Article Figures & Data
Figures
- Figure 1.
PP2A catalyzes the dephosphorylation of pS129 α-Syn in a methylation-dependent manner. A, In vitro dephosphorylation reactions performed by incubating phosphorylated α-Syn with PP1 (top) or PP2A (middle) demonstrate that PP2A, but not PP1, dephosphorylates α-Syn in a concentration-dependent manner. B, Quantification of the effects of PP1 and PP2A on α-Syn dephosphorylation. *p < 0.05; ***p < 0.005. C, Western blotting of lysates from SH-SY5Y cells stably expressing α-Syn treated for 1 h with 250 nm okadaic acid (OA) or 1 μm tautomycetin (Tc) with p-S129 α-Syn antibody shows that inhibition of PP2A but not PP1 modulates α-Syn phosphorylation (top). Total levels of α-Syn are unaffected by OA or Tc treatment (bottom). Ctl, Control. D, Tautomycetin is active on PP1 in SH-SY5Y cells. Cells incubated with ethanol (control) or 1 μm Tc for 20 min were lysed and in vitro dephosphorylation reactions were performed using 32P-labeled phosphorylase a. Activities are defined as follows: PP1, the fraction sensitive to 2 μm OA but insensitive to 5 nm OA; PP2A, the fraction sensitive to 5 nm OA; and residual fraction, insensitive to 2 μm OA. ***p < 0.005. E, Knockdown of PP2A subunits results in increased p-S129 α-Syn. SH-SY5Y cells expressing α-Syn were transfected with siRNA of nontargeting (NT), catalytic C subunit, or Bα subunit. Cell lysates were subjected to Western blotting for p-S129 α-Syn, total α-Syn, PP2A C subunit, and PP2A B subunit. Representative data from three separate experiments are shown. F, Quantification of p-S129 α-Syn compared to total α-Syn with knockdown of PP2A subunits. **p < 0.01. G, Western blots with pS129 α-Syn antibody of in vitro dephosphorylation reactions performed by incubating phosphorylated α-Syn with PP2A AC dimer (AC, top) or methylated AC dimer (Me-AC, middle) indicate that methylation increases the ability of PP2A AC to dephosphorylate α-Syn at Ser129. H, Quantification of the effects of methylation of AC dimers on PP2A activity toward phosphorylated α-Syn. I, Heterotrimeric assemblies of PP2A ABαC subunits (ABαC, top) increase activity to dephosphorylate α-Syn subsequent to methylation (Me-ABαC, middle). J, Quantification of the effects of methylation of ABαC heterotrimers on PP2A activity toward phosphorylated α-Syn. K, Different regulatory B subunits strongly influence the ability of PP2A to dephosphorylate α-Syn at Ser129. L, Quantification of the effects of B subunit composition on PP2A activity toward phosphorylated α-Syn.
- Figure 2.
EHT inhibits PP2A demethylation. A, In vitro PP2A demethylation is inhibited by EHT. In vitro assay performed with PP2A AC dimers methylated and labeled using PP2A methyl transferase, S-[methyl-3H]-adenosyl-l-methionine, and incubated with varying concentrations of EHT and 400 nm PP2A methyl esterase. Amount of 3H remaining in PP2A was assessed using liquid scintillation counting. B, EHT inhibits demethylation of PP2A in cultured neurons. Primary hippocampal neurons were incubated with varying concentrations of EHT for 4 h and harvested for Western blotting. Demethylation of PP2A (deMe-PP2A) was inhibited in a dose-dependent manner by EHT (top and graph, *p < 0.05), while total levels of PP2A (middle) and α-tubulin (bottom) were unchanged (n = 3 experiments).
- Figure 3.
EHT administration modulates PP2A methylation and prevents α-Syn phosphorylation and aggregation in transgenic mice. A, Western blots of cortical tissue lysates from wild-type mice. RIPA insoluble fraction was solubilized with 1% SDS and probed with antibodies to demethylated PP2A (deMe-PP2A) and total PP2A-C subunit. B, Western blots for PP2A as in Figure 2A with lysates from α-SynTg mice. These samples were also probed with phospho-S129-α-Syn antibody and total α-Syn antibody (Syn 1) showing reduction of high molecular weight aggregation. C, Quantification of band intensities in Figure 2A showing significant reduction with 0.1% EHT administration (n = 3 for each bar). D–F, Quantification of band intensities in Figure 2B showing significant decrease in demethylated PP2A, phospho-S129-α-Syn, and aggregated α-Syn (n for control feed = 4; low dose = 4; high dose = 3). G, Western blots of fractionated brain lysates with phospho-S129-α-Syn antibody and LB509 (human monoclonal α-Syn antibody) show the phosphorylated transgene expressed primarily in the cytoplasmic fraction, while the nuclear marker HDAC1 is in the nuclear fraction. WT, Wild type. H–K, Immunohistochemistry with phospho-S129-α-Syn antibody of cortical tissue from α-SynTg mice treated with control, 0.01% EHT, and 0.1% EHT, respectively, demonstrating significant decrease in the number of immunoreactive neurons and neurites (arrows). Scale bar, 100 μm. L, Quantification of the number of cortical phospho-S129-α-Syn-immunoreactive neurons (control feed, n = 6; low dose, n = 5; high dose, n = 4). *p < 0.05, **p < 0.001.
- Figure 4.
Improved dendritic arborization and neuronal activity by EHT. A–D, Confocal micrographs of cortical sections stained with MAP2. Scale bar, 25 μm. WT, Wild type. E–H, c-fos-immunoreactive neurons in the hippocampus. Arrows point to some of the c-fos-positive neurons. Scale bar, 100 μm. I, Quantification of MAP2-immunoreactive dendrites by ImageJ (n for each of the four groups = 5). *p < 0.05, ***p < 0.001. J, Quantification of c-fos-positive neurons in the dentate gyrus (control fed wild-type mice, n = 4; control fed α-SynTg, n = 6; low dose α-SynTg, n = 4; high dose α-SynTg, n = 4).**p < 0.01, ***p < 0.001. K–N, c-fos-immunoreactive neurons in the dentate gyrus of the hippocampus in wild-type mice. Arrows point to some of the c-fos-positive neurons. No difference was found with EHT administration (control fed, n = 4; low dose, n = 3; high dose, n = 3). Scale bar, 100 μm.
- Figure 5.
Repression of glial activation by EHT administration. A–H, Immunohistochemistry for GFAP in cortex (top) and hippocampus (middle) demonstrating significantly more intense staining in α-SynTg mice compared to wild-type littermates and reduction with EHT treatment. Scale bar, 100 μm. I–L, Staining with the microglial marker CD11b showing the same trend. M, Western blotting of cortical tissue lysates (soluble fraction) with GFAP, iNOS, and LB509 (human monoclonal α-Syn antibody). N, Quantification of GFAP band intensities relative to β-actin shown in M. O, Quantification of iNOS band intensities shown in M. *p < 0.05.
- Figure 6.
Amelioration of α-Syn-mediated behavioral deficits by EHT administration. A, Performance on the rotarod at 3 months of age (control fed wild-type mice, n = 9; control fed α-SynTg, n = 12; low dose wild-type, n = 10; low dose α-SynTg, n = 13; high dose wild-type, n = 9; high dose α-SynTg, n = 12). B, Performance on the rotarod at 6 months. (control fed wild-type mice, n = 9; control fed α-SynTg, n = 12; low dose wild-type, n = 10; low dose α-SynTg, n = 11; high dose wild-type, n = 9; high dose α-SynTg, n = 10). C, Nesting behavior scored at 9 months (control fed wild-type mice, n = 4; control fed α-SynTg, n = 4; low dose wild-type, n = 4; low dose α-SynTg, n = 6; high dose wild-type, n = 6; high dose α-SynTg, n = 5). *p < 0.05; **p < 0.01.
