GSK-3β dysregulation contributes to parkinson's-like pathophysiology with associated region-specific phosphorylation and accumulation of tau and α-synuclein

Abstract

Aberrant posttranslational modifications (PTMs) of proteins, namely phosphorylation, induce abnormalities in the biological properties of recipient proteins, underlying neurological diseases including Parkinson's disease (PD). Genome-wide studies link genes encoding α-synuclein (α-Syn) and Tau as two of the most important in the genesis of PD. Although several kinases are known to phosphorylate α-Syn and Tau, we focused our analysis on GSK-3β because of its accepted role in phosphorylating Tau and to increasing evidence supporting a strong biophysical relationship between α-Syn and Tau in PD. Therefore, we investigated transgenic mice, which express a point mutant (S9A) of human GSK-3β. GSK-3β-S9A is capable of activation through endogenous natural signaling events, yet is unable to become inactivated through phosphorylation at serine-9. We used behavioral, biochemical, and in vitro analysis to assess the contributions of GSK-3β to both α-Syn and Tau phosphorylation. Behavioral studies revealed progressive age-dependent impairment of motor function, accompanied by loss of tyrosine hydroxylase-positive (TH+ DA-neurons) neurons and dopamine production in the oldest age group. Magnetic resonance imaging revealed deterioration of the substantia nigra in aged mice, a characteristic feature of PD patients. At the molecular level, kinase-active p-GSK-3β-Y216 was seen at all ages throughout the brain, yet elevated levels of p-α-Syn-S129 and p-Tau (S396/404) were found to increase with age exclusively in TH+ DA-neurons of the midbrain. p-GSK-3β-Y216 colocalized with p-Tau and p-α-Syn-S129. In vitro kinase assays showed that recombinant human GSK-3β directly phosphorylated α-Syn at a single site, Ser129, in addition to its known ability to phosphorylate Tau. Moreover, α-Syn and Tau together cooperated with one another to increase the magnitude or rate of phosphorylation of the other by GSK-3β. Together, these data establish a novel upstream role for GSK-3β as one of several kinases associated with PTMs of key proteins known to be causal in PD.

Figure 1

Behavioral and histochemical analyses of hGSK-3β-S9A overexpressing and WT mice. TG and WT mice aged 4–6 (TG N=15; WT N=18), 9 (TG N=13; WT N=18), 12 (TG N=12; WT N=10), and 15 months (TG N=10; WT N=10) were used to measure motor impairment and motor co-ordination. (a) Rotorad tests, (b) Wire-hang tests, (c) pole tests (d) gait tests. Comparisons between age-matched WT and TG mice, as well as between TG mice of different ages were made using Welch's t test or one-way ANOVA, and significance was measured as *P<0.05 or **P<0.01 or ***P<0.001. (e) Unbiased stereological counts of TH-stained SN were conducted as described before, using four mice at 15 months of age and both TH+ and TH− neurons were counted. (f) Scale bars on the upper panels represent 750 μm, whereas on lower panels, the scale bars are 100 μm. Dashed line denotes SN and VTA border with dentate gyrus (DG) and hippocampus (CA1) regions labeled for comparison. See also Supplementary Figure S1A and D

Figure 2

Phospho-isoforms of GSK-3β in different brain regions and at different ages. Midbrain, striatum, and frontal cortex were analyzed from TG and WT mice at 4–6 (N=6 each for TG and WT), 9 (N=8 each for TG and WT) and 15 months (N=8 each for TG and WT) for the differentially phosphorylated isoforms of GSK-3β. (a, b) midbrain (MB). (c, d) striatum (STR). (e, f) frontal cortex (FC). Immunohistochemical analyses from 15-month-old WT or TG mice (N=4 each) were conducted using 10-micron-thick sections. (g) GSK-3β/TH. (h) p-GSK-3β-Y216/TH. IHC of paraffin-embedded human non-diseased (control, CT) and PD postmortem SN showing α-Syn or p-GSK-3β-Y216 expression. (i) All WT values of proteins in western blots were normalized to GAPDH and set at 100%, denoted by the dashed line, and showed <10% variation at each data point. TG values were expressed relative to the WT control from within the same age group, after normalization to GAPDH, used as the loading control. Comparisons between age-matched WT and TG mice were made using Welch's t test or one-way ANOVA, and significance was measured as *P<0.05 or **P<0.01 or ***P<0.001. Scale bar denotes 10 μm in (g,h). In (i), scale bars correspond to 200 μm and * shows Lewy bodies, ** neurites, and *** aggregates. See also Supplementary Table S2

Figure 3

Regional and age-specific accumulation of differentially phosphorylated Tau epitopes in WT and TG mice. The midbrain (a, b), striatum (c, d), and frontal cortex (e, f) from TG and WT mice (N=8 each) at 15 months of age were analyzed by immunoblotting for various tau-phosphorylated epitopes. (g) IHC of TG and WT mice brain. (h) IHC of paraffin-embedded human non-diseased (control) and PD postmortem SN for pTau(Ser396/Ser404). Comparisons between age-matched WT and TG mice were made using Welch's t test or one-way ANOVA, and significance was measured as *P<0.05 or **P<0.01 or ***P<0.001. Scale bar on the IHC in (g) denotes 10 μm. In (h), scale bars correspond to 200 μm and * shows Lewy bodies, ** neurites, and *** aggregates. See also Supplementary Figure S2B, S3A and G, and Supplementary Table S2

Figure 4

Regional and age-specific accumulation of phosphorylated α-Syn at S129 in WT and TG mice. (a,b) α-Syn and TH levels were examined in midbrain (MB), striata (STR), and frontal cortex (FC) tissue from 15-month-old WT and TG mice (N=5). (c) IHC comparison of α-Syn immunoreactivity in TH+ neurons in age-matched 15-month-old WT and TG mice. (d,e) p-α-Syn-S129 levels were examined in midbrain (MB), striata (STR), and frontal cortex (FC) tissue from 4–6, 9, and 15-month-old WT and TG mice (N=5). (f) IHC comparison of p-α-Syn-S129 immunoreactivity in TH+ neurons in age-matched 15-month-old WT and TG mice. (g) Co-localization of p-α-Syn-S129 immunoreactivity with p-GSK3B-Y216 in the SN of age-matched 15-month-old WT and TG mice. Comparisons between age-matched WT and TG mice were made using Welch's t test or one-way ANOVA, and significance was measured as *P<0.05 or **P<0.01 or ***P<0.001. Scale bar on the IHC in (g) denotes 10 μm. See also Supplementary Figures S2A, S3A, and G, and Supplementary Figures S5A and B

Figure 5

In vivo phosphorylation of α-Syn and Tau-2N4R by GSK-3β. Recombinant human proteins (hα-Syn, hGSK-3β, and hTau-2N4R) were purified as described in Methods (Supplementary Information), and purity was confirmed by coomassie blue (CB) staining of gels. For the in vitro reactions (a, b), 74.21 ng (3.34 nM) of recombinant hGSK-3β was incubated at room temperature for 24 h with either 1.45 μg (3.34 μM) of hα-Syn or 4.59 μg (3.34 μM) hTau-2N4R, in the absence or presence of 1 μM of the hGSK-3β inhibitor, TDZD-8. Proteins were analyzed by CB staining to determine purity and by immunoblots to determine phosphorylated epitopes. MS analysis of in vitro kinase reactions (c) hα-Syn and hGSK-3β 0-h reaction (upper panel). hα-Syn and hGSK-3β with TDZD8 24-h reaction (middle panel). hα-Syn and hGSK-3β 24-h reaction (lower panel). Calculated mass difference (black) comparing 0-h reaction (control) (upper panel) to 24-h reaction (lower panel) with theoretical mass difference of single phosphorylation (red) shown for reference. Each reaction was analyzed by western blots from three separate and independent studies

Figure 6

GSK-3β phosphorylation rate accelerated by dual influence of α-Syn and Tau. (a–f) Time course experiments. A total of 74.21 ng of hGSK-3β was incubated with substrate (hα-Syn, 1.45 μg a–c, or hTau, 4.59 μg d–f) in the absence (closed circles) or presence (open circles) of trace levels of either hTau (0.046 μg or 100 fM) or hα-Syn (0.015 μg or 1 pM). Reactions were conducted for the following times: lanes 1 and 2=control; lane 3=0 min; lane 4=7.5 min; lane 5=15 min; lane 6=30 min; lane 7=60 min; lane 8=120 min; lane 9=240 min; lane 10=480 min; lane 11=1440 min. Each reaction was analyzed by western blots from three separate and independent studies. p-hTau (Ser396/Ser404), p-hα-Syn-S129, and p-hGSK-3β-Y216 was normalized to total unphosphorylated forms. Comparisons between data were made using Welch's t test, and significance was measured as *P<0.05 or **P<0.01 or ***P<0.001. See also Supplementary Figures S4A and D

Figure 7

Model of how GSK3β may contribute to the pathobiology of PD through the dual accumulation of phosphorylated α-Syn (S129) and Tau (various epitopes). (i) GSK3β is a central regulator of many diverse yet essential cellular signaling networks. GSK3β is therefore highly regulated through various physical interactions and biochemical modifications. (ii) Activation of GSK3β is thought to occur through known and yet-discovered avenues including activation by cellular stresses such as oxidative stress. (iii) Activated GSK3β is known to hyperphosphorylate Tau at a number of sites in both AD and PD. Through the use of in vitro kinase assays together with analysis of the hGSK3β-S9A TG mouse model, this study provides evidence that α-Syn phosphorylation is enhanced via activated GSK3β (p-GSK3β-Y216). Two possible modes of GSK3β mediated effects on α-Syn phosphorylation are possible. One, directly, with α-Syn being a substrate for phosphorylation by p-GSK3β-Y216. Two, indirectly, through p-GSK3β-Y216-mediated alterations to cellular signaling pathways leading to the increased expression, activation, or interactions of kinases known to phosphorylate α-Syn such as casein kinase-2, polo-like kinases-2 and 3, and the G-protein coupled receptor kinases-1–5. Other possible indirect means include p-GSK3β-Y216-mediated reductions in the cells ability to efficiently degrade proteins through the proteasomal and autophagic pathways. In this study, α-Syn and Tau are also shown to be capable of enhancing the rate or magnitude of phosphorylation of the other by GSK3β. (iv) Pathological and phenotypic changes associated with the phosphorylation and accumulation of Tau and α-Syn include declines in motor skills, decrease in SN volume, decreased DA, and TH+ neuronal loss