Abstract
Lewy bodies are made from insoluble, phosphorylated α-synuclein, but the earliest changes that precipitate such pathology still remain conjecture. In this study, we quantify and identify relationships between the levels of the main pathologic form of phosphorylated α-synuclein over the course of Parkinson’s disease in regions affected early through to end-stage disease. Brain tissue samples from 33 cases at different disease stages and 13 controls were collected through the Australian Network of Brain Banks. 500 mg of frozen putamen (affected preclinically) and frontal cortex (affected late) was homogenized, fractionated and α-synuclein levels evaluated using specific antibodies (syn-1, BD Transduction Laboratories; S129P phospho-α-synuclein, Elan Pharmaceuticals) and quantitative western blotting. Statistical analyses assessed the relationship between the different forms of α-synuclein, compared levels between groups, and determined any changes over the disease course. Soluble S129P was detected in controls with higher levels in putamen compared with frontal cortex. In contrast, insoluble α-synuclein occurred in Parkinson’s disease with a significant increase in soluble and lipid-associated S129P, and a decrease in soluble frontal α-synuclein over the disease course. Increasing soluble S129P in the putamen correlated with increasing S129P in other fractions and regions. These data show that soluble non-phosphorylated α-synuclein decreases over the course of Parkinson’s disease, becoming increasingly phosphorylated and insoluble. The finding that S129P α-synuclein normally occurs in vulnerable brain regions, and in Parkinson’s disease has the strongest relationships to the pathogenic forms of α-synuclein in other brain regions, suggests a propagating role for putamenal phospho-α-synuclein in disease pathogenesis.
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Introduction
α-Synuclein is an unstructured soluble 17–18 kDa protein making up 1% of all cytosolic proteins found in neurons [21] with approximately 15% transiently membrane bound [24]. It forms two types of pathologic inclusions in neurodegenerative disorders; protease-resistant insoluble α-synuclein fibrils in neuronal Lewy bodies and neurites in Parkinson’s disease (PD) and dementia with Lewy bodies, and less protease-resistant α-synuclein fibrils in oligodendroglial inclusions in multiple system atrophy (MSA) [8, 41]. A recent study comparing the membrane-associated form of α-synuclein across these disorders showed a massive increase largely restricted to the pathologically affected basal ganglia regions in MSA, while widespread changes in PD were not observed [41]. Surprisingly, in PD there was no increase in membrane-associated α-synuclein in either the putamen (which has an early loss of dopamine terminals) or frontal cortex (which accumulates Lewy bodies and neurites in the late stages of the disease) highlighting that idiopathic PD is not a simple, global over-expression of the protein in the regions affected by the disease process [41]. As this study assessed only a single form of α-synuclein (membrane-associated α-synuclein), and did not assess the dynamics that may occur over the course of PD, it still remains unclear which forms of α-synuclein may play a role in the slow propagation of Lewy bodies and neurites that characterize PD.
The toxic forms of α-synuclein remain contentious. Lewy bodies are made of protease-resistant insoluble filaments, but their toxicity is questioned [30]. The recent identification of Lewy bodies in grafted fetal neurons in patients with PD has suggested the propagation of toxicity through the extrusion and uptake of small α-synuclein aggregates in a prion-like process [2]. The strong finding that genetic multiplications cause PD has many believing that toxicity is caused by simply increasing the cellular monomeric form of α-synuclein [14, 38], but evidence for such an increase in idiopathic PD is limited and not without challenge [11, 46]. Recent studies suggest that soluble oligomers underlie neuronal death [7], while others suggest increased binding of the oligomers to membranes are necessary for toxicity [39, 42]. The concept that such membrane-associated forms of α-synuclein play a large role in idiopathic PD is directly challenged by the study discussed above [41]. Importantly, α-synuclein can be phosphorylated at serine 129 (S129), and most α-synuclein in Lewy bodies is phosphorylated at this site [1, 15], suggesting its phosphorylation state has an important pathogenic role. To date there have been no studies analyzing the different major forms of α-synuclein over the course of PD where Lewy body formation is known to progress to regions not initially involved in the disease. The quantitative assessment of the forms of α-synuclein in such regions involved late in the disease process should provide the required in vivo evidence for the earliest and progressive change involved in Lewy body formation in PD.
Materials and methods
Cases
Approval was obtained to assess brain tissue from 33 PD cases and 13 controls collected with consent through the Australian Network of Brain Banks. Controls had no significant neuropathology and no evidence of neurological or psychiatric disease. PD cases fulfilled the UK Parkinson’s Disease Society Brain Bank Diagnostic Criteria [20] and were levodopa-responsive and medicated throughout the disease course. The PD cases were pathologically classified into three groups according to simplified diagnostic criteria [17]; brainstem or stage IV disease (N = 14), limbic or stage V disease (N = 9) and neocortical or stage VI disease (N = 10). No case had brain injury due to non-degenerative mechanisms (such as head trauma, infarction or sepsis) or other significant neuropathological changes. Demographic details of the groups are given in Table 1.
Tissue sampling and fractionation
Half the hemisphere was freshly sectioned and frozen blocks stored at −80°C prior to fractionation. One gram of the posterior putamen and 500 mg of the mid-superior frontal cortex from each case were homogenized in 4 ml of homogenization buffer [0.32 M sucrose, 20 mM Tris–HCl pH 7.4, 5 mM EDTA, 1× protein inhibitor cocktail (EDTA free; Roche), 1× phosphatase inhibitor (Roche)], sonicated 2 × 10 s on ice and then cleared by centrifugation at 16,000g for 10 min at 4°C. The supernatant was labeled the TBS-soluble extract (S1). The pellet was washed twice with homogenization buffer, resuspended in 2 ml homogenization buffer with 5% SDS, sonicated for 2 × 10 s and centrifuged at 100,000g for 30 min at 24°C. The supernatant was labeled the SDS-soluble fraction (S2). The pellet was rinsed twice with homogenization buffer with 5%SDS, then resuspended in 400 μl 8%SDS/8 M urea for 2 h at room temperature and termed the urea-soluble/SDS-insoluble fraction (S3). All protein concentrations were analyzed using a Nanodrop1000 (Thermo scientific).
Quantitative western immunoblotting
For each gel, a previously tested sample of known antibody reactivity (20 μg/well) was run against the unknown samples as an internal control. Aliquots containing equal amount of protein samples (TBS-soluble, 20 μg; SDS-soluble and urea-soluble, 10 μg respectively) were dissolved in an equal volume aliquot of 2× Laemmli buffer (4% SDS, 20% glycerol, 0.004% bromophenol blue, 0.125 M Tris–HCl pH 6.8, 5% mercaptoethanol), boiled at 95°C for 5 min, loaded onto a 10% SDS-polyacrylamide gel and then subjected to electrophoresis with constant voltage of 100 V. After separation, protein was transferred by electrophoresis to a 0.22 mm nitrocellulose filter membrane by the application of 100 V for 1 h. Blots were blocked with 5% milk in TBST buffer (10 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.05% Tween-20) and probed using the primary antibodies suspended in 5% milk in TBST buffer at 4°C overnight. Mouse monoclonal anti-α-synuclein antibodies were applied [syn-1, 42/α-synuclein, BD Transduction Laboratories, 1:1,000; anti-phospho-α-synuclein (S129P) from Elan Pharmaceuticals, South San Francisco, California, 1:12,500] at 4°C overnight. Membranes were washed three times with TBST buffer and probed with goat anti-mouse horseradish peroxidase-conjugated secondary antibodies (Bio-Rad, 1:7,000) suspended in 5% milk in TBST buffer, applied for 1 h at room temperature. After washing, chemiluminescence was produced using an ECL kit (Amersham Biosciences). Films were scanned and the intensity of each band was quantified with Quantity One software (Bio-Rad) and expressed as arbitrary units relative to the internal control for comparison across groups.
Statistical analyses
SPSS 18 (IBM, Chicago, USA) was used for all analyses and significance established when P < 0.05. There were no correlations between the levels of the different α-synuclein species in the different regions analyzed and postmortem delay (P > 0.05). Wilcoxon Signed Rank and Median tests were used to describe the relationships between different forms of α-synuclein in different control regions. Mann–Whitney U tests were used to describe any overall difference in the levels of the different forms of α-synuclein between PD and controls. Stepwise multiple regression analyses were used to determine any relationships to disease stage and between the different forms of α-synuclein changing over the disease course.
Results
Forms of α-synuclein expressed in the putamen and frontal cortex in controls
In controls monomeric α-synuclein was the dominant species in all tissue fractions (Fig. 1). In both the putamen and frontal cortex there was more TBS-soluble α-synuclein (55–65% of relative α-synuclein amounts) compared with SDS-soluble α-synuclein (30–37% of relative α-synuclein amounts, median tests P = 0.03) and very low levels of urea-soluble α-synuclein (<10% of relative α-synuclein amounts; Fig. 1a). There was no difference between the distribution of the forms of α-synuclein between the regions examined (Wilcoxon Signed Rank test P = 0.6).
In controls S129 phosphorylation was observed in a small proportion of both the TBS-soluble (Fig. 1m; 3–8% of relative α-synuclein amounts) and SDS-soluble (5–10% of relative α-synuclein amounts) protein fractions. Because of the low relative levels of S129 phosphorylated α-synuclein, both monomeric as well as a 25 kDa species was revealed (Fig. 1m). The mobility of this higher molecular weight species is consistent with the mono-ubiquitinated α-synuclein species observed previously [1, 18], which must be in low overall abundance compared with the total amount of monomeric α-synuclein, but is a larger fraction of monomeric S129 phosphorylated α-synuclein (Fig. 1j, m). No S129 phosphorylated α-synuclein was observed in the urea-soluble fractions in controls. There was a striking regional variation in the distribution of the forms of phosphorylated α-synuclein at S129 in controls (Wilcoxon Signed Rank test P = 0.03), with much greater phosphorylation of the TBS-soluble compared with SDS-soluble protein in the putamen (65 vs. 35% of relative putamen S129P) than in frontal cortex (both fractions 49–51% of relative frontal S129P). This was largely due to greater phosphorylation (and monoubiquitination) of the TBS-soluble α-synuclein in the putamen compared with the frontal cortex (3.3× more S129P α-synuclein, Wilcoxon Signed Rank test P = 0.02).
Changes observed in Parkinson’s disease
As may have been expected, the most striking change observed in PD is the amount of higher molecular weight urea-soluble species of α-synuclein in both regions analyzed (Fig. 1b) and the overall increase in α-synuclein in the urea-soluble fraction (Fig. 1c, 7.0–8.6× increase from control levels, Mann–Whitney U tests P < 0.001). Surprisingly, this large increase in urea-soluble α-synuclein was not reflected by an increase in its phosphorylation at S129, with only a few cases having comparatively weak immunoreactive blots (data not shown). Significantly more urea-soluble α-synuclein was observed in the frontal cortex compared with the putamen in PD (Fig. 1c, 67 vs. 44% of relative control α-synuclein amounts, Wilcoxon Signed Rank test P = 0.035), with the amount of insoluble frontal α-synuclein being highly variable between cases (Fig. 1c).
There was no increase in either the TBS-soluble or SDS-soluble α-synuclein levels in the PD putamen samples compared with controls (Fig. 1f, l, Mann–Whitney U tests P > 0.14). There was a small but significant increase in frontal TBS-soluble α-synuclein levels (Fig. 1k, l, 1.14 ± 0.12× increase, Mann–Whitney U test P = 0.047), but no difference between the levels in the frontal cortex versus the putamen due to the large variation in amounts (Wilcoxon Signed Rank test P = 0.64). There was a larger and less variable increase in the SDS-soluble α-synuclein in the frontal cortex in PD (Fig. 1e, f, 1.4 ± 0.1× increase, Mann–Whitney U test P < 0.001) differentiating this region from the putamen (1.8 ± 0.6× difference in SDS-soluble α-synuclein between the frontal cortex vs. the putamen, Wilcoxon Signed Rank test P = 0.001).
There was a striking increase in the levels of S129 phosphorylated α-synuclein in both the TBS-soluble and SDS-soluble fractions in the putamen (Fig. 1i, o, 5.8–18×, Mann–Whitney U tests P < 0.001) and frontal cortex (Fig. 1g–i, m, o, 21–28×, Mann–Whitney U tests P < 0.001) with significantly more S129 phosphorylated α-synuclein in the frontal cortex compared with the putamen in PD (Fig. 1i, o, 1.7–3.6×, Wilcoxon Signed Rank test P < 0.001).
To determine the relationships between the different α-synuclein species in patients with PD, stepwise multiple regression analyses were performed using either the TBS-soluble S129 phosphorylated putamen α-synuclein (high levels found in controls, see above) or the urea-soluble frontal syn-1 α-synuclein (high levels in PD, see above), covarying for age at onset and disease duration. In PD, the species relating most to changes in the levels of TBS-soluble phosphorylated S129 α-synuclein in the putamen were SDS-soluble phosphorylated S129 α-synuclein in the putamen and TBS-soluble phosphorylated S129 α-synuclein in the frontal cortex (Fig. 2a, P < 0.001, β coefficients for putamen SDS-soluble = 0.51 and frontal TBS-soluble = 0.49). To identify changes most associated with Lewy bodies (made of insoluble α-synuclein), the species relating most to changes in the levels of urea-soluble α-synuclein in the frontal cortex were determined to be frontal TBS-soluble phosphorylated S129 α-synuclein and frontal SDS-soluble α-synuclein (Fig. 2b, P < 0.001, β coefficients for TBS-soluble S129P = 0.78 and SDS-soluble syn-1 = 0.33). In addition, disease duration affected this relationship (more frontal urea-soluble α-synuclein with increasing disease duration, β coefficient = 0.27).
Changes over the disease course
As disease duration does not directly reflect disease stage (see Table 1), the amount of α-synuclein in the different fractions was assessed by disease stage. The urea-soluble and SDS-soluble syn-1 and TBS-soluble and SDS-soluble S129P in PD appeared to increase with disease stage (Fig. 3). To determine the regional changes relating most to the different stages of PD, stepwise multiple regression analysis was performed in the PD cases, covarying for age at onset and disease duration. The factors relating most to disease stage were a decrease in the levels of TBS-soluble and an increase in the SDS-soluble syn-1 in the frontal cortex (Fig. 2c, P < 0.001, β coefficients for TBS-soluble = −0.74 and SDS-soluble = 0.29, 36% reduction in TBS-soluble and 24% increase in SDS-soluble), with increasing levels of SDS-soluble syn-1 relating to Lewy body formation (see above).
Discussion
There have been no previous studies assessing the changes in α-synuclein phosphorylation and solubility with increasing pathological stage of PD. Our findings confirm the recent observations by Tong and colleagues [41] that there is only a small increase over control levels in the TBS-soluble and SDS-soluble α-synuclein levels in PD, although we have found that variation in these measures occurs with greater pathological severity and with increasing disease duration. In the frontal cortex where the deposition of Lewy bodies and neurites is a later pathological event, the initially ~15% higher cytosolic (TBS-soluble) levels of α-synuclein that precedes Lewy body formation normalizes to control levels as the disease progresses. This small transient increase in the cytosolic levels of α-synuclein in PD appears to precipitate a large shift in the location of intracellular α-synuclein increasing its membrane (SDS) association by around 40% and changing the ratio of cytosolic:membrane-associated α-synuclein from around 2:1 to 1:1 by late disease stages. Such a shift in soluble α-synuclein may be reflected in the α-synuclein levels in cerebrospinal fluid, which are significantly decreased in patients with PD [19, 28, 43]. Importantly, this intracellular shift and increased accumulation of membrane-associated α-synuclein over time directly related to both increased cytosolic S129 phosphorylation of the protein and to its increased insolubility (in the urea-soluble fraction), as would be expected for Lewy body formation. The amount of cytosolic α-synuclein that is phosphorylated at S129 progressively rises from its basal level of around 5% to between 30 and 100% depending on the region and severity of pathology in the patients with PD. These data suggest that the factors driving Lewy body formation in sporadic PD are large increases in both the cytosolic phosphorylation of α-synuclein and its membrane associations rather than primarily any large increase in the soluble cytosolic amount of the protein, as previously hypothesized [10, 26, 36].
Our finding of no substantial increase in cytosolic α-synuclein protein levels in brain regions affected by Lewy body formation in patients with sporadic PD confirms with findings from recent studies using a variety of techniques [see Table 1 in 41, 45]. This contrasts with the slow increase in the SDS membrane-associated levels of α-synuclein and has important implications for the pervasive concept that an increase in cytosolic α-synuclein concentration is the main pathogenic mechanism for PD [10, 26, 36]. The relatively stable amount of soluble α-synuclein measured in the present and previous PD studies is surprising, and perhaps suggests that regulatory mechanisms are required to maintain such normal α-synuclein cytosolic levels when insoluble intracellular pathogenic depositions of the protein are forming. Both increased and decreased expression levels of α-synuclein mRNA have been documented in sporadic PD [16, 37] and the amount of cytosolic α-synuclein may also be held relatively constant through micro-RNA regulation [12] or methylation [23]. Intracellular and extracellular transport mechanisms may be involved, although the reduction in soluble CSF levels in PD [19, 28, 43] suggests that cellular excretion is not a major regulatory mechanism, leaving mechanisms involved in the membrane localization of the protein as potentially important for disease pathogenesis. Understanding the cellular regulation of the gene and any additional non-transcriptional mechanisms regulating the levels of the major cytosolic α-synuclein species may assist further with understanding disease pathogenesis.
Despite no substantive change in the relative amount of soluble cytosolic levels of α-synuclein, the small changes noted over time were significantly correlated with an increase in the amount of SDS membrane-associated protein that occurred with increasing Braak stage of Lewy body formation. This intracellular shift in protein location not only related to disease onset and progression in the frontal cortex but also related to the large increase in insoluble α-synuclein observed in the PD tissue fractions over time, suggesting that membrane localization is a key event in Lewy body formation. Membrane-associated α-synuclein is documented to play a critical role in protein aggregation and cellular degeneration [4, 22, 24]. In vitro studies indicate that membrane-associated α-synuclein accelerates fibril formation [22] and seeds the aggregation of cytosolic α-synuclein [24], confirming it can initiate and precipitate inclusion formation [4]. α-Synuclein translocates to mitochondrial membranes with increasing intracellular acidification [9] and patients with PD have significantly increased accumulation of mitochondrial α-synuclein and decreased complex 1 activity compared with controls [11]. Such cellular deficits impact on α-synuclein’s interaction with synaptic vesicles to decrease neurotransmitter release through lowering the recycling pool of synaptic vesicles [29]. Overall, the increase in membrane-associated α-synuclein observed would significantly impact on cell function, in addition to increasing its aggregation propensity.
Importantly, this intracellular shift and increase in membrane-associated α-synuclein over time is directly related to increased cytosolic S129 phosphorylation of the protein which is also directly related to the amount of insoluble protein found in the tissue. Our data show a dramatic rise in the relative proportion of cytosolic S129 phosphorylated α-synuclein over the disease course, which suggests that such cytosolic phosphorylation is important for its membrane association and subsequent accumulation into the protease-resistant fibrillar forms found in PD. This is consistent with the diffuse non-fibrillar neuronal cytoplasmic staining using S129 specific immunohistochemsitry in regions with Lewy body formation [34]. It is well known that α-synuclein is extensively phosphorylated at S129 in PD [1, 15, 31] and that such phosphorylation makes the protein more acidic, possibly increasing its capacity for membrane translocation [9]. A proportion of the cytosolic S129 phosphorylated α-synuclein appears to be monoubiquitinated according to its molecular weight [1, 18], although the extent of S129 α-synuclein phosphorylation is not influenced by its monoubiquitination, rather the monoubitquitination of α-synuclein has been shown to increase the stability of the monomer [18]. Pulldown assays show that S129 phosphorylated α-synuclein binds to different cellular proteins compared to non-phosphorylated α-synuclein [27]. The phosphorylated form of the protein selectively binds to enzymes and signaling proteins involved in serine/threonine phosphorylation [27], although its role in such signaling remains unexplored. It also selectively binds to clathrin heavy chain and subunits involved in clathrin-mediated endocytosis of vesicles destined for the recycling pool [27], an association likely to impact on neurotransmitter release [29]. In addition, it binds to cytosolic proteins that form the presynaptic web required for synapse stability [27]. Presynaptic phosphorylated α-synuclein binding proteins include spectrins and spectrin-interacting proteins, cytoplasmic actins, non-muscle myosins as well as MAP1B and neurofilament L [27], with many of these proteins found in Lewy bodies [25]. The presynaptic web is essential for stability during vesicle exocytosis and membrane retrieval, dissolving at high basic pH and being more stable at lower acidic pH, bridging between the synaptic cell adhesion and the microtubule cytoskeleton [32, 33]. While phosphorylated α-synuclein immunoreactive dot-like structures were identified some time ago in cases with Lewy body formation [34], it has only recently been recognized that these structures are synaptic accumulations of the phosphorylated protein [35, 40]. These data suggest that increased phosphorylation of cytosolic α-synuclein may increase its signaling capacity as well as synaptic and membrane stability.
The substantial Lewy body-related increase in cytosolic phosphorylation of α-synuclein in the frontal cortex was directly related to the amount of S129 phosphorylated α-synuclein in the earlier affected putamen in the same cases, a region we have found to contain a significantly higher basal control level of S129 phosphorylated α-synuclein compared with the frontal cortex. These data support the concept that α-synuclein pathology may propagate between different brain regions [3, 6], but suggests that the species involved may be the S129 phosphorylated form of the protein rather than insoluble α-synuclein. The regional difference in the degree of α-synuclein phosphorylation in controls also suggests that its signaling and synaptic functions following S129 phosphorylation [27] are more necessary in the basal ganglia compared with the cortex. Whether other regions predisposed to α-synuclein pathology also have high S129 phosphorylation levels remains to be determined. Synaptic damage in the putamen occurs preclinically in PD [5] with limited Lewy body formation found in this region [6] despite extensive α-synuclein synaptic and neuritic pathology [13]. This higher level of α-synuclein phosphorylation in the putamen may predispose this region to the synaptic degeneration that precedes the motor signs of PD and allow the slow propagation of the pathology through interconnected regions.
The present pathological study has used cross-sectional data from different PD cases with different anatomical severities to interpret the progression of pathological changes over the disease course. The sample size at each stage is relatively small but also fairly homogeneous, as significant coexisting and age-related neuropathologies were excluded in all cases. All cases had typical PD using the UK Parkinson’s Disease Society Brain Bank Diagnostic Criteria [20] and were levodopa-responsive with average disease durations of 8 years or more. As may have been expected, cases with stage IV disease had shorter disease durations than stage V, although this did not hold true for cases with stage VI disease. No case had clinical dementia with Lewy bodies, or had the more rapid clinical phenotype often seen in such cases [44]. The same extraction methods were used in all cases, and the relative α-synuclein levels directly compared using the same techniques. The methods and results used are similar to those recently published by Tong et al. [41] where they showed that the most substantial changes occurred in the nigrostriatal pathway in the early disease cases they assessed. As previously stated, there have been no studies assessing the changes in the relative α-synuclein levels or phosphorylation status over the different stages of PD. Using the same methods across the different stages of PD, we have been able to show that soluble non-phosphorylated α-synuclein decreases in PD vulnerable brain regions, becoming increasingly phosphorylated and insoluble over the course of PD. In addition, the levels of S129 phosphorylated α-synuclein in the nigrostriatal pathway relates to the pathogenic forms of α-synuclein in frontal brain regions, suggesting a propagating role of putamenal S129 phosphorylated α-synuclein in PD pathogenesis.
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Acknowledgments
Human brain tissue samples were received from the Australian Brain Bank Network which is supported by the National Health and Medical Research Council of Australia (NHMRC), specifically from the Sydney Brain Bank (also supported by Neuroscience Research Australia and the University of New South Wales), from the NSW Tissue Resource Centre [also supported by the Schizophrenia Research Institute, the National Institute of Alcohol Abuse and Alcoholism (NIH (NIAAA) R24AA012725, and the University of Sydney], from the South Australian Brain Bank (also supported by the Flinders Medical Centre Foundation), from the Victorian Brain Bank Network (also supported by Neurosciences Australia, the University of Melbourne, the Mental Health Research Institute of Victoria, the Alfred Hospital, and the Victorian Forensic Institute of Medicine), from the Queensland Brain Bank and from the Western Australia Brain Bank Network. This work was also supported by the NHMRC (510186) and the National Basic Research Program of China (2006CB500700), NSFC fund (30771062). Gai has a NHMRC Senior Research Fellowship 535014. Halliday has a NHMRC Senior Principal Research Fellowship 630434. We would like to thank Heather McCann for laboratory assistance and Heidi Cartwright for the preparation of the figures.
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Zhou, J., Broe, M., Huang, Y. et al. Changes in the solubility and phosphorylation of α-synuclein over the course of Parkinson’s disease. Acta Neuropathol 121, 695–704 (2011). https://doi.org/10.1007/s00401-011-0815-1
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DOI: https://doi.org/10.1007/s00401-011-0815-1