Sensitive western blotting for detection of endogenous Ser129-phosphorylated α-synuclein in intracellular and extracellular spaces

α-Synuclein deposited in Lewy bodies, a pathological hallmark of Parkinson’s disease (PD), is highly phosphorylated at serine 129 (Ser129). In contrast, there is very little Ser129-phosphorylated α-synuclein in the normal brains. This difference suggests that Ser129-phosphorylation is involved in neurodegenerative processes of PD. However, the role of this modification remains unclear. One limiting factor for relevant biochemical analyses is that it is difficult to detect endogenous Ser129-phosphoryated α-synuclein by western blotting, because α-synuclein monomers detached from the transferred membrane during incubation. Here, we reported that combination fixation of the transferred membrane with 4% paraformaldehyde and 0.01 ~ 0.1% glutaraldehyde produced an approximately 10-fold increase in the sensitivity for Ser129-phosphorylated α-synuclein monomers, allowing detection of endogenous proteins even in conditioned medium, human cerebrospinal fluid, and extracts from cell lines and human brain. This method may enable more detailed biochemical analyses for α-synuclein transmission between intra and extracellular spaces under physiological and pathological conditions.


Results
Concentration dependent effect of membrane fixation with paraformaldehyde on detection of total and Ser129-phosphorylated α-synuclein monomers by western blotting. To assess whether transferred membrane fixation with paraformaldehyde improves sensitivity to detect total α -synuclein monomers, including non-phosphorylated and phosphorylated forms, in a paraformaldehyde concentration dependent manner, we compared the signals for total α -synuclein monomers with increasing paraformaldehyde concentrations. When extracts from SH-SY5Y cells stably expressing wild-type α -synuclein (wt-aS/SH#4) were analyzed by western blotting with anti-α -synuclein monoclonal antibody (Syn-1), the total α -synuclein monomer signals were enhanced by membrane fixation with paraformaldehyde in a concentration dependent manner, up to 4% paraformaldehyde (Fig. 1A, Supplementary figure 1). Fixation with 4% paraformaldehyde produced a 3-fold increase in signal, compared with 0.4% paraformaldehyde fixation. Western blotting using anti-human α -synuclein monoclonal antibody (LB509) also resulted in signal enhancement with high concentrations of paraformaldehyde, although to a lesser extent (Fig. 1A, Supplementary figure 1). Western blotting with anti-human α -synuclein monoclonal antibody (211) demonstrated that the signals for total α -synuclein monomers were enhanced by membrane fixation with paraformaldehyde in a concentration dependent manner, similar to the effects observed for LB509 antibody (Fig. 1A, Supplementary figure S1). Next, we tested whether this method produced similar results for detection of Ser129-phosphorylated α -synuclein. Western blotting using rabbit monoclonal antibody (EP1536Y), which is specific to Ser129-phosphorylated α -synuclein, produced Ser129-phosphorylated α -synuclein monomer signals that were enhanced by increasing paraformaldehyde concentrations up to 4% (Fig. 1A, Supplementary figure S1). The 4% paraformaldehyde fixation condition produced a 4-fold increase in the signals compared to 0.4% paraformaldehyde fixation. The Ser129-phosphorylated α -synuclein signals were also enhanced by increasing the paraformaldehyde concentration when different anti-Ser129-phosphorylated α -synuclein monoclonal antibody, psyn#64 was used (Fig. 1A, Supplementary figure S1).
To elucidate whether fixation with a higher concentration of paraformaldehyde more effectively detects endogenous α -synuclein monomers, parental SH-SY5Y cells lysates were analyzed by western blotting. Western blotting with Syn-1 antibody and 4% paraformaldehyde fixation detected endogenous total α -synuclein monomers more clearly, compared with the conventional western blotting method without fixation (Fig. 1B, Supplementary figure S1). Signals for endogenous total α -synuclein monomers were enhanced by increasing paraformaldehyde concentrations with 211 or LB509 antibody (Fig. 1B,  Supplementary figure S1). In addition, western blotting with EP1536Y antibody demonstrated that 4% paraformaldehyde fixation enabled detection of endogenous Ser129-phosphorylated α -synuclein monomers, although these signals were weak (Fig. 1B, Supplementary figure S1).
To test time dependency of the paraformaldehyde fixation on detection of endogenous α -synuclein monomers, we treated the transferred membrane with 4% paraformaldehyde for 10, 30, or 60 min. Western blotting of parental SH-SY5Y cell lysates with Syn-1, 211, or LB509 antibody demonstrated that the signals were increased in a time dependent manner (Fig. 1C, Supplementary figure S1). Western blotting with EP1536Y antibody also showed the time dependent enhancement of signals for endogenous Ser129-phosphorylated α -synuclein monomers (Fig. 1C, Supplementary figure S1). Effect of membrane fixation with glutaraldehyde on α-synuclein monomer detection in western blotting. Next, we tested whether fixation of the transferred membrane with glutaraldehyde affects α -synuclein detection. Western blotting using wt-aS/SH#4 cell lysates with Syn-1 antibody demonstrated that glutaraldehyde fixation enhanced the signals for α -synuclein monomers at low concentrations of 0.001 ~ 0.01% ( Fig. 2A, Supplementary figure S2). Higher concentrations of glutaraldehyde diminished the signals. In contrast, western blotting with 211 or LB509 antibody demonstrated that glutaraldehyde fixation enhanced the signals in a concentration dependent manner, up to 0.1% Figure 1. The concentration dependent effect of transferred membrane fixation with paraformaldehyde on α-synuclein monomer detection by western blotting (WB). After SDS-PAGE, the transferred membrane was treated with PBS containing the indicated concentrations of paraformaldehyde for 30 min (A,B) or 4% paraformaldehyde for indicated durations (C). For the membrane without paraformaldehyde fixation, this step was omitted, and was immediately followed by the blocking step. It should be noted that WB with anti-β -actin antibody as a loading control was not performed because the β -actin signals were altered by the fixation. Although equal amounts of protein samples were simultaneously loaded on the gel, the transferred membrane was cut into several strips on the protein marker lanes between sample lanes to test fixation conditions. The strips were spliced and reconstituted to be one original membrane according to the protein markers, and then we simultaneously detected signals by CCD camera. Solid lines indicated cropping boundaries of strips (please note that this editing is done in the blot panels in . (A) Cell lysates (5 μ g/lane) of SH-SY5Y cells stably expressing wild-type α -synuclein (wt-aS/SH#4) were loaded on SDS-PAGE and analyzed by WB with anti-total α -synuclein antibody (Syn-1, 211, or LB509). WB was also performed with anti-Ser129 phosphorylated α -synuclein specific antibody (EP1536Y or psyn#64). (B) Cell lysates (10 μ g/lane) of parental SH-SY5Y cells were analyzed by WB with the same set of antibodies, except psyn#64. (C) Time dependent effect of the 4% paraformaldehyde fixation. Cell lysates (10 g/lane) of parental SH-SY5Y cells were analyzed by WB with Syn-1, 211, LB509, or EP1536Y. The transferred membrane was treated with the fixation solution for 10, 30, or 60 min. Left panels show representative blots. The graphs shows the signal intensity (means ± SD) as a percentage of the maximum observed on the blots. glutaraldehyde ( Fig. 2A, Supplementary figure S2). Western blotting with glutaraldehyde fixation using EP1536Y antibody remarkably enhanced the signals for Ser129-phopshorylated α -synuclein monomers, similar to effects with LB509 antibody ( Fig. 2A, Supplementary figure S2). Western blotting of parental SH-SY5Y cells with Syn-1 antibody resulted in endogenous total α -synuclein monomer signals that peaked at 0.01% glutaraldehyde fixation ( Effect of combination fixation with paraformaldehyde and glutaraldehyde on α-synuclein monomer detection in western blotting. To assess whether glutaraldehyde fixation additively enhances the signals for total and Ser129-phoshorylated α -synuclein monomers, we investigated the effects of glutaraldehyde addition on signal detection in wt-aS/SH#4 cells and parental SH-SY5Y cells. Paraformaldehyde fixation was set at a constant concentration of 4%. In western blotting with Syn-1 antibody, this constant 4% paraformaldehyde fixation enabled us to detect signals representing overexpressed and endogenous total α -synuclein monomers (Fig. 3A,B, Supplementary figure S3). When we compared the signals for α -synuclein monomers in combination fixation with paraformaldehyde and glutaraldehyde to those in single fixation with paraformaldehyde, there was no significant change in the signals for overexpressed total α -synuclein monomers by adding 0.01% glutaraldehyde (144.0 ± 11.3%, n = 3, P = 0.053) (Fig. 3A, Supplementary figure S3). The signals for endogenous α -synuclein monomers were significantly enhanced by adding 0.01% glutaraldehyde (198.7 ± 27.9%, n = 3, P = 0.001) (Fig. 3B,  Supplementary figure S3). Western blotting with EP1536Y antibody demonstrated that signals for overexpressed and endogenous Ser129-phosphorylated α -synuclein monomers were enhanced by adding glutaraldehyde in a concentration dependent manner, up to 0.1% (Fig. 3A,B, Supplementary figure S3). The signals for overexpressed Ser129-phosphorylated α -synuclein monomers were significantly enhanced by adding 0.01% glutaraldehyde (248.7 ± 36.3%, n = 3, P < 0.001) and 0.1% glutaraldehyde (235.7 ± 15.9%, n = 3, P < 0.001) (Fig. 3A, Supplementary figure S3). The signals for endogenous Ser129-phosphorylated α -synuclein monomers were significantly enhanced by adding 0.01% glutaraldehyde (239.7 ± 35.2%,  antibody. The transferred membrane was treated with PBS containing 4% paraformaldehyde and 0%, 0.001%, 0.01%, or 0.1% glutaraldehyde for 30 min. For the membrane without paraformaldehyde and glutaraldehyde fixation, this step was omitted, and was immediately followed by the blocking step. B. Effect of combination fixation for endogenous α -synuclein detection. Cell lysates (10 μ g/lane) of parental SH-SY5Y cells were analyzed by WB with Syn-1 or EP1536Y. The graphs of (A,B) show the signal intensity (means ± SD) as a percentage of the 4% paraformaldehyde single fixation. P values were estimated by one-way ANOVA with a Bonferroni correction (**P < 0.01). In the Syn-1 signals for overexpressed total α -synuclein, P values were estimated by one-way ANOVA with the Games-Howell post hoc test, because variances were unequal. (C) Time dependent effect of combination fixation with 4% paraformaldehyde and 0.01% glutaraldehyde. Cell lysates (10 μ g/lane) of parental SH-SY5Y cells were analyzed by WB with Syn-1, 211, LB509, or EP1536Y antibody. The transferred membrane was treated with combination fixation solution for 10, 30, or 60 min. Left panels show representative blots. The graph of (C) shows the signal intensity (means ± SD) as a percentage of the maximum observed on the blots. (D) Comparison of the sensitivity of α -synuclein monomer detection between the conventional and combination fixation methods. After partially phosphorylating E. coli derived recombinant α -synuclein at Ser129 by casein kinase 2 (CK2), the indicated amounts of proteins were analyzed by WB with Syn-1 or EP1536Y. The transferred membranes were treated with or without combination fixation with 4% paraformaldehyde and 0.01% glutaraldehyde. Note that phosphorylated proteins contain non-phosphorylated ones, so that they are shown as amounts of total proteins. n = 3, P = 0.002) and 0.1% glutaraldehyde (337.0 ± 35.3%, n = 3, P < 0.001) (Fig. 3B, Supplementary figure S3). These findings indicate that glutaraldehyde plus paraformaldehyde fixation produced additive effects on detection of endogenous total and Ser129-phosphorylated α -synuclein monomers. In the present study, combination fixation with 4% paraformaldehyde and 0.01 or 0.1% glutaraldehyde produced the most sensitive detection of endogenous total α -synuclein monomers using Syn-1 antibody, and endogenous Ser129-phosphorylated α -synuclein monomers using EP1536Y antibody.
To test time dependency of combination fixation on detection of endogenous α -synuclein monomers, we treated the transferred membrane with 4% paraformaldehyde and 0.01% glutaraldehyde for 10, 30, or 60 min. Western blotting of parental SH-SY5Y cell lysates with Syn-1, 211, or LB509 antibody demonstrated that the signals were increased in a time dependent manner (Fig. 3C, Supplementary figure S3). The signals by Syn-1 antibody peaked at 30 min. Western blotting with EP1536Y antibody also showed the time dependent enhancement of signals for endogenous Ser129-phosphorylated α -synuclein monomers during incubation (Fig. 3C, Supplementary figure S3). In the present study, we treated the transferred membrane for 30 min with the combination of 4% paraformaldehyde and 0.01% glutaraldehyde, because this seemed to be optimal and convenient for simultaneously detecting the signals for total and Se129-phosphorylated α -synuclein.
To assess sensitivity to detect α -synuclein monomers by membrane fixation, we investigated the signals resulting from western blotting using purified recombinant α -synuclein proteins that were partially phosphorylated by incubation with casein kinase 2 (CK2). However, it should be noted that we were unable to quantify the signals for Ser129-phosphorylated α -synuclein because we used a mixture of proteins, including non-phosphorylated and Ser129-phosphorylated forms, as standards. Western blotting with Syn-1 antibody using the conventional method without fixation detected signal indicating 250 pg of total α -synuclein, whereas combination fixation with 4% paraformaldehyde and 0.01% glutaraldehyde detected signal indicating 25 pg of total α -synuclein (Fig. 3D, Supplementary figure S3). Using EP1536Y antibody, the conventional method faintly visualized Ser129-phosphorylated α -synuclein signals indicating 2,500 or 5,000 pg of total α -synuclein. The combination of 4% paraformaldehyde and 0.01% glutaraldehyde enabled us to detect signal for Ser129-phosphorylated α -synuclein as a component of 250 pg of total α -synuclein (Fig. 3D, Supplementary figure S3). These findings indicate that combination fixation led to an approximately 10-fold increase in the sensitivity to detect both total and Ser129-phosphorylated α -synuclein.

Effect of paraformaldehyde or glutaraldehyde fixation on detection of other proteins by western blotting.
To test whether paraformaldehyde fixation effectively detects proteins other than α -synuclein, and whether signal enhancement by paraformaldehyde fixation is influenced by the molecular size of the target molecule, we investigated signals for Cu/Zn superoxide dismutase (SOD1), β -actin, heat shock protein 40 (Hsp40), and nicastrin (NCT) using western blotting with wt-as/SH#4 cell lysates. Signals for all of these proteins were enhanced by paraformaldehyde fixation (Fig. 4A, Supplementary  figure S4). However, the effects can be classified into two groups. In one group, signal enhancement peaked at 0.4% paraformaldehyde and signals declined with higher concentrations of paraformaldehyde. This group included SOD1, Hsp40, and NCT (Fig. 4A, Supplementary figure S4). In the second group, signals increased in a paraformaldehyde concentration dependent manner, such as for β -actin (Fig. 4A,  Supplementary figure S4).
We then tested the effects of glutaraldehyde fixation on detection of these proteins. The effects were classified into three groups. The first group included proteins for which the glutaraldehyde fixation had no enhancing effect. This result was observed for SOD1 and Hsp40 (Fig. 4B, Supplementary figure  S4). The second group demonstrated slightly enhanced signal at a low concentration of 0.001%, and included NCT (Fig. 4B, Supplementary figure S4). The third group had signals that increased in a glutaraldehyde concentration dependent manner up to 0.1% glutaraldehyde, and included β -actin (Fig. 4B,  Supplementary figure S4). These findings demonstrate that paraformaldehyde fixation has the potential to enhance signal detection for target proteins of varying molecular sizes. Glutaraldehyde fixation was effective for limited proteins or antibodies, although it sometimes yields remarkable enhancement.
To further assess how this combination fixation affects detection of α -synuclein oligomers, we investigated a change in the signal by using in vitro oligomerized α -synuclein proteins in western blotting with In left panels, recombinant α -synuclein (2.5 ng/lane) proteins treated with or without CK2 were analyzed by WB with Syn-1 or EP1536Y antibody. In middle panels, parental SH-SY5Y cell extracts (10 μ g/lane) treated with or without calf intestine alkaline phosphatase were analyzed by WB. In right panels, cell lysates (5 μ g/lane) of wt-aS/SH#4 or SH-SY5Y cells stably expressing S129A α -synuclein were analyzed by WB.
Scientific RepoRts | 5:14211 | DOi: 10.1038/srep14211 211 antibody. The α -synuclein monomer signals were clearly enhanced by the combination of 4% paraformaldehyde and 0.01% glutaraldehyde fixation (Fig. 5C, Supplementary figure S5). The high molecular weight signals were also slightly enhanced by this fixation (Fig. 5C, Supplementary figure S5). This combination fixation was more effective for detecting monomers than oligomers.

Effect of membrane fixation on detection of endogenous α-synuclein in the extracts of cell lines, rat brains, and human brains.
To assess how our membrane fixation method applies to biochemical experiments on α -synuclein, we investigated the signals for endogenous α -synuclein in cell lysates of HEK293, SH-SY5Y, and CHO-K1 cells. The conventional method of western blotting using Syn-1 antibody without fixation did not detect signals for endogenous total α -synuclein monomers, whereas the combination of 4% paraformaldehyde and 0.01% glutaraldehyde fixation enabled us to visualize these signals in HEK293 and SH-SY5Y cells (Fig. 6A, Supplementary figure S6). No obvious signals were observed in CHO-K1 cells. Western blotting with EP1536Y antibody indicated that the conventional method did not detect signals for endogenous Ser129-phosphorylated α -synuclein monomers, whereas the combination of 4% paraformaldehyde and 0.01% glutaraldehyde fixation allowed us to detect these signals in HEK293 and SH-SY5Y cells (Fig. 6A, Supplementary figure S6). We then assessed signals for endogenous α -synuclein in rat striatum homogenates. Western blotting with Syn-1 antibody using the conventional method detected signals for endogenous total α -synuclein monomers, whereas the combination of 4% paraformaldehyde and 0.01% glutaraldehyde fixation remarkably enhanced these signals (Fig. 6A, Supplementary figure S6). Western blotting with EP1536Y antibody using the conventional method produced a faint signal for endogenous Ser129-phosphorylated α -synuclein monomers (Fig. 6A, Supplementary figure S6). The combination of 4% paraformaldehyde and 0.01% glutaraldehyde fixation resulted in clearer visualization of these signals (Fig. 6A, Supplementary figure S6). When we examined signals for endogenous total α -synuclein in human cerebral cortex homogenates, western blotting with Syn-1 or LB509 antibody demonstrated that the combination of 4% paraformaldehyde and 0.01% glutaraldehyde fixation further enhanced the signals for endogenous total α -synuclein monomers, compared with the conventional method (Fig. 6A, Supplementary figure S6). Western blotting with EP1536Y antibody using the conventional method did not detect any signals for endogenous Ser129-phosphorylated α -synuclein monomers (Fig. 6A, Supplementary figure S6). However, the combination of 4% paraformaldehyde and 0.01% glutaraldehyde fixation clearly visualized the signals for endogenous Ser129-phosphorylated α -synuclein monomers in the human brain tissue (Fig. 6A,  Supplementary figure S6).
To confirm the effects of 4% paraformaldehyde and 0.01% glutaraldehyde combination on endogenous α -synuclein monomer detection, we compared signals yielded by this combination fixation with 0.4% paraformaldehyde fixation, which is the original method described by Lee and Kamitani 11 . Western blotting with Syn-1 antibody using combination fixation enhanced signals for endogenous total α -synuclein monomers in parental SH-SY5Y cells and human cerebral cortex more intensively than 0.4% paraformaldehyde fixation (Fig. 6B, Supplementary figure S6). Western blotting with EP1536Y antibody using 0.4% paraformaldehyde fixation alone did not result in signal detection for endogenous Ser129-phosphorylated α -synuclein monomers in parental SH-SY5Y cells or human cerebral cortex (Fig. 6B, Supplementary figure  S6). However, combination fixation enabled detection of signals for endogenous Ser129-phosphorylated α -synuclein monomers (Fig. 6B, Supplementary figure S6). These findings indicate that combination fixation effectively assesses the endogenous expression of Ser129-phosphorylated α -synuclein monomers in extracts from cell lines and mammalian brains.

Effect of membrane fixation on detection of endogenous α-synuclein in conditioned medium (CM) and cerebrospinal fluid (CSF).
To clarify whether the membrane fixation method effectively detects endogenous α -synuclein in the extracellular space, we investigated the signals by western blotting using the CM from parental HEK 293, SH-SY5Y, and CHO-K1 cells. Western blotting with Syn-1 antibody using the conventional method failed to detect the signals for endogenous total α -synuclein monomers, whereas combination fixation with 4% paraformaldehyde and 0.01% glutaraldehyde enabled us to detect the clear signals in the CM obtained from HEK293 and SH-SY5Y cells (Fig. 7A, Supplementary  figure S7). By this combination fixation, the endogenous total α -synuclein monomer was also observed as a faint signal in CM from CHO-K1 cells (Fig. 7A, Supplementary figure S7), although the endogenous total α -synuclein monomer was undetectable in cell lysates of CHO-K1 cells (Fig. 6A, Supplementary  figure S6). In addition, western blotting with EP1536Y antibody demonstrated that combination fixation enabled us to detect the signals for endogenous Ser129-phosphorylated α -synuclein monomers in CM from SH-SY5Y cells (Fig. 7A, Supplementary figure S7). However, no obvious signals were observed for endogenous Ser129-phosphorylated α -synuclein monomers in the CM from HEK293 and CHO-K1 cells (Fig. 7A, Supplementary figure S7). When the post-fixative transferred membranes were incubated only with secondary antibodies, we found no obvious signals (Fig. 7A, Supplementary figure S7). These findings indicate that the signals detectable in western blotting with the Syn-1 or EP1536Y antibody were not non-specific effects resulting from treatment with the secondary antibodies.
Finally, we tested whether combination fixation with 4% paraformaldehyde and 0.01% glutaraldehyde was applied to detect the signals for endogenous α -synuclein monomers in human CSF. Western blotting with Syn-1 antibody using the conventional method did not detect the signals for endogenous total α -synuclein monomers, whereas the combination fixation enabled us to detect clear signals in CSF (Fig. 7B, Supplementary figure S7). Western blotting with EP1536Y antibody also demonstrated that combination fixation allowed us to detect the signals from endogenous Ser129-phosphorylated α -synuclein monomers in CSF (Fig. 7B, Supplementary figure S7). When the post-fixative transferred membranes were incubated only with our secondary antibodies, there were no obvious signals; therefore, providing evidence for the specificity of signals enhanced by the fixation (Fig. 7B, Supplementary figure S7).

Discussion
Our data demonstrate that transferred membrane fixation with paraformaldehyde and glutaraldehyde enabled signal detection for endogenous Ser129-phosphorylated α -synuclein monomers by western blotting. Glutaraldehyde had a remarkable effect on detection, and the most effective combination was 4% paraformaldehyde and 0.01 ~ 0.1% glutaraldehyde in western blotting with EP1536Y antibody. The sensitivity of detection was increased approximately 10-fold in recombinant α -synuclein proteins phosphorylated at Ser129. Although the signals for endogenous Ser129-phosphorylated α -synuclein monomers were very faint (lower than the detection limit by conventional western blotting without fixation), this simple method allowed us to reproducibly visualize these signals in cell line lysates (HEK293 and SH-SY5Y cells) and in extracts from the rat striatum and human cerebral cortex. Lee and Kamitani reported that 0.4% paraformaldehyde fixation in western blotting using EP1536Y antibody effectively enabled visualization of signals for endogenous Ser129-phosphorylated α -synuclein monomers in human cell lines, including Daoy, SK-MEL28, MeWo, and WM266-4 cells; however, it did not enable signal detection in HEK293 and SH-SY5Y cells 11 . The 4% paraformaldehyde and 0.01% glutaraldehyde combination fixation further improved signal sensitivity in lysates from HEK293 and SH-SY5Y cells, was loaded on SDS-PAGE and analyzed by WB with Syn-1 or EP1536Y antibody. To assess signal specificity, the post-fixative transferred membranes were incubated only with anti-mouse or anti-rabbit secondary antibody. As positive controls, recombinant α -synuclein proteins were subjected to WB along with samples. (B) 10 μ L of human CSF (1/2 volume of total) were analyzed by WB with Syn-1 or EP1536Y antibody. The post-fixative transferred membranes were also incubated only with anti-mouse or anti-rabbit secondary antibody.
We found that our paraformaldehyde or glutaraldehyde fixation method enhanced signals for different molecules (SOD1, β -actin, Hsp40, and NCT), despite their molecular sizes. However, the enhancing effects were classified into two groups. In the first group, the signal enhancing effect peaked at low concentrations of fixative agents (0.4% paraformaldehyde or 0.001% glutaraldehyde), and declined at higher concentrations. Excessive fixation with paraformaldehyde or glutaraldehyde may inhibit epitope accessibility of antibody on the blot membranes. In the second group, the enhancing effects increased in a concentration dependent manner, up to 8% paraformaldehyde or 0.1% glutaraldehyde. These findings suggest that paraformaldehyde or glutaraldehyde fixation effectively enhances signals for proteins other than α -synuclein, and that it is necessary to optimize the fixation effects in each molecule or antibody.
Our findings do not explain the mechanism of action for improved immunoreactivity to α -synuclein antibody. Lee and Kamitani reported that α -synuclein monomers easily detach from the transferred membrane during incubation, using conventional western blotting protocols 11 . Similarly, Newman et al. demonstrated that α -synuclein monomer bands disappeared by washing the transferred PVDF membrane overnight in PBS containing 0.1% (v/v) Tween 20 14 . One hypothesis is that paraformaldehyde fixation may generate intermolecular covalent bonds between α -synuclein monomers themselves or with other proteins on the blot membranes, causing firm adherence to the membrane 14 . However, the enhancing effect of paraformaldehyde fixation was observed by western blotting using purified α -synuclein proteins, indicating that this effect is not due to intermolecular bonds between α -synuclein monomers and other proteins. Alternatively, Newman et al. demonstrated that treatment of cells or cell lysates with the reducible amine-reactive crosslinker DSP enhanced α -synuclein monomer signals by western blotting 14 . This enhancement occurred even in samples that underwent reductive cleavage of DSP cross-links by β -mercaptoethanol 14 . The authors proposed that DSP/β -mercaptoethanol treatment neutralized positive charges and increased the hydrophobicity of α -synuclein, resulting in enhanced adhesion to the blot membranes 14 . The effects of paraformaldehyde or glutaraldehyde fixation may occur by a mechanism analogous to DSP/β -mercaptoethanol treatment, because paraformaldehyde and glutaraldehyde react with amino groups and increase hydrophobicity 15 . According to this hypothesis, 0.4% paraformaldehyde membrane fixation may be insufficient to reduce the charges on α -synuclein. Furthermore, the strong crosslinking ability of glutaraldehyde may greatly reduce the hydrophilicity of Ser129-phosphorylated α -synuclein monomers, thereby resulting in higher sensitivity than conventional or 0.4% paraformaldehyde fixation methods.
Ser129-phosphorylation is the dominant α -synuclein modification found in LBs and Lewy neuritis 4,5 . This suggests that abnormal elevation of Ser129-phosphorylation accelerates α -synuclein aggregation or that it is a secondary reaction to eliminate misfolded and insoluble α -synuclein. However, the exact role of this modification remains unknown. To address this issue, it is necessary to clarify the stage of α -synuclein aggregation at which abnormal phosphorylation acceleration occurs. Expression levels of Ser129-phosphorylated α -synuclein monomers in radioimmunoprecipitation assay-soluble fractions increase prior to detectable LB pathology in the human cingulate and temporal cortices 16 . In addition, the levels of Ser129-phosphorylated α -synuclein in TBS soluble and moderately insoluble fractions increase in brain samples from patients with PD 17 . These findings suggest that increased Ser129-phosphorylation is an early event in the process of α -synuclein aggregation 16,17 . Our previous paper reported that Ser129-phosphorylated α -synuclein was targeted to the proteasome degradation pathway, in addition to dephosphorylation 18 . In addition, Oueslati, et al. demonstrated that Ser129-phosphorylation by PLK2 induced autophagic α -synuclein clearance 9 . This phenomenon required both phosphorylation at Ser129 and formation of α -synuclein and PLK2 complexes 9 . Although the role of Ser129-phosphorylation remains controversial, these findings suggest that the state of Ser129-phosphorylation affects the metabolic fate of α -synuclein under physiological conditions 9,16-18 . In contrast, alterations in Ser129-phosphorylated α -synuclein proteins have been reported as more sensitive biomarkers for diagnosis of PD than total α -synuclein protein [19][20][21] . A previous report demonstrated that melanoma cells release microvesicles that attach Ser129-phosphorylated α -synuclein on the membranes 22 . These findings suggest that the regulatory system of intracellular Ser129-phosphorylated α -synuclein protein and the associated release mechanism may be involved in the pathological process of PD. To resolve these questions, it is necessary to detect the levels of endogenous total and Ser129-phosphorylated α -synuclein proteins in intra-and extracellular spaces more stably, using western blotting. Combination fixation with paraformaldehyde and glutaraldehyde seems to be a potent method for performing more quantitative biochemical experiments, and may facilitate studies to elucidate these outstanding questions.
Measurement of α -synuclein levels in the blood plasma and CSF is proposed as a useful biomarker for PD diagnosis 19,20,23 . Ser129-phosphorylated α -synuclein in CSF weakly correlates with the severity of PD and, when combined with total α -synuclein concentrations, contributed to distinguishing PD from multiple system atrophy and progressive supranuclear palsy 23 . In addition, Ser129-phosphorylated α -synuclein in blood plasma is reported to be a more promising diagnostic biomarker than total α -synuclein 19,20 . These results were obtained using sandwich ELISA data. The transferred membrane fixation method may be applied to measurement of Ser129-phosphorylated α -synuclein and total α -synuclein using western blotting. This technique provides assessment of antibody specificity and elucidates the state of