Reduction of Immunoreactivity Against the C-Terminal Region of the Intracellular α-Synuclein by Exogenous α-Synuclein Aggregates: Possibility of Conformational Changes

Abstract

Background: The formation of intracellular aggregates containing α-synuclein (α-syn) is a main pathological feature of Parkinson disease. The propagation of α-syn aggregation via cell-to-cell transmission has been implicated in the progression of Parkinson disease.

Objective: Our aim is to clarify the molecular mechanisms underlying the formation of intracellular aggregation by extracellular α-syn.

Methods: We investigated the effects of exogenous α-syn aggregates on intracellular α-syn immunoreactivity in α-syn-overexpressing SH-SY5Y cells using two antibodies to distinct epitopes of α-syn. To obtain α-syn aggregates, α-syn solution was aged with continuous agitation.

Results: Immunoreactivity against the acidic C-terminal domain of the intracellular α-syn was reduced by exposure to agedα-syn, whereas that against the hydrophobic non-amyloid component region was not changed. The reduction in immunoreactivity was not suppressed by protease inhibitors but was mimicked by neutralization of the negative charges on the C-terminal of the intracellular α-syn induced by spermine or extracellular acidification.

Conclusions: These results suggest that the reduction in immunoreactivity is attributed not to proteolytic cleavage but to a conformational change at the C-terminus of the intracellular α-syn. The conformational change at the C-terminus of the intracellular α-syn might be involved in an initial step of fibril formation by exogenous α-syn aggregates.

Keywords

α-synuclein conformational change fibril Parkinson disease proteolysis

ABBREVIATIONS

α-syn

α-synuclein

NAC

non-Aβ component

PAGE

polyacrylamide gel electrophoresis

Parkinson disease

INTRODUCTION

Parkinson disease (PD) is a common neurodegenerative disorder that is characterized by a selective loss of dopaminergic neurons and by the appearance of Lewy bodies in the substantia nigra. In the pathological state, α-synuclein (α-syn), the major component of Lewy bodies, misfolds and forms oligomers that grow into protofibrils and, finally, forms fibrils [1]. The process of α-syn aggregation is responsible for neuronal dysfunction and death, although it is still unclear which are the toxic forms of the protein [2].

α-Syn pathology is believed to spread in a temporal and topological manner [3]. Recent studies have shown that exogenous α-syn fibrils induce the formation of Lewy body-like intracellular inclusions in cultured cells and mice [4, 5]. In fact, Kordower et al. [6] reported that embryonic neurons transplanted into the striatum of a patient with PD had Lewy body-like inclusions. These findings suggest that the spread of pathogenic α-syn by cell-to-cell transmission occurs via a seeding mechanism.

Several experiments in cell-free systems indicate that the C-terminus of α-syn plays a critical role in the initiation of aggregation and fibrillogenesis [7, 8]. The sequence of α-syn, which consists of 140-amino acids, can be divided into three domains, namelyN-terminal amphiphilic region (residues 1–60), a middle hydrophobic region that contains the amyloidogenic non-Aβ component (NAC) region (residues 61–95) and a C-terminal acidic region (residues 96–140). It has been suggested that shielding of the amyloidogenic NAC region by the C-terminal region within the protein prevents self-assembly [9]. However, the intracellular initiation step in fibril formation by exogenous α-syn fibrils has not been completely clarified. Previous studies using tagged α-syn indirectly detected the conformational changes and aggregation in intracellular α-syn [10, 11]. Because α-syn is a relatively small protein, the tagged protein may alter the native protein conformation. In this study, we investigated structural changes in intracellular untagged α-syn by exogenous α-syn aggregates by double-staining immunocytochemistry for two distinct epitopes.

MATERIALS AND METHODS

Materials

Recombinant α-syn was purchased from rPeptide (Bogart, GA, USA). Thioflavine S, E-64d, and spermine were purchased from Sigma–Aldrich Co.(St. Louis, MO, USA). MDL-28170 and MG-132 were obtained from Calbiochem (San Diego, CA, USA). Pepstatin A was purchased from Peptide Institute Inc. (Osaka, Japan). Hoechst 33258 was obtained from Wako (Osaka, Japan).

Cell culture

Human neuroblastoma SH-SY5Y cells stably expressing α-syn were established, as reported previously [12]. Cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 1% non-essential amino acids and were incubated at 37°C in a humidified atmosphere of 95% air and 5% CO₂.

Preparation of aged a-syn and quantification of fibril formation

Recombinant human α-syn was freshly dissolved at a concentration of 25μM in 20 mM Tris-HCl buffer (pH 7.4) containing 100 mM NaCl, which was defined as fresh α-syn. The α-syn solution was aged at 37°C for 5 days with continuous shaking (2,400 rpm), which was defined as aged α-syn. To measure the formation of α-syn fibril, thioflavin S assay was performed. Aged α-syn (25μM) was diluted to a concentration of 1μM with 50 mM glycine-NaOH buffer (pH 8.5) containing 10μM thioflavin S. Fluorescence was measured with excitation and emission wavelengths of 440 and 535 nm, respectively. Before the treatment of the cells, aged α-syn was sonicated with 15 intermittent pulses (1 s on and 1 s off) using an ultrasonic disruptor.

Polyacrylamide gel electrophoresis (PAGE) and western blotting

Denatured proteins were separated using the commonly used Laemmli-SDS-PAGE, as described previously [13]. Denatured low-molecular-weight proteins were separated using the Tricine-SDS-PAGE, according to the method described by Schägger and von Jagow [14]. For determination of aggregation status under non-denaturing conditions, the blue native PAGE was performed, as described previously [15]. The following antibodies were used to detect target proteins on the western blot: anti-α-syn monoclonal antibody (1 : 1000 dilution, 6103787, Syn-1, BD Transduction labs, San Jose, CA, USA), anti-α-syn polyclonal antibody (1 : 1000 dilution, S3062, Sigma–Aldrich), anti-β-actin monoclonal antibody (1 : 100,000 dilution, A1978, AC-15, Sigma–Aldrich) and horseradish peroxidase-conjugated secondary antibodies (GE Healthcare, Little Chalfont,UK).

Immunocytochemistry

Following fixation with 4% paraformaldehyde for 30 min, cells were incubated with 0.2% Triton X-100 for 15 min, with primary antibodies for 2 h, and with secondary antibodies for 1 h. For nuclear staining, cells were incubated with Hoechst 33258 (100μg/mL) for 15 min. The following antibodies were used for immunocytochemistry: anti-α-syn monoclonal antibody (1 : 1000 dilution, 6103787, Syn-1, BD Transduction labs); anti-α-syn polyclonal antibody (1 : 1000 dilution, S3062, Sigma–Aldrich); anti-α-syn monoclonal antibody (1 : 1000 dilution, S5566, Syn211 Sigma–Aldrich); anti-β-actin monoclonal antibody (1 : 1,000 dilution, A1978, AC-15 Sigma–Aldrich); and Alexa Fluor-conjugated secondary antibodies (Life technologies, Carlsbad, CA, USA). Unless otherwise noted, images of immunostained cells were obtained using a BZ-8100 fluorescence microscope (Keyence, Osaka, Japan). Confocal images were obtained using an A1RMP multiphoton confocal microscope (Nikon, Tokyo, Japan).

Statistics

The statistical differences between three or more groups were analyzed by one-way analysis of variance and post-hoc multiple comparisons using Turkey’s test. Statistical significance was defined as p < 0.05. Data are expressed as mean±SEM.

RESULTS

Aged α-syn aggregates did not induce cell injury in α–syn-overexpressing SH–SY5Y cells

The expression level of α-syn in SH-SY5Y cells was negligible in comparison to α-syn-overexpressing cells (Fig. 1A). Although a previous study has shown that α-syn oligomers are detected by size exclusion chromatography inα-syn-overexpressing SH-SY5Ycells [12], cellular overexpression of α-syn by itself did not lead to aggregation that resembled Lewy bodies (Fig.1B). To examine the effect of extracellular α-syn aggregates on endogenous α-syn, preformed α-syn aggregates were prepared from recombinant α-syn peptide. Recombinant α-syn freshly prepared was defined as fresh α-syn. Fibril formation was detected after 48 h of incubation of fresh α-syn with agitation and reached a plateau between 48 and 72 h. However, no fibrils were seen without agitation (Fig.1C). The α-syn solution incubated for 5 days with agitation was defined as aged α-syn. To determine the polymeric state of aged α-syn, blue native PAGE, a technique that allows the separation of protein complexes under non-denaturing conditions, was performed. While monomeric α-syn migrated around 19 kDa, aged α-syn remained in the well because of high molecular weight (Fig. 1D). However, treatment of α-syn-overexpressing SH-SY5Ycells with agedα-syn failed to induce cytotoxicity in our experimental conditions (data not shown).

Changes in α-syn immunoreactivity by exogenous α-syn aggregates

To examine the effect of extracellular α-syn aggregates on the intracellular aggregate formation, α-syn-overexpressing SH-SY5Ycells were stained using two antibodies to distinct epitopes of α-syn. Perrin et al. [16] have reported that the epitope for the monoclonal antibody Syn-1 (BD Transduction labs) is localized within residues 91–99 of α-syn. The polyclonal antibody S3062 (Sigma–Aldrich) is produced using amino acids 111–132 of α-syn as the immunogen. Untreated α-syn-overexpressing SH-SY5Ycells were immunostained with both antibodies in a similar pattern. However, in α-syn-overexpressing SH-SY5Ycells treated with aged α-syn, the staining pattern with anti-α-syn (111–132) antibody was granular, while that with anti-α-syn (91–99) antibody remained diffuse. This change in the staining pattern was not observed in α-syn-overexpressing SH-SY5Ycells treated with fresh α-syn (Fig. 2A). Similarly, when another monoclonal antibody Syn211 (Sigma–Aldrich) that has the epitope located within amino acids 121–125 of α-syn [17] was used, diffuse immunoreactivity decreased and punctual immunoreactivity appeared after treatment with aged α-syn (Fig. 2B). When SH-SY5Ycells were treated with aged α-syn, the punctual immunoreactivity for α-syn appeared, although immunoreactivity detected with anti-α-syn (111–132) antibody was more intense than that detected with anti-α-syn (91–99) antibody (Fig. 2C). This result suggests that the punctual immunoreactivity for α-syn was derived from exogenous α-syn aggregates. Confocal imaging showed that some puncta immunoreactive for anti-α-syn (111–132) antibody appeared to be outside the cells, others existed inside the cells (Fig. 2D). To clarify the localization of the puncta immunoreactive for anti-α-syn (111–132) antibody, the cell shapes of SH-SY5Y cells were determined using anti-β-actin antibody. A high-magnification image using confocal laser scanning microscopy demonstrated that the puncta immunoreactive for anti-α-syn (111–132) antibody could exist in both the nucleus and the cytoplasm (Fig. 2E).

Reduction of the intracellular α-syn C-terminal domain immunoreactivity by exogenous α-syn aggregates

We focused on the finding that the diffuse immunoreactivity detected with anti-α-syn (111–132) antibody was decreased by aged α-syn, even though that detected with anti-α-syn (91–99) antibody was not changed. Because treatment of SH-SY5Y cells with aged α-syn induced only puncta, but not diffuse, immunoreactivity (Fig. 2C), diffuse immunoreactivity was considered as endogenous α-syn signals. In double-staining immunocytochemistry for two distinct epitopes of α-syn, fluorescence intensity in the cells exclucing the puncta was measured (Fig. 3A). The ratio of α-syn (111–132) to α-syn (91–99) fluorescence intensity was significantly decreased by treatment with aged α-syn. On the other hand, fresh α-syn did not affect the ratio of fluorescence intensity (Fig. 3B). The agedα-syn-induced reduction in immunoreactivity against the C-terminal domain of intracellular α-syn was observed in concentration- and time-dependent manners (Fig. 3C and D).

Exogenous α-syn aggregates did not cleave the C-terminus of the intracellular α-syn

The reduction in α-syn C-terminus immunoreactivity may be attributed to proteolytic cleavage at the C-terminus. Various proteases are known to cleave α-syn [18]. Thus, the effects of several protease inhibitors on the reduction of immunoreactivity were examined. E-64d is an inhibitor of cysteine proteases, including cathepsin B. Pepstatin A is an inhibitor of aspartic proteases, including cathepsin D. MDL-28170 is a selective inhibitor of calpain-1/2 and cathepsin B. MG-132 is a specific proteasome inhibitor. Because treatment with MG-132 for 48 h caused cytotoxicity, α-syn-overexpressing SH-SY5Ycells were exposed to aged α-syn in the presence or absence of these protease inhibitors for 24 h. These inhibitors did not suppress the reduction in the intracellular α-syn C-terminal immunoreactivity induced by the aged α-syn (Fig. 4A and B). In addition, Tricine-SDS-PAGE revealed that exposure to aged α-syn had no effect on the apparent molecular weight of intracellular α-syn. Truncated α-syn, which was identified by either anti-α-syn (91–99) or anti-α-syn (111–132) antibody, was not changed by aged α-syn (Fig. 4C). Sevlever et al. [19] have reported that α-syn truncated by cathepsin D was detected byanti-α-syn (91–99) antibody, but not by antibody against the C-terminus of α-syn. When cell lysates from α-syn-overexpressing SH-SY5Ycells were incubated in sodium acetate solution (pH 4) for 4 h according to the previous report [19], truncated α-syn was detected by anti-α-syn (91–99) antibody, but not by anti-α-syn (111–132) antibody. The degradation of α-syn was partially blocked by E-64d and pepstatin A (protease inhibitors: PI) (Fig. 4D).

Cation reduced the intracellular α-syn C-terminus immunoreactivity

Another possible mechanism of the reduction of α-syn C-terminus immunoreactivity is a conformational change at the C-terminal domain. Spermine, a biogenic polyamine, and low pH are known to neutralize C-terminal negative charges of α-syn in cell-free systems, resulting in conformational changes at the C-terminus [8 , 21]. Treatment with spermine or lowering the extracellular pH to 4 significantly reduced the α-syn C-terminus immunoreactivity in α-syn-overexpressing SH-SY5Ycells (Fig. 5A). Figure 5B showed the spermine concentration-dependent and pH-dependent reduction of α-syn C-terminus immunoreactivity. Figure 5C showed the time-dependent reduction of α-syn C-terminus immunoreactivity. Treatment with spermine (1 mM) for 10 h caused obvious cytotoxicity (data not shown).

DISCUSSION

This study demonstrated that exogenous α-syn aggregates selectively decreased the intracellularα-syn C-terminus immunoreactivity. The most likely explanation for this finding is that decreased immunoreactivity is because of proteolytic cleavage or a conformational change at the C-terminal domain. C-terminal truncated forms of α-syn have been identified in healthy and PD brains [22]. A number of enzymes have been implicated in proteolysis of α-syn and generation of C-terminal truncated fragments [19]. In fact, cell-free fibrillization studies have shown that C-terminal truncated fragments enhance the rate of fibrillization [7, 8]. However, several protease inhibitors used in the present study did not suppress the decrease in C-terminus immunoreactivity. Furthermore, C-terminal truncated forms of α-syn did not appear in α-syn-overexpressing SH-SY5Ycells after exposure to aged α-syn aggregates. Therefore, we rejected the possibility that exogenous α-syn aggregates cleaved the C-terminus of the intracellular α-syn under our experimental conditions. Recently, Tsujimura et al. [11] have reported that exogenous α-syn fibrils induced fluorescent aggregates in a cathepsin B-dependent manner in cells overexpressing C-terminal ECFP-fused α-syn. Therefore, exogenous α-syn fibrils might cleave the N-terminus of intracellular α-syn.

Because the C-terminal region of α-syn, which contains 10 glutamate and 5 aspartate residues, has an acidic nature, the C-terminus interacts with cationic compounds, including polyamines and protons. Under physiological conditions, the C-terminus is believed to populate an extended conformational state because of a high density of negative charges, resulting in the shielding of the amyloidogenic NAC region. Once the C-terminal negative charges are neutralized, the C-terminus populates a compacted conformational state and loses the ability to shield the NAC region [8 , 23]. In fact, cell-free fibrillization studies have shown that polyamines and acidification promote aggregation of α-syn [8, 24]. We showed that exposure to spermine and extracellular acidification reduced C-terminus immunoreactivity in α-syn-overexpressing SH-SY5Ycells. It has been reported that spermine is transported into the cells through an organic cation transporter 2 [25]. Intracellular pH is known to be decreased via inhibition of the Na⁺/Ca^2 + exchanger 1 by extracellular acidification [26]. Therefore, exposure to spermine and extracellular acidification are likely to cause the conformational change at the C-terminal domain of intracellular α-syn, resulting in the reduction in C-terminus immunoreactivity. Taken together, we speculate that exogenous α-syn aggregates also induce a conformational change at the C-terminal domain of the intracellular α-syn.

The mechanisms by which exogenous α-syn aggregates induced the conformational change in the intracellular α-syn remain to be investigated. We found that exogenous α-syn aggregates existed both outside and inside the cells. In addition, because it is likely that aged α-syn used in the present study contain oligomers, protofibrils, and fibrils, we cannot conclude which form of α-syn induced the conformational change. Nevertheless, exogenous α-syn aggregates might neutralize the negative charges on the C-terminal of the intracellular α-syn. Pachecoet al. [27] have recently reported that α-syn oligomers increase intracellular Ca^2 + levels in hippocampal neurons through the formation of ion-permeable pores. In fact, the binding of Ca^2 + to the C-terminus of α-syn enhanced the oligomerization of α-syn [28]. Furthermore, it has been reported that increased intracellular Ca^2 + promoted α-syn aggregation[29].

Unfortunately, we admit the lack of direct evidence of the conformational change in the intracellular α-syn. We could not exclude the possibility that spermine and acidification reduced the intracellular α-syn C-terminus immunoreactivity without the conformational change. Besides, due to crossreactivity of antibodies, endogenous and exogenous α-syn was not completely distinguished in this study. To overcome these technical problems, fluorescence resonance energy transfer (FRET) measurement using fluorescence lifetime imaging microscopy (FLIM) is useful. Outeiro et al. [10] suggest that dopamine induces conformational change in the intracellular α-syn using N- and C-terminally small peptides-tagged α-syn.

Our findings suggest that intracellular α-syn adopts an aggregation-prone conformation by exogenousα-syn aggregates. However, the present study did not show any intracellular aggregation and cell damage by exogenous α-syn aggregates, although a previous study has reported that introduction of preformed α-syn fibrils into SH-SY5Y cells with a transfection reagent caused aggregate formation and cell death [30]. The phenomenon observed in this study might be just an initiation step of intracellular aggregation. A study in cultured neurons has reported that treatment with preformed α-syn fibrils induced intracellular aggregation and cell death from 7 days onward [31]. Moreover, in mice, intrastriatal inoculation of preformed α-syn fibrils induced neurodegeneration of dopaminergic neurons from 90 days onward [32]. Incorporation of α-syn fibrils into cells as fibril nuclei may be required to form intracellular aggregation.

The present study investigated the influence of exogenous α-syn aggregates on the intracellularα-syn. We found that exogenous α-syn aggregates selectively decreased the α-syn C-terminal domain immunoreactivity. It is suggested that exogenous α-syn aggregates induce the conformational change at the C-terminal domain of the intracellular α-syn. Further understanding of the mechanisms of intracellular α-syn aggregation by exogenous α-syn aggregates will provide insights into a role of cell-to-cell α-syn propagation in the pathologic processes of PD.

CONFLICT OF INTEREST

The authors have no conflict of interest to report.

Footnotes

ACKNOWLEDGMENTS

This work was supported by supported by a grant from the Smoking Research Foundation, Japan. The authors would like to thank Enago for the English language review.

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