Mechanisms underlying extensive Ser129-phosphorylation in α-synuclein aggregates
© The Author(s). 2017
Received: 17 April 2017
Accepted: 4 June 2017
Published: 15 June 2017
Parkinson’s disease (PD) is characterized neuropathologically by intracellular aggregates of fibrillar α-synuclein, termed Lewy bodies (LBs). Approximately 90% of α-synuclein deposited as LBs is phosphorylated at Ser129 in brains with PD. In contrast, only 4% of total α-synuclein is phosphorylated at Ser129 in brains with normal individuals. It is unclear why extensive phosphorylation occurs in the pathological process of PD. To address this issue, we investigated a mechanism and role of Ser129-phosphorylation in regulating accumulation of α-synuclein. In CHO cells, the levels of Ser129-phosphorylated soluble α-synuclein were maintained constantly to those of total α-synuclein in intracellular and extracellular spaces. In SH-SY5Y cells and rat primary cortical neurons, mitochondrial impairment by rotenone or MPP+ enhanced Ser129-phosphorylation through increased influx of extracellular Ca2+. This elevation was suppressively controlled by targeting Ser129-phosphorylated α-synuclein to the proteasome pathway. Rotenone-induced insoluble α-synuclein was also targeted by Ser129-phosphoryation to the proteasome pathway. Experiments with epoxomicin and chloroquine showed that proteasomal targeting of insoluble Ser129-phosphorylated α-synuclein was enhanced under lysosome inhibition and it reduced accumulation of insoluble total α-synuclein. However, in a rat AAV-mediated α-synuclein overexpression model, there was no difference in the number of total α-synuclein aggregates between A53T mutant and A53T plus S129A double mutant α-synuclein, although Ser129-phosphorylated α-synuclein-positive aggregates were increased in rats expressing A53T α-synuclein. These findings suggest that Ser129-phosphorylation occurs against stress conditions, which increases influx of extracellular Ca2+, and it prevents accumulation of insoluble α-synuclein by evoking proteasomal clearance complementary to lysosomal one. However, Ser129-phosphorylation may provide an ineffective signal for degradation-resistant aggregates, causing extensive phosphorylation in aggregates.
KeywordsParkinson’s disease α–Synuclein Phosphorylation Aggregation Mitochondrial impairment Proteasome pathway
Although Parkinson’s disease (PD) is the most common movement disorder, there is currently no treatment for slowing or stopping disease progression. Identification of a promising target for PD therapies needs to elucidate how nigral dopaminergic neurons are lost and how Lewy bodies (LBs) and Lewy neurites are formed in surviving neurons, because these are key features of PD [6, 10]. LBs and Lewy neurites are aggregates of fibrillar α-synuclein (α-syn) . As a characteristic point of LBs, approximately 90% of α-syn deposited in LBs is extensively phosphorylated at Ser129 [1, 7]. In sharp contrast, only 4% or less of total α-syn is phosphorylated at this residue in brains from individuals without PD [1, 7]. This disparity suggests that the levels of Ser129-phosphorylated α-syn are tightly regulated under physiological conditions, and extensive Ser129-phosphorylation occurs in conjunction with LB formation and dopaminergic neurodegeneration in PD. In a Drosophila model of PD, co-expression of α-syn and Drosophila G-protein-coupled receptor kinase 2 (Gprk2) was shown to generate Ser129-phosphorylated α-syn and enhance α-syn toxicity . In a rat recombinant adeno-associated virus (rAAV)-based model, co-expression of A53T α-syn and human G-protein-coupled receptor kinase 6 (GRK6) accelerated α-syn-induced degeneration of dopaminergic neurons . Conversely, the co-expression of wild-type α-syn and polo-like kinase 2 (PLK2) attenuated a loss of dopaminergic neurons . Although the effect of Ser129-phosphorylation is still under debate, these findings show that Ser129-phosphorylation modulates α-syn toxicity. One simple explanation that links extensive α-syn phosphorylation in LBs with neurodegeneration is that Ser129-phosphorylation enhances α-syn aggregation and exerts a toxic or protective effect against neuronal damage. However, most in vitro studies have shown that Ser129-phosphorylation has no accelerating effect on the fibril formation of α-syn. The mechanism of extensive phosphorylation of α-syn in LBs remains unclear.
To address the issue, we first assessed how levels of phosphorylated α-syn are maintained in intra- and extracellular spaces using Chinese hamster ovary (CHO) cells. We then investigated how external stimulants affect α-syn phosphorylation in SH-SY5Y cells and primary rat cortical neurons. As external stimulants, we focused on intracellular Ca2+ and mitochondrial impairment, because previous studies demonstrated that α-syn phosphorylation by GRK5 is activated by Ca2+ and calmodulin (CaM), and α-syn can bind to CaM in a calcium-dependent manner [13, 15]. Mitochondrial complex I inhibition via administration of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) or rotenone causes a loss of dopaminergic neurons, and mitochondrial complex I dysfunction is found in PD patients, indicating the involvement of mitochondrial impairment in dopaminergic neurodegeneration of PD [11, 19, 21]. We also focused on the proteasome pathway as the competitive machinery, because soluble Ser129-phosphorylated α-syn was shown to degrade by the proteasome pathway . The present study describes the regulation and dysregulation mechanisms of Ser129-phosphorylaion as an interplay among calcium, mitochondrial impairment, and proteasome clearance.
Materials and methods
Plasmid cDNA and reagents
Human wild-type α-syn cDNA was subcloned into the pcDNA3.1(+) vector (Thermo Fisher Scientific). Reagents were purchased from Sigma unless otherwise stated.
Cell lines, rat primary cortical neuron cultures, and transfection
Human dopaminergic neuroblastoma SH-SY5Y cells (ECACC #94030304) were maintained in Ham’s F-12/Eagle’s minimum essential medium supplemented with 15% fetal bovine serum (FBS, Thermo Fisher Scientific), 2 mM l-glutamine (Thermo Fisher Scientific) and 1 × non-essential amino acids. SH-SY5Y cell lines stably expressing wild-type α-syn (wt-aS/SH #4) and Ser129-phosphorylation-incompetent S129A mutant α-syn (S129A-aS/SH #10) were used as described previously . CHO cells were maintained in Ham’s F-12 supplemented with 10% FBS. Primary cortical neuron cultures were prepared from Sprague-Dawley rats. Neurons were isolated from the neocortex of embryonic day 18 rats and dissociated cells were plated at a density of 1 × 106 cells on poly-D-lysine-coated 6-well plates. Neurons were maintained in serum-free neurobasal medium supplemented with B27 and GlutaMAX (Thermo Fisher Scientific) . At intervals of 2 days, half of the plating medium was renewed. At 21 days in vitro (DIV), neurons were harvested for experiments. For transient transfection, cells were transfected with cDNA by using LipofectAMINE Plus reagent (Thermo Fisher Scientific) according to the manufacture’s protocol. The cells were harvested at 48 h post-transfection.
To assess effects of intracellular Ca2+, at 16 h after plating wt-aS/SH cells onto 6-well plates, we checked the cells to be ∼80% confluent, and then the cells were incubated in fresh medium containing the indicated concentrations of calcium ionophore A23187 with or without the indicated concentrations of Ca2+ chelators (EGTA or BAPTA-AM) or CaM inhibitors (W-7 or calmidazolium chloride). In rat primary cortical neurons, they were cultured for 21 DIV and then incubated in fresh medium containing the indicated concentrations of A23187 with or without the indicated concentrations of EGTA, BAPTA-AM, or W-7. For mitochondrial complex I inhibition, at 16 h after plating parental SH-SY5Y cells or wt-aS/SH cells onto 6-well plates, we checked the cells to be ∼80% confluent, and then the cells were incubated in fresh medium containing the indicated concentrations of MPP+ iodide or rotenone with or without the indicated concentrations of EGTA, BAPTA-AM, or W-7. At 21 DIV, rat primary cortical neurons were also treated with the indicated concentrations of rotenone with or without EGTA, BAPTA-AM, or W-7. As a vehicle control, cells were treated with the same concentration of DMSO, which was used for dissolving chemicals other than EGTA.
To assess the metabolic fates of proteins in the cells, we performed experiments using the de novo protein synthesis inhibitor cycloheximide (CHX) . At 16 h post-plating wt-aS/SH cells onto 6-well plates, we confirmed the cells to be ∼80% confluent. The cells were incubated in fresh medium containing 100 μM CHX for the indicated times. To test the effect of mitochondrial impairment and inhibition of the proteasome pathway on the metabolic fates of target proteins, we pre-incubated the cells with 10 μM rotenone or 0.1% DMSO for 8 h and treated them with CHX in the presence or absence of 10 μM MG132 for the indicated times.
To assess the relationship between the proteasome and lysosome pathways on the expression of α-syn, we treated cells with lysosome inhibitor chloroquine in the absence or presence of proteasome inhibitor epoxomicin (Peptide Institute Inc., Japan). At 16 h post-plating wt-aS/SH cells onto 6-well plates, we confirmed the cells to be ∼80% confluent. The cells were incubated in fresh medium containing 100 μM chloroquine with or without 100 nM epoxomicin for 24 h for the indicated times.
Preparation of protein extracts and conditioned media
For preparation of cell lysates, cultured cells were suspended in buffer A (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 10% glycerol, 1 × protease inhibitor cocktail [Roche Diagnostic], 1 mM EDTA, 5 mM NaF, 1 mM Na3VO4, 1 × phosSTOP [Roche Diagnostic]), sonicated at 30 W for 1 s 5 times, and kept on ice for 30 min. After centrifugation at 12,000×g for 30 min, the resulting supernatant was collected and stored at −80 °C until use. Protein concentrations were measured by the BCA assay (Thermo Fisher Scientific).
For preparation of the conditioned media (CM), cells were plated onto 10 cm dishes. When cells were 90% confluent, we exchanged the growth media with 6 mL of Opti-MEM (Thermo Fisher Scientific). After further 24 h incubation, CM was collected and centrifuged at 6,000×g for 5 min to remove cell debris. Immediately, 6 mL of CM was added with 1/4 volume of 100% trichloroacetic acid (TCA), incubated for 30 min on ice, and centrifuged at 14,000×g for 5 min. The resultant pellet was washed three times with 300 μL of cold acetone, air dried, and dissolved in 100 μL of Laemmli’s sample buffer containing 2.5% β-mercaptoethanol.
For analyzing the levels of insoluble α-syn, 1% Triton X-100 cell lysates were separated by centrifugation at 100,000×g for 30 min . Supernatants were collected as soluble fractions. Resultant pellets were washed with buffer A by centrifugation at 100,000×g for 30 min. The pellets were added with the solution containing 8 M urea and 2% SDS, sonicated at 30 W for 1 s 10 times, and then centrifuged at 100,000×g for 30 min. Resultant supernatants were used as insoluble fractions.
Samples were denatured at 95 °C for 5 min in Laemmli’s sample buffer containing 2.5% β- mercaptoethanol. Equal protein amounts of denatured samples were subjected to SDS-PAGE on 13.5% polyacrylamide gels and then transferred to PVDF membranes (Immobilon-P, Millipore). The transferred membrane was incubated in phosphate-buffered saline (PBS) (10 mM phosphate, 137 mM NaCl, 2.7 mM KCl) containing 4% paraformaldehyde (PFA) with 0.1% glutaraldehyde for detecting Ser129-phosphorylated α-syn for 30 to 60 min or without glutaraldehyde for detecting other proteins as described previously . After incubation, the membrane was washed in Tris-buffered saline (TBS, 25 mM Tris-HCl, pH 7.4, 137 mM NaCl, 2.7 mM KCl) containing 0.05% (v/v) Tween 20 (TBS-T) for 10 min 3 times. The membrane was blocked by TBS-T containing 5% skim milk for 30 min, incubated in TBS-T containing 2.5% skim milk and primary antibody overnight in the cold room, and further incubated in the same buffer containing the corresponding secondary antibody overnight in the cold room. When we detected phosphorylated α-syn, 50 mM NaF was added to TBS-T containing skim milk. To visualize the signal, membranes were treated with ECL plus (Thermo Fisher Scientific) for detection of total α-syn, including non-phosphorylated and Ser129-phosphorylated forms, and phosphorylated α-syn. Other proteins were detected by supersignal West Pico chemiluminescent substrate (Thermo Fisher Scientific). Signals were recorded using a CCD camera, VersaDog 5000 (Bio-Rad). Levels of total α-syn and Ser129-phosphorylated α-syn were estimated by measuring band intensities with Quantity One software (Bio-Rad). For more quantitative estimation of the expression levels of total α-syn and Ser129-phosphorylated α-syn, we used purified recombinant α-syn proteins and Ser129-phosphorylated α-syn proteins as standards [8, 12]. A set of diluted standards was subjected to SDS-PAGE along with samples. After quantifying band intensities of samples, we corrected their relative intensities by plotting them on the standard curve. The following antibodies were used: anti-α-syn (Syn-1, mouse monoclonal, which recognizes total α-syn independently of Ser129-phosphorylation, BD Transduction Laboratories), anti-Ser129-phosphorylated α-syn (EP1536Y, rabbit monoclonal, Abcam), and anti-β-actin (AC-15, mouse monoclonal, Sigma).
rAAV-based rat model of Parkinson’s disease
The experiments using rats had been approved by the Animal Subjects Committee of Yamagata University . We used rats expressing familial PD-linked A53T mutant or A53T plus S129A double mutant α-syn by unilaterally injecting a rAAV2 vector in the rat substantia nigra. These rats were the same as ones reported in our previous paper . Methods concerning rAAV particles preparation and immunohistochemistry were described in this paper . We analyzed the brain sections that were already immunostained with anti-human α-syn (LB509, 1: 200; Zymed Laboratories) and anti-Ser129-phosphorylated α-syn antibodies (EYPSYN-01, 1: 200; courtesy of Eisai). Briefly, the brains fixed by 4 ~ 8% PFA/PBS were coronally sectioned on a freezing microtome at a thickness of 30 μm. Sections were collected in 10 series to be regularly spaced at intervals of 300 μm from each other. For counting the number of α-syn aggregates in the striatum, we analyzed the injected sides of 4 to 6 sections around bregma by the optical fractionator method using Stereo Investigator software (MicroBrightField) . The region of interest was traced and sampled using an Olympus BX50 microscope at a magnification of 4× and 10×, respectively. By setting the x-y sampling grid size equal to the counting frame size (330 × 330 μm), we scanned the whole area of the striatum on the section. We counted the number of α-syn aggregates larger than 5 μm in diameter. The size of aggregates was judged by measuring the maximum diameter with a “quick measure circle” tool or a “grid indicator” tool (5 μm × 5 μm/ one square) in this software.
We performed each experiment at least three times. Data are expressed as mean ± standard deviation and n represents the number of samples. Homogeneity of variances was checked with Levene’s test. If the variances were homogenous, comparisons were performed by one-way analysis of variance (ANOVA) with Bonferroni’s post hoc test. In case of nonhomogeneity of variances, comparisons were performed by Welch-ANOVA with Games-Howell post hoc test (SPSS version 17, IBM). P values <0.05 were considered statistically significant.
Regulation of Ser129-phosphorylated α-syn in intra- and extracellular spaces
Effects of calcium on Ser129-phosphorylation of α-syn
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 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
Role of Ser129-phosphorylation in α-syn solubility change by mitochondrial complex I inhibition
Ser129-phosphorylation-mediated α-syn clearance in the proteasome and lysosome pathways
Effect of Ser129-phosphorylation on α-syn aggregate formation in a rat AAV-mediated α-syn overexpression model
To determine the mechanisms and biological role of Ser129-phosphorylation in α-syn aggregate formation, we first examined the regulation of Ser129-phosphorylation in normal soluble α-syn. Our results showed that α-syn was phosphorylated at Ser129 in proportion to the levels of total α-syn. This phenomenon could be explained by two possibilities: 1) responsible kinases are constitutively active in a substrate-dose dependent manner; or 2) this regulation system is maintained by dephosphorylating or degrading excess amounts of Ser129-phosphorylated α-syn in reaction to total α-syn levels. To further explore these possibilities, we examined how the regulation mechanism of Ser129-phosphorylation was disrupted. Our results showed that the intracellular Ca2+ concentration was a key factor in kinase-modulated Ser129-phosphorylation. In support of this, Ser129-phosphorylation required CaM function, which controls a variety of kinases in a Ca2+-dependent manner [16, 22]. In mitochondrial complex I inhibition by rotenone or MPP+, Ser129-phosphorylation was enhanced through an increased influx of extracellular Ca2+. However, the CHX-chase experiments showed that rotenone-induced Ser129-phosphorylated α-syn was targeted to the proteasome pathway at the same rate as normally phosphorylated α-syn. It should be noted that in the present experimental condition, ATP-dependent proteasome activity was not lost by mitochondrial impairment. These findings suggested that proteasomal targeting played a role in suppressively controlling Ser129-phosphorylated α-syn levels. Then, we examined the role of Ser129-phosphorylation in insoluble α-syn accumulation. Chronic treatment with a low concentration of rotenone for 5 days induced small amounts of insoluble α-syn. CHX-chase experiments with MG132 showed that insoluble Ser129-phosphorylated α-syn was targeted to the proteasome pathway. This finding suggested that proteasomal targeting also played a role in suppressing accumulation of insoluble Ser129-phosphorylated α-syn.
These findings were consistent with results from a previous yeast study showing that Ser129-phosphorylation and sumoylation push α-syn aggregates into the proteasome pathway and autophagy-lysosome pathway, respectively, and Ser129-phosphorylation rescues autophagy-lysosome clearance of α-syn by promoting proteasomal clearance when sumoylation is impaired . This previous study also showed that Ser129-phosphorylation pushed soluble α-syn monomers into autophagy-lysosomal and proteasome pathways, which was inconsistent with the present results. Our data showed that Ser129-phosphorylation pushed soluble α-syn through the proteasome pathway. This inconsistency could be a result of a difference in yeast and mammalian cell models. Another study reported that PLK2 overexpression selectively induces autophagic clearance of soluble α-syn . However, our results were inconsistent with this finding. We did not overexpress kinases for assessing the effects of Ser129-phosphorylation, which is physiologically mediated by a set of endogenous kinases in cells. Overexpression of each kinase may exert different effects on the degradation of Ser129-phosphorylated α-syn, because PLK2-mediated autophagic clearance of α-syn has also been shown to require binding of PLK2 to α-syn .
The present data also raised a question as to relationship between effects of Ser129-phosphorylation on proteasomal targeting of soluble and insoluble α-syn proteins and extensive phosphorylation in α-syn aggregates. To address this, we assessed α-syn aggregates in a rat rAAV model expressing A53T α-syn with or without the S129A mutation. The present data showed that Ser129-phosphorylation had no impact on α-syn aggregate accumulation despite extensive phosphorylation. A previous study demonstrated that fibrillar α-syn proteins were phosphorylated by casein kinase (CK) I or CK II, and they were not good substrates for phosphatases in vitro . Because Ser129-phosphorylation has a role in removing excess amounts of α-syn, α-syn aggregates may continuously undergo phosphorylation. However, it may be ineffective against α-syn proteins, which are degradation-resistant (Fig. 9). Ser129-phosphorylation of α-syn should consider two different aspects: 1) Ser129-phosphorylation may have a protective effect complementary to the lysosome pathway to degrade excess amounts of α-syn; 2) extensive targeting of Ser129-phosphorylated α-syn and degradation-resistant proteins may put a burden on the proteasome pathway. Further studies focused on the relationship between Ser129-phosphorylation and degradation system would provide insight into the potential of modulating the amounts of Ser129-phosphorylation as a new strategy for PD therapy.
We report that a role of Ser129-phosphorylation in regulating α-syn expression levels is associated with extensive phosphorylation in α-syn aggregates. The levels of Ser129-phosphorylated α-syn were suppressively maintained to be constant to those of total α-syn in intracellular and extracellular spaces. Although mitochondrial impairment by rotenone or MPP+ enhanced Ser129-phosphorylation through increased influx of extracellular Ca2+, this elevation was suppressively controlled by targeting Ser129-phosphorylated α-syn to the proteasome pathway. This targeting was seen in insoluble α-syn induced by rotenone. Additionally, proteasomal targeting of insoluble Ser129-phosphorylated α-syn was promoted under lysosome inhibition. This complementary action prevented accumulation of insoluble total α-syn. However, in a rat AAV-mediated α-syn overexpression model, Ser129-phosphorylation did not affect α-syn aggregate formation. Taken together, we propose a model that extensive phosphorylation in α-syn aggregates was consequently generated by an ineffective action of Ser129-phosphorylation for removing degradation-resistant α-syn aggregates.
This work was supported in part by a grant from Takeda Research Foundation (HS) and a Gran-in-Aid for Scientific Research (C) (No. 898720) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (SA).
SA designed experiments, interpreted data, and wrote the manuscript. SA and AS performed experiments using cultured cells. SK prepared rat primary cortical neurons. HS prepared rAAV-based animal samples. TK supervised the project and revised the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
All applicable institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution at which the studies were conducted.
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