for total tau using Tau-5 and for phosphorylated tau using a set of phosphoepitope-specific, anti-tau antibodies. A subset of control cells was treated for 1 h with 100 nm OA before harvesting. Representative blots are shown. HA-tagged C expression levels are shown for reference.

Figure 3.

PP2A methylation-, SAM-, and SAH- dependent modulation of tau phosphorylation in N2a cells. A, Representative immunoblots showing that stable expression of Myc-tagged PME-1 is associated with PP2A demethylation, decreased Bα subunit expression, and increased tau phosphorylation. The arrow indicates Myc-tagged PME-1 recognized by the anti-PME-1 antibody. B, Representative immunoblots showing that stable expression of HA-tagged PPMT is associated with enhanced expression levels of PPMT, methylated C and Bα subunits, and tau dephosphorylation. The arrow indicates HA-tagged PPMT recognized by the anti-PPMT antibody. C, Tau-transfected N2a cells were treated with SAM or SAH and analyzed by immunoblotting for tau phosphorylated at the PHF-1 epitope and for total tau using Tau 5 antibodies. D, Concomitant incubation with 10 nm OA or expression of the L309Δ mutant in N2a cells prevent tau dephosphorylation induced by 100 μm SAM. Bottom rows in C and D show the relative amounts of phosphorylated tau (n = 3; *p < 0.001).

Figure 4.

PP2A-, SAM-, and SAH- dependent modulation of APP phosphorylation in N2a cells. A–F, Representative immunoblots showing the relative levels of APP phosphorylated on Thr-668 and total APP in N2a cells. A, Endogenous APP migrates as several bands corresponding to mature and immature APP isoforms (Latasa et al., 1998). The fastest migrating band corresponds to immature APP₆₉₅. The slowest migrating APP₇₅₁ and APP₇₇₀ isoforms are immunoreactive to antibodies raised against the KPI domain of APP. B, Incubation of N2a cells for 1 h with increasing OA concentrations induces dose-dependent APP phosphorylation. C, Phosphorylated APP levels are altered after deregulation of PP2A, PPMT, or PME-1. D, Incubation of N2a cells with 100 μm SAM or SAH affects APP phosphorylation. E, Concomitant incubation for 18 h with 10 nm OA prevents SAM-induced APP dephosphorylation. F, Incubation of N2a–Wt C cells with 100 μm SAM promotes APP dephosphorylation. SAM cannot induce similar effects in N2a–L309Δ cells.

Figure 5.

PP2A activity and methylation regulate APP secretion and Aβ production in N2a cells. A, Secreted sAPP isoforms migrate as a triplet band recognized by the 22C11 antibody (Nakagawa et al., 2006). sAPP₇₅₁ and sAPP₇₇₀ isoforms are immunoreactive to the anti-KPI–APP antibody (Latasa et al., 1998). B, Steady-state levels of sAPPα and sAPPβ were measured after 1 h incubation of N2a clones in conditioned medium. A subset of control N2a cells was incubated for 1 h with 100 nm OA. Intracellular APP was detected with anti-APP 22C11 antibody. The bottom panel shows the relative amounts of sAPPα and sAPPβ determined after normalization for intracellular protein concentration (mean ± SD; n = 3; p < 0.001 in all conditions). C, The extracellular release of sAPPα was measured in the same cells analyzed for APP in Figure 4, C and F, 4 h after incubation in the conditioned media. D, Representative experiment showing the concomitant release of sAPPβ and Aβ₄₀ peptides measured 24 h after incubation of cells in conditioned medium. Aβ₄₀ levels (mean ± SEM) were quantified by ELISA.

Figure 6.

Effects of the high-methionine, low-folate diet on Hcy, methylation metabolites, PPMT, PP2A methylation, and tau and APP phosphorylation in Cbs^+/+ and Cbs^+/− mice. A–D, Groups of 1-month-old heterozygous Cbs^+/− (black bars) and wild-type Cbs^+/+ (white bars) mice were fed for 8 months on a control or HM/LF diet. Data are mean ± SD; n = 7 mice per group. A, Hcy and methylation metabolites were determined in plasma and brain tissue from the mice (*p < 0.01 compared with same genotype). B–D, Equivalent aliquots (∼14 μl) of total brain extracts from the same mice were simultaneously analyzed by gel electrophoresis and Western blotting. Note that the representative panels shown were assembled from the same exposed autoradiograph. B, Relative levels of PPMT in each mouse group (*p < 0.001; **p < 0.05). C, Relative levels of methylated C (*p < 0.046; **p = 0.046). D, Relative levels of Thr-668-phosphorylated APP (*p < 0.001). E, Equivalent amounts of proteins (10 μg) from boiled mouse brain homogenates were analyzed for tau phosphorylated at the PHF-1 epitope after normalization for total tau levels (*p < 0.001).