Regulation of Ser129-phosphorylated α-syn in intra- and extracellular spaces
We compared the levels of Ser129-phosphorylated α-syn with those of total α-syn, including non-phosphorylated and phosphorylated forms, by transfecting either an empty vector or three different amounts of α-syn cDNA into CHO cells. We used CHO cells in this experiment to achieve transient transfection with relatively high efficiency. In cell lysates, the levels of total or Ser129-phosphorylated α-syn elevated with increasing amounts of transfected α-syn cDNA (Fig. 1a). Expression of all samples was assessed, revealing that the levels of Ser129-phosphorylated α-syn positively correlated with those of total α-syn from endogenous proteins (n = 12, r
2 = 0.885) (Fig. 1a). The same correlation in levels between Ser129-phosphorylated α-syn and total α-syn was observed in conditioned media (CM) (n = 9, r
2 = 0.908), although endogenous α-syn was undetectable (Fig. 1b). These findings showed that Ser129-phosphorylation was modulated at a constant rate in proportion to levels of total α-syn, and this relationship was also observed in secreted α-syn.
Effects of calcium on Ser129-phosphorylation of α-syn
To test the effect of intracellular Ca2+ on Ser129-phosphorylation of α-syn, we incubated SH-SY5Y cell lines, which stably expressed wild-type α-syn (wt-aS/SH #4) [8], for 8 h in media containing 5 μM calcium ionophore A23187. The levels of Ser129-phosphorylated α-syn significantly increased after 4 h incubation (1.99 ± 0.48-fold increase at 4-h incubation, P = 0.033; 3.40 ± 0.45-fold increase at 8-h incubation, P = 0.001, n = 5, each group), compared with vehicle control cells (Fig. 2a). Cells were then incubated with various concentrations of A23187 for 4 h. Phosphorylated α-syn levels significantly increased (1.64 ± 0.31-fold increase at 2.5 μM, P = 0.019; 2.03 ± 0.17-fold increase at 5.0 μM, P < 0.001; 2.20 ± 0.40-fold at 10 μM, P = 0.004, n = 6, each group) (Fig. 2b). However, the levels of total α-syn were not altered by A23187 (Fig. 2a and b). The A23187-mediated Ser129-phosphorylation of α-syn (2.39 ± 0.14-fold increase) was significantly inhibited by the addition of EGTA (1.36 ± 0.04-fold increase at 0.5 mM, P = 0.015; 1.00 ± 0.39-fold increase at 1.0 mM, P = 0.007, n = 3, each group) (Fig. 2c). Additionally, A23187-mediated Ser129-phosphorylation of α-syn (2.03 ± 0.04-fold increase) was significantly inhibited by adding BAPTA-AM (1.66 ± 0.07-fold increase at 1.0 μM, P = 0.015; 1.31 ± 0.05-fold increase at 10 μM, P < 0.001, n = 3, each group) (Fig. 2d). EGTA and BAPTA-AM have been shown to chelate extracellular and intracellular Ca2+, respectively [3, 9]. The present findings showed that A23187-mediated Ser129-phosphorylation was caused by raising intracellular Ca2+ concentrations from extracellular sources. A23187-mediated Ser129-phosphorylation of α-syn (2.09 ± 0.05-fold increase) was significantly blocked by the addition of CaM inhibitor W-7 from 0.05 μM (1.61 ± 0.05-fold increase, P = 0.002) and different CaM inhibitor calmidazolium chloride from 5.0 μM (1.47 ± 0.07-fold increase, P = 0.030, n = 3, each group), compared with vehicle control cells (Fig. 2e and f). These findings showed that CaM was involved in A23187-mediated Ser129-phosphorylation.
To test whether A23187 enhanced Ser129-phosphorylation of α-syn in neurons, we examined the effects of A23187 in rat primary cortical neurons. The levels of Ser129-phosphorylated α-syn significantly increased with 0.25 μM A23187 (2.58 ± 0.59-fold increase, P = 0.013, n = 5), compared with vehicle control cells (Additional file 1: Figure S1a). However, the levels of total α-syn remained unaltered. Unlike wt-aS/SH cells, the A23187 effect was attenuated at a higher concentration (Additional file 1: Figure S1A). A23187-mediated Ser129-phosphorylation of α-syn (1.78 ± 0.13-fold increase) was significantly inhibited by 0.5 mM EGTA (1.28 ± 0.17-fold increase, P = 0.023, n = 4, each group), and A23187-mediated Ser129-phosphorylation of α-syn (3.30 ± 0.28-fold increase) was significantly inhibited by BAPTA-AM (2.42 ± 0.31-fold increase at 0.5 μM, P = 0.010; 2.00 ± 0.20-fold increase at 1.0 μM, P < 0.001, n = 5, each group) (Additional file 1: Figure S1b and c). Additionally, A23187-mediated Ser129-phosphorylation of α-syn (1.56 ± 0.10-fold increase) was blocked by 20 μM W-7 (0.99 ± 0.20-fold increase, P = 0.021, n = 3, each group) (Additional file 1: Figure S1d). These findings showed that A23187 similarly enhanced Ser129-phosphorylation of α-syn via increased influx of extracellular Ca2+ and CaM in primary cortical neurons.
Effects of mitochondrial complex I inhibition on Ser129-phosphorylation of α-syn
To assess the effects of mitochondrial complex I inhibition on Ser129-phosphorylation of α-syn, we incubated wt-aS/SH cells in media containing either 1 mM MPP+ or 5 μM rotenone for 16 h. In MPP+-treated cells, the levels of Ser129-phosphorylated α-syn significantly increased and peaked after 12 h (2.01 ± 0.21-fold increase, P = 0.034, n = 3), compared with vehicle control cells (Fig. 3a). In rotenone-treated cells, Ser129-phosphorylated α-syn levels significantly increased and peaked after 8 h (1.84 ± 0.28-fold increase, P = 0.008, n = 3) (Fig. 3a). After incubation for 12 h, Ser129-phosphorylated α-syn levels significantly increased from 1 mM of MPP+ (2.30 ± 0.42-fold increase, P = 0.006, n = 3) or 5 μM of rotenone (1.82 ± 0.14-fold increase, P = 0.030, n = 3) in a dose-dependent manner (Fig. 3b). Total α-syn levels remained unchanged by MPP+ or rotenone (Fig. 3a and b).
To identify the mechanism of mitochondrial complex I inhibition-mediated Ser129-phosphorylation of α-syn, we treated wt-aS/SH cells with 1 mM MPP+ in the presence or absence of Ca2+ chelators. MPP+-mediated Ser129-phosphorylation of α-syn (2.38 ± 0.96-fold increase,) was significantly inhibited by BAPTA-AM (1.08 ± 0.06-fold increase at 10 μM, P = 0.001; 1.04 ± 0.20-fold increase at 20 μM, P = 0.008, n = 3, each group) (Fig. 4a). To assess whether MPP+-mediated Ser129-phosphorylation of α-syn was due to Ca2+ leakage from damaged mitochondria, cells were treated with extracellular Ca2+ chelator EGTA. MPP+-mediated Ser129-phosphorylation of α-syn (1.49 ± 0.04-fold increase) was significantly inhibited by EGTA (1.06 ± 0.15-fold increase at 0.1 mM, P = 0.013; 0.89 ± 0.11-fold increase at 0.5 mM, P = 0.001; 0.85 ± 0.23-fold increase at 1.0 mM, P = 0.016, n = 5, each group) (Fig. 4b). Similarly, rotenone-mediated Ser129-phosphorylation of α-syn (1.83 ± 0.19-fold increase) was significantly inhibited by 20 μM BAPTA-AM (0.97 ± 0.18-fold increase, P = 0.001, n = 3, each group) (Fig. 4c). Rotenone-mediated Ser129-phosphorylation of α-syn (1.72 ± 0.08-fold increase, n = 3) was significantly inhibited by EGTA (1.19 ± 0.05-fold increase at 0.5 mM, P = 0.001; 1.08 ± 0.13-fold increase at 1.0 mM, P < 0.001, n = 3, each group) (Fig. 4d). Additionally, Ser129-phosphorylation of α-syn by MPP+ (2.04 ± 0.06-fold increase) was inhibited by W-7 (1.34 ± 0.10-fold increase at 5.0 μM, P = 0.025; 0.86 ± 0.09-fold increase at 50 μM, P < 0.001, n = 3, each group) (Fig. 4e). Ser129-phosphorylation of α-syn by rotenone (1.79 ± 0.08-fold increase) was inhibited by 50 μM W-7 (1.20 ± 0.06-fold increase, P < 0.001, n = 3, each group) (Fig. 4f). These findings showed that MPP+ and rotenone-mediated Ser129-phosphorylation of α-syn was the result of increased intracellular Ca2+ concentrations from extracellular sources, and CaM could modulate this effect.
To determine whether mitochondrial complex I inhibition enhances Ser129-phosphorylation of α-syn in neurons, we examined the effect of rotenone in rat primary cortical neurons. Ser129-phosphorylated α-syn levels significantly increased by rotenone treatment (2.31 ± 0.59-fold increase at 1.0 nM, P < 0.001; 1.68 ± 0.09-fold increase at 10 nM, P = 0.036, n = 4, each group), compared with vehicle control cells (Additional file 2: Figure S2a). The effect of rotenone on Ser129-phosphorylation peaked at 1.0 nM. However, total α-syn levels remained unchanged. The rotenone-mediated Ser129-phosphorylation of α-syn (1.68 ± 0.09-fold increase) was significantly inhibited by BAPTA-AM (1.16 ± 0.19-fold increase at 0.5 μM, P = 0.050; 0.80 ± 0.12-fold increase at 1.0 μM, P = 0.004, n = 5, each group). The rotenone-mediated Ser129-phosphorylation of α-syn (1.54 ± 0.11-fold increase) was significantly inhibited by 0.5 mM EGTA (1.17 ± 0.04-fold increase, P = 0.005, n = 5, each group) (Additional file 2: Figure S2b and c). Additionally, rotenone-mediated Ser129-phosphorylation of α-syn (1.89 ± 0.14-fold increase) was significantly inhibited by 20 μM W-7 (1.24 ± 0.15-fold increase, P = 0.020, n = 3, each group) (Additional file 2: Figure S2d). These findings showed that rotenone enhanced Ser129-phosphorylation of α-syn by increased influx of extracellular Ca2+ and CaM in primary cortical neurons.
Role of the proteasome pathway in mitochondrial complex I inhibition-mediated Ser129-phosphorylation of α-syn
To assess whether mitochondrial complex I inhibition enhances Ser129-phosphorylation of α-syn by impairing the degradation system, we investigated the metabolic fates of α-syn using cycloheximide (CHX)-chase experiments in wt-aS/SH cells. As previously reported, the levels of Ser129-phosphorylated α-syn rapidly decreased (Fig. 5a). When cells were pretreated with 10 μM rotenone for 8 h followed by CHX-chase experiments, Ser129-phosphorylated α-syn levels similarly decreased, although the starting levels were higher with rotenone treatment (Fig. 5a). This finding suggested that the rotenone-enhanced Ser129-phosphorylation was independent of degradation. To determine the degradation effect of rotenone-mediated Ser129-phosphorylation of α-syn, we performed CHX-chase experiments using cells treated with proteasome inhibitor MG132 in the absence or presence of rotenone. As previously reported, in the absence of rotenone, the rapid decrease in Ser129-phosphorylated α-syn was blocked (Fig. 5a). In the presence of rotenone, Ser129-phosphorylated α-syn attenuation was also blocked by MG132 (Fig. 5a). The levels of total α-syn remained unchanged in the CHX-chase experiments. To further compare the metabolic fates of α-syn in the absence or presence of rotenone, Ser129-phosphorylated α-syn levels were simultaneously assessed in vehicle control and rotenone-treated cell lysates on the same gel. The attenuation rate of Ser129-phosphorylated α-syn was comparable between vehicle control and rotenone-treated cells (Fig. 5b). These findings showed that rotenone did not impair the proteasome pathway under this experimental condition, and the rotenone-induced increase in Ser129-phosphorylation was suppressively controlled by pushing Ser129-phosphorylated α-syn along the proteasome pathway.
Role of Ser129-phosphorylation in α-syn solubility change by mitochondrial complex I inhibition
To determine the role of Ser129-phosphorylation in the accumulation of insoluble α-syn, we first examined whether detergent-insoluble α-syn proteins were present in rotenone-treated cells. Wt-aS/SH cells were incubated in low concentrations of rotenone (10 nM and 50 nM) for 5 days. The cells were then separated into 1% Triton X-100-soluble and 1% Triton X-100-insoluble fractions by centrifugation at 100,000×g for 30 min. The 1% Triton X-100-insoluble fractions were resolved in a solution containing 8 M urea / 2% SDS. Western blots of 1% Triton X-100-soluble fractions showed that 10 and 50 nM rotenone elevated Ser129-phosphorylated α-syn levels without altering total α-syn levels. When we analyzed 1% Triton X-100-insoluble fractions, 50 nM rotenone generated insoluble total α-syn (Fig. 6a). The levels of insoluble total α-syn increased by 3.89 ± 0.84-fold (P = 0.011, n = 4) as compared with vehicle control cells (Fig. 6a). Although the Ser129-phosphorylated α-syn signals were very faint, insoluble Ser129-phosphorylated α-syn levels also increased by 14.40 ± 6.23-fold (P = 0.004, n = 4) (Fig. 6a). We then performed CHX-chase experiments using cells pretreated with 50 nM rotenone for 5 days, followed by incubation in media containing DMSO or 10 nM MG132 for 8 h. In the absence of MG132, insoluble Ser129-phosphorylated α-syn levels rapidly attenuated before 120 min (Fig. 6b). MG132 significantly blocked this attenuation (Fig. 6b). However, insoluble total α-syn proteins remained unaltered during the observation time in the absence or presence of MG132 (Fig. 6b). These findings showed that Ser129-phosphorylation also pushed rotenone-induced insoluble α-syn through the proteasome pathway.
Ser129-phosphorylation-mediated α-syn clearance in the proteasome and lysosome pathways
To analyze Ser129-phosphorylation-mediated targeting of insoluble α-syn proteins in the degradation pathway, we examined the relationship between the proteasome and lysosome pathways. Wt-aS/SH cells were incubated in media containing 10 nM of selective proteasome inhibitor epoxomicin or 100 μM of chloroquine for 16 h. As shown in Fig. 7a (left panels), epoxomicin did not affect the levels of 1% Triton X-100-insoluble Ser129-phosphorylated α-syn, but did increase 1% Triton X-100-soluble Ser129-phosphorylated α-syn levels. Additionally, epoxomicin did not affect 1% Triton X-100-insoluble total α-syn levels. Chloroquine treatment failed to induce expression of insoluble Ser129-phosphorylated α-syn, but insoluble total α-syn did accumulate (Fig. 7a, middle panels). When cells were co-incubated in media containing epoxomicin and chloroquine, insoluble Ser129-phosphorylated α-syn proteins were generated in conjunction with the accumulation of insoluble total α-syn (Fig. 7a
right panels). These findings showed that proteasomal targeting of insoluble Ser129-phosphorylated α-syn was more activated under lysosome inhibition. To further test the effect of Ser129-phosphorylation on the metabolism of insoluble α-syn proteins, we compared insoluble total α-syn levels between cells expressing wild-type α-syn (wt-aS/SH cells) and Ser129-phosphorylation incompetent S129A mutant α-syn (S129A-aS/SH cells). In the wt-aS/SH cells, epoxomicin and chloroquine treatment yielded insoluble total α-syn (11.00 ± 1.17-fold increase as compared with vehicle control cells, n = 5) more abundantly than chloroquine single treatment (6.51 ± 0.82-fold increase, P < 0.001, n = 5) (Fig. 7b). Additionally, epoxomicin and chloroquine treatment elevated insoluble total α-syn levels in wt-aS/SH more greatly than S129A-aS/SH cells (7.49 ± 1.27-fold increase, P < 0.001, n = 5) (Fig. 7b). S129A-aS/SH cells exhibited no Ser129-phosphorylated α-syn signals in the insoluble fractions (Fig. 7b). These findings showed that Ser129-phosphorylation prevented insoluble α-syn accumulation by evoking proteasomal clearance complementary to lysosomal clearance.
Effect of Ser129-phosphorylation on α-syn aggregate formation in a rat AAV-mediated α-syn overexpression model
To determine whether Ser129-phosphorylation affects the formation of α-syn aggregates in vivo, we quantified the number of α-syn aggregates in the striatum of the rat AAV-mediated α-syn overexpression model. These samples were obtained from our previous study [18]. In rats expressing A53T mutant α-syn, the striatal α-syn aggregates were extensively phosphorylated at Ser129 (Fig. 8). The number of Ser129-phosphorylated α-syn-positive aggregates accounted for more than 50% of total α-syn aggregates at 2 and 4 weeks (62.1% in 2 weeks and 55.7% in 4 weeks) after viral injection. Additionally, the number of total α-syn aggregates increased to 265.3 ± 33.4/mm3 at 2 weeks (n = 5) and 591.9 ± 34.6/mm3 at 4 weeks (P = 0.001, n = 3). Rats expressing A53T plus S129A double-mutant α-syn had 285.8 ± 103.9/mm3 α-syn aggregates at 2 weeks (n = 4) and 585.4 ± 103.3/mm3 at 4 weeks (n = 3). There was no significant difference in the number of total α-syn aggregates between rats expressing A53T single mutant and A53T plus S129A double-mutant α-syn (P = 1.000). These findings showed that Ser129-phosphorylation had no impact on the accumulation of α-syn aggregates in vivo.