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J. Biol. Chem., Vol. 277, Issue 21, 19213-19219, May 24, 2002

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Biochemical Characterization of the Core Structure of -Synuclein Filaments^*

Hirotomo Miake^§, Hidehiro Mizusawa^§, Takeshi Iwatsubo^¶, and Masato Hasegawa^¶**

From the  Department of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, the ^§ Department of Neurology and Neurological Science, Graduate School of Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, and the  Department of Molecular Neurobiology, Tokyo Institute of Psychiatry, Tokyo Metropolitan Organization for Medical Research, 2-1-8 Kamikitazawa, Setagaya-ku, Tokyo 156-8585, Japan

Received for publication, November 2, 2001, and in revised form, March 11, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Intracellular filamentous aggregates comprised of -synuclein such as Lewy bodies and glial cytoplasmic inclusions are the defining hallmarks of a subset of neurodegenerative diseases including Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. We have analyzed biochemical and structural properties of -synuclein filaments assembled in vitro or extracted from brains of patients with multiple system atrophy and found that both types of filaments are insoluble to detergents and partially resistant to proteinase K digestion. Immunoelectron microscopy and immunoblot analysis showed that both amino and carboxyl termini of -synuclein in in vitro assembled filaments were degraded by proteinase K treatment, whereas the central portion of -synuclein is resistant to proteinase K and retains filamentous structures. Protein sequencing and mass spectrometric analyses of the proteinase K-resistant, minimal fragment of 7 kDa revealed that amino acid residues 31-109 of -synuclein constitute the core unit of the filaments. These observations suggest that the central half of the -synuclein polypeptide, containing five tandem repeats as well as a part of the carboxyl-terminal acidic region, forms the core structure of -synuclein filaments, which is coated by the amino- and carboxyl-terminal portions at the periphery.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Filamentous cytoplasmic inclusion bodies comprised of -synuclein in neurons or glial cells are the hallmark lesions of a group of neurodegenerative diseases collectively referred to as synucleinopathies (1). In Parkinson's disease (PD)¹ and dementia with Lewy bodies (DLB), -synuclein is deposited as Lewy bodies and Lewy neurites that accumulate in cell bodies or neuronal processes (2-4), whereas filamentous -synuclein aggregates are predominantly found in oligodendrocytes as glial cytoplasmic inclusions (GCIs) in multiple system atrophy (MSA) (5-7). The following evidence strongly implicates the deposition of -synuclein in the pathogenesis of these neurodegenerative disorders. 1) Two missense mutations (A53T and A30P) in the -synuclein gene that cosegregate with the onset of PD have been identified in kindreds of autosomal dominantly inherited familial PD (8, 9). 2) Immunohistochemical and biochemical analysis of PD, DLB, and MSA brains have revealed widespread deposition of -synuclein in the brains of patients with either sporadic or familial forms of PD, as well as in DLB and MSA (10, 11), in which -synuclein has been shown to form the major filamentous component of inclusion bodies (6, 10, 12). 3) Recombinant -synuclein proteins assemble into filaments in vitro that closely resemble those found in LB and GCIs, whereas other members of synuclein family proteins, i.e. -synuclein and -synuclein, neither deposit in brains nor assemble into filaments (13-15). 4) Missense mutations (A53T and A30P) identified in familial PD have been shown to increase the propensity of -synuclein to form filaments or oligomers (16-20).

-Synuclein is a 140-amino acid, heat-stable protein, harboring seven imperfect tandem repeat sequences in the amino-terminal half (Fig. 1A), followed by a hydrophobic central region (referred to as the NAC portion) and an acidic carboxyl terminus. -Synuclein is abundantly expressed in neurons as a cytosolic protein that is localized to presynaptic termini, although it has been shown that a proportion of -synuclein is associated with membranes (21, 22). Circular dichroism spectra analysis of recombinant proteins revealed that -synuclein is a natively unfolded protein with little ordered secondary structure (23). Further structural analyses have shown that full-length or carboxyl-terminal-truncated recombinant -synuclein can assemble into straight filaments 5-10 nm wide that closely resemble filaments isolated from PD, DLB, or MSA brains (13-15). X-ray fiber diffraction and electron diffraction analyses have shown that a transition from random coil to a cross--sheet structure underlies the assembly of -synuclein into filaments (15). Recent studies have shown that residues 71-82 of -synuclein, which are absent in -synuclein, play a crucial role in its assembly into filaments (24). However, -synuclein also harbors a hydrophobic stretch similar to that of -synuclein (and especially a homologous portion to residues 71-82 of -synuclein) although -synuclein poorly assembles into filaments (14, 15).

To learn more about the mechanisms underlying the assembly of the natively unfolded -synuclein protein into -sheet-rich filaments, we studied the biochemical properties of -synuclein filaments, especially their structural stability to protease digestion. Here we have shown that -synuclein filaments assembled in vitro or recovered from MSA brains that are morphologically similar to each other share the following biochemical characteristics: (i) insolubility in detergents (Triton X or Sarkosyl) but high solubility in urea or SDS, and (ii) resistance of a subdomain of -synuclein against proteinase K treatment. We propose that the proteinase K-resistant 7-kDa fragment comprised of residues 31-109 of -synuclein may represent the core unit of -synuclein filaments, which contributes to the structural stability of these filaments.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Antibodies-- Monoclonal antibody LB509 was raised against isolated Lewy bodies, and the epitope was localized to residues 121/122 of -synuclein (25). Syn102 was raised against recombinant -synuclein with an epitope within residues 131-140 (7, 26). No. 36 is an antiserum against a synthetic peptide corresponding to residues 1-10 of -synuclein. NAC1 raised against a synthetic peptide corresponding to residues 75-91 of -synuclein is a gift from Dr. Jäkälä (27).

Extraction of Dispersed -Synuclein Filaments from MSA Brains-- -Synuclein filaments derived from glial cytoplasmic inclusions were extracted from frozen cerebella from patients with MSA or from normal individuals as controls. 0.5-1 g of brain tissue was homogenized in 10 volumes of buffer A (50 mM Tris-HCl, pH 7.5, 1 mM EGTA, 1 mM dithiothreitol) and centrifuged at 1,000 × g for 10 min. The supernatants were ultracentrifuged at 350,000 × g for 20 min, and the resulting pellets were sequentially extracted by homogenization followed by ultracentrifugation in buffer A containing 1% Triton X-100 and then in 10% sucrose and 0.5 M NaCl. The pellets were homogenized in buffer A containing 1% Sarkosyl, 10% sucrose, and 0.5 M NaCl and incubated at 37 °C for 1 h. After centrifugation at 27,000 × g for 20 min, the supernatants were further centrifuged at 350,000 × g for 20 min. The resulting pellets were suspended in a 0.5-fold volume of 50 mM Tris-HCl buffer (pH 7.5) and subjected to proteinase K treatment and immunoelectron microscopic analysis of -synuclein filaments. This Sarkosyl-insoluble fraction was further homogenized in buffer A containing 8 M urea, and the urea-soluble fraction was obtained by ultracentrifugation. Each supernatant fraction was denatured in SDS sample buffer, separated by 15% Tris/Tricine gels, and analyzed by immunoblotting as described (11).

In Vitro Assembly and Ultrastructural and Biochemical Analyses of Recombinant -Synuclein into Filaments-- Recombinant -synuclein was expressed in Escherichia coli BL21 and purified by boiling treatment and then Q-Sepharose ion exchange chromatography followed by separation by reverse phase-HPLC on an Aquapore RP300 column as described (28). For assembly, recombinant -synuclein was prepared at a concentration of 4 mg/ml in 50 µl of 30 mM Tris-HCl (pH 7.5) and incubated at 37 °C with shaking at 250 rpm in an incubator. After incubation for 48 h, aliquots (0.5-1 µl) were placed on 400-mesh carbon-coated grids and negatively stained with 2% lithium phosphotungstate and observed by JEOL-1200EX. Immunoelectron microscopic analysis was performed as described (10). Briefly, after blocking with 10% calf serum, the grids were incubated with primary antibodies (Syn102, NAC1, or no. 36) diluted at appropriate concentrations for 2 h, followed by incubation with secondary antibodies conjugated with 5-nm gold particles (Sigma). The grids were stained with 2% lithium phosphotungstate prior to observation by electron microscopy. For differential solubilization of -synuclein filaments, aliquots of assembly mixtures were dispersed by sonication in 10 volumes of buffer A and then centrifuged at 350,000 × g for 20 min. The resulting pellets were extracted with 10 volumes of buffer A containing 1% Triton X-100. After centrifugation, the Triton-insoluble pellets were homogenized in 1% Sarkosyl using sonication. The Sarkosyl-insoluble pellets were further extracted in 8 M urea. Each supernatant fraction was dissolved in SDS sample buffer and analyzed by SDS-PAGE.

Proteinase K Treatment of -Synuclein Filaments-- Sarkosyl-insoluble -synuclein filaments extracted from brains of patients with MSA or Tris-soluble -synuclein from control brains were treated with 1, 100, 500, or 1,000 µg/ml proteinase K at 37 °C for 30-60 min. The protein concentration of each fraction was adjusted to 2 mg/ml. In vitro assembled -synuclein filaments and unassembled recombinant -synuclein were treated with proteinase K at various concentrations of 2-1,000 µg/ml at 37 °C for 30-60 min. The reaction was stopped by boiling for 5 min. After centrifugation, the resulting pellets were dissolved in 8 M urea containing 2% SDS and analyzed by immunoblotting with LB509, Syn102, NAC1, or no. 36. Proteinase K-treated filaments were also analyzed by immunoelectron microscopy.

Protein Chemical Analysis of Proteinase K-resistant Core of -Synuclein-- In vitro assembled -synuclein filaments and unassembled -synuclein in soluble form were treated with 10 µg/ml proteinase K for 30 min. The digests were dissolved in SDS sample buffer, separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and visualized by staining with Coomassie Brilliant Blue. The amino-terminal sequences of the major three bands migrating at 7, 8, and 9 kDa were directly analyzed by a protein sequencer (ABI492 protein sequencer) as described (29). Analysis of the proteinase K-resistant 7-kDa fragment was performed by treating the assembled and unassembled -synuclein with 500 µg/ml proteinase K for 60 min, followed by boiling for 5 min, and solubilization in 6 M guanidine HCl. The digests were separated on an Aquapore RP300 column (2.1 × 30 mm, Applied Biosystems) by HPLC (Hewlett-Packard, Model 1100) with a linear gradient of 0-48% acetonitrile in 0.1% trifluoroacetic acid for 16 min at a flow rate of 0.2 ml/min. Aliquots of the fractions were lyophilized, subjected to SDS-PAGE, and analyzed by immunoblotting with NAC1. Mass spectral analysis was performed by a Voyager-DE Pro MALDI-TOF mass spectrometer (PerSeptive Biosystems).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Previous studies have shown that recombinant -synuclein forms filaments that closely resemble those isolated from brains of patients with PD, DLB, and MSA in vitro. To verify the morphological and biochemical characteristics of synthetic -synuclein filaments, we have compared their ultrastructure and solubility with those isolated from brains of patients with MSA (Fig. 1B). Incubation of purified recombinant -synuclein at 37 °C for 48 h with continuous shaking resulted in the formation of abundant filaments (Fig. 1C). These filaments were 50-700 nm long and 5-10 nm wide, as previously documented (15). These filaments appeared as straight filaments that were very similar to those extracted from MSA brains.

View larger version (43K): [in this window] [in a new window]   Fig. 1.   Ultrastructural and biochemical characteristics of -synuclein filaments derived from MSA brains and assembled in vitro from recombinant proteins. A, sequence alignment of human -, -, and -synuclein. The underlined sequences indicate five to seven tandem repeat sequences found in each synuclein. Amino acid residues of - or -synucleins identical to those in -synuclein are in bold letters. A30P and A53T mutations linked to familial PD also are shown. B and C, immunoelectron microscopic observation of dispersed filaments extracted from MSA brains (B, LB509/10 nm immunogold) and negatively stained electron micrograms from assembled recombinant -synuclein protein in vitro (C). Scale bar represents 100 nm. D and E, differential solubilization profiles of -synuclein from control and MSA brains (D), as well as from in vitro assembled filaments (assembled) and controls (unassembled) (E) revealed by immunoblotting with LB509. Buffers used for extraction are shown below the lanes. Molecular mass standards are shown in kilodaltons.

To biochemically characterize the -synuclein filaments, we performed differential extraction of -synuclein from brain tissues and in vitro assembled filaments. Frozen tissues from control and MSA brains were sequentially extracted with Tris-HCl buffer, 1% Triton X-100, 1% Sarkosyl, and 8 M urea. Extracted proteins were separated by SDS-PAGE and analyzed by immunoblotting with LB509. A 15-kDa polypeptide strongly immunoreactive for LB509 was detected in Tris-soluble and Triton-soluble fractions from both control and MSA brains. The amounts of -synuclein in these fractions were slightly smaller in MSA brains compared with control brains. In contrast, LB509 immunoreactive polypeptides migrating at similar positions to normal -synuclein were detected in the Sarkosyl-insoluble, urea-soluble fraction of MSA brain (Fig. 1D), whereas no -synuclein immunoreactivities were observed in the same fractions of control brains. Immunoelectron microscopic observation of the Sarkosyl-insoluble fraction from MSA brains showed filaments that were labeled by multiple anti--synuclein antibodies. The extraction patterns of -synuclein aggregates in MSA brains were similar to that observed by Dickson et al. (11) as well as to those in other synucleinopathies including DLB (30) and Hallervorden-Spatz disease,² suggesting that filamentous -synuclein aggregates deposited in synucleinopathy brains exhibit similar insolubility profiles (i.e. Sarkosyl-insoluble and urea-soluble). The amounts of Sarkosyl-insoluble -synuclein appeared to correlate with the density of GCIs as judged by semiquantitative evaluation of the amount of -synuclein-positive aggregates by immunostaining of the smears of the brain homogenates (data not shown).

Similarly, in vitro assembled -synuclein filaments were sequentially extracted by Tris-HCl, 1% Triton X-100, 1% Sarkosyl, and 8 M urea, together with unassembled -synuclein incubated without shaking and analyzed by immunoblotting. As shown in Fig. 1E, unassembled -synuclein was totally recovered in Tris-HCl and Triton X-soluble fractions, without any immunoreactive substances detected in Sarkosyl-insoluble fractions. In sharp contrast, a ~15-kDa protein as well as additional polypeptides migrating at ~25-30 kDa in a Sarkosyl-insoluble, urea-soluble fraction of in vitro assembled -synuclein showed strong immunoreactivity for LB509, the latter presumably representing -synuclein dimers. Electron microscopic observation of the Sarkosyl-insoluble fraction of in vitro assembled -synuclein confirmed the preservation of filamentous structures after extraction with 1% Triton X and 1% Sarkosyl. Taken together, synthetic -synuclein filaments share a number of biochemical as well as morphological characteristics (i.e. solubility profiles, molecular size, and ultrastructure) with those recovered from inclusion bodies in synucleinopathy brains.

We next examined the structural stability of -synuclein filaments by treating them with proteinase K. Tris-soluble fractions from control brains containing abundant normal -synuclein and Sarkosyl-insoluble fractions from MSA brains rich in insoluble -synuclein filaments were treated with 1 µg/ml proteinase K, and digestion of -synuclein was monitored by immunoblotting with LB509. Immunoreactivities for -synuclein in Tris-soluble fractions disappeared after proteinase K treatment for 30 min. In contrast, the amount as well as banding patterns of -synuclein in the Sarkosyl-insoluble fraction remained almost unchanged until 60 min of treatment (Fig. 2A). Soluble -synuclein added to Sarkosyl-insoluble fractions from control brains was also readily degraded by proteinase K treatment (data not shown), indicating that the stability of Sarkosyl-insoluble -synuclein to proteinase K is not due to interference by contaminants in this fraction. These results suggest that the filamentous form of -synuclein in Sarkosyl-insoluble fractions is resistant to proteinase K digestion.

View larger version (43K): [in this window] [in a new window]   Fig. 2.   Proteinase K treatment of Sarkosyl-insoluble -synuclein from MSA brains and in vitro assembled filaments from recombinant -synuclein. A, Tris-soluble fractions from control human brain (left panel) and Sarkosyl-insoluble fractions from MSA brain (right panel) were treated with 1 µg/ml proteinase K for 0, 30, and 60 min and analyzed by immunoblotting with LB509. B, Sarkosyl-insoluble fractions from MSA brain prior to (0 min) and after treatment with 100 µg/ml proteinase K for 30 min were analyzed by immunoblotting with NAC1 (left panel) and LB509 (right panel). C, Sarkosyl-insoluble fractions from MSA brain prior to (0 min) and after treatment with 100, 500, and 1,000 µg/ml proteinase K for 30 min were analyzed by immunoblotting with NAC1. D, in vitro assembled filaments from recombinant -synuclein were treated with 1 µg/ml proteinase K for 0, 30, and 60 min and analyzed by immunoblotting with Syn102, LB509, and NAC1. E, in vitro assembled filaments from recombinant -synuclein and unassembled controls were treated with 10 µg/ml proteinase K for 30 min and analyzed by immunoblotting with Syn102, LB509, and NAC1. Note that ~7-9-kDa bands were detected after proteinase K treatment by NAC1 both in Sarkosyl-insoluble fractions of MSA brain (B, arrowheads) and assembled -synuclein (E, arrowheads), but Syn102 or LB509 failed to label these polypeptides. F, unassembled and assembled recombinant -synuclein treated by proteinase K (10 µg/ml, 30 min) were separated by SDS-PAGE and stained with Coomassie Brilliant Blue. Amino acid sequences derived from polypeptides migrating at 9, 8, and 7 kDa, as well as the corresponding amino-terminal positions in human -synuclein, are shown at the right of the panel.

Sarkosyl-insoluble -synuclein from MSA brains was further analyzed by immunoblotting with an additional anti--synuclein antibody NAC1, which recognizes the central hydrophobic region (i.e. NAC domain, residues 75-91) of -synuclein, using higher concentrations of proteinase K (Fig. 2B). Prior to proteinase K treatment, NAC1 reacted with the major ~15-kDa polypeptide (migrating at the same position as full-length -synuclein), as well as with additional minor bands migrating at ~6-12 kDa. After treatment with 100 µg/ml proteinase K, NAC1 exclusively reacted with ~7-9-kDa polypeptides (Fig. 2B, arrowheads), whereas -synuclein immunoreactive bands migrating at higher molecular weight ranges (including full-length -synuclein) were almost completely abolished. The ~7-9-kDa bands were not recognized by LB509, the epitope of which is located around residues 121/122 at the carboxyl terminus of -synuclein. The 7-kDa band was still detected after the treatment with 500 µg/ml proteinase K with NAC1, although it disappeared by the 1,000 µg/ml proteinase K treatment (Fig. 2C), suggesting that these ~7-9-kDa polypeptides corresponded to the core portion of -synuclein filaments that acquired high resistance to protease digestion. To further characterize the proteinase K-resistant -synuclein fragments, in vitro assembled filaments were treated with 1 µg/ml proteinase K for 30-60 min and analyzed by immunoblotting with Syn102, LB509, and NAC1. Immunoreactivities of Syn102 or LB509 were almost completely abolished by the proteinase K treatment for 60 min, whereas ~7-11-kDa bands were detected in the assembled filament fraction with NAC1 (Fig. 2D). Similar results were obtained in the experiment with 10 µg/ml proteinase K treatment for 30 min. NAC1 detected ~7-9-kDa polypeptides in the assembled filament fraction (Fig. 2E, arrowheads), exhibiting a similar pattern to those observed in the proteinase K-treated Sarkosyl-insoluble fractions of MSA brains (Fig. 2B, arrowheads), whereas antibodies to the carboxyl terminus of -synuclein (i.e. LB509 and Syn102) only labeled the ~10-15-kDa bands. Unassembled -synuclein digested by 10 µg/ml proteinase K did not yield any immunoreactive fragments, suggesting that -synuclein was totally degraded in this condition (Fig. 2E).

To further analyze these proteinase K-resistant -synuclein fragments by protein chemical methods, larger amounts (18 µg) of -synuclein proteins in assembled or unassembled states were treated with proteinase K (10 µg/ml), separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and stained with Coomassie Brilliant Blue. The amino-terminal sequences of the three major bands migrating at 7, 8, and 9 kDa detected in proteinase K-treated -synuclein filaments were directly analyzed by a protein sequencer. Analysis of amino acid sequences of the 7- and 8-kDa bands revealed an identical amino-terminal sequence, GKTKEGVLYV, that corresponded to residues 31-40 of -synuclein. The 9-kDa band gave two sequences, i.e. AEKTKQGVAE and EKTKQGVAEA, which corresponded to residues 19-28 and 20-29 of -synuclein, respectively (Fig. 2F). Thus, both the 7- and 8-kDa proteinase K-resistant fragments consisted of polypeptides starting at the same amino terminus (i.e. residue 31 of -synuclein) but ending at different carboxyl-terminal positions.

To investigate the relationship between the proteinase K-resistant ~7-9-kDa fragments and the integrity of -synuclein filaments, we examined the ultrastructure as well as immunoreactivities of -synuclein filaments prior to and after proteinase K treatment by immunoelectron microscopy (Fig. 3). Prior to proteinase K treatment, the filaments were positively labeled by antibodies against the amino- (Fig. 3A, no. 36) and carboxyl- (Fig. 3E, Syn102) terminal portions of -synuclein, whereas NAC1 failed to label them (Fig. 3C). After proteinase K treatment, these filaments still retained their filamentous nature, but the mean diameters were decreased by ~20% (untreated, 12.3 ± 0.5 µm; proteinase K-treated, 9.8 ± 2.3 µm), and immunoreactivities for the amino (no. 36) and carboxyl (Syn102) termini were abolished. In sharp contrast, the filaments became immunoreactive for NAC1, which recognizes the mid-portion of -synuclein. These results suggest that both the amino- and carboxyl-terminal regions of -synuclein are structurally labile and cleaved off from the filaments by proteinase K digestion, whereas the central region containing the hydrophobic NAC portion represents the core structure of filaments that is resistant to protease treatment.

View larger version (108K): [in this window] [in a new window]   Fig. 3.   Immunoelectron microscopic analysis of assembled -synuclein filaments with or without proteinase K treatment. In vitro assembled filaments from recombinant -synuclein prior to (A, C, E) or after proteinase K treatment (B, D, F) were immunolabeled by no. 36 (A and B), NAC1 (C and D), and Syn102 (E and F), followed by decoration with 5-nm gold particles and observed by negative staining. Scale bar represents 100 nm.

To unequivocally define the structure of the minimal fragment that constitutes the protease-resistant core of -synuclein filaments, in vitro assembled -synuclein filaments were treated with various concentrations of proteinase K ranging from 2 to 1,000 µg/ml. The NAC1 immunoreactive 7-kDa polypeptide remained undigested even by treatment with 1,000 µg/ml proteinase K, whereas other fragments including the 6-kDa species disappeared with increasing concentrations of proteinase K (Fig. 4A). This strongly suggested that the 7-kDa fragment corresponds to the highly stable, protease-resistant core unit of the -synuclein filaments. To determine the exact structure of the proteinase K-resistant 7-kDa polypeptide, assembled -synuclein filaments and unassembled -synuclein proteins were treated with 500 µg/ml proteinase K, and the digests were dissolved in 6 M guanidine HCl and separated by reverse phase-HPLC. When the HPLC profiles of unassembled and filamentous -synuclein were carefully compared after treatment with 500 µg/ml proteinase K, one peak was unique to assembled filament digests (Fig. 4B, peak 9); other peaks were derived from fragments of proteinase K, because identical peaks were observed by incubation without -synuclein (data not shown). Immunoblotting of these HPLC fractions with NAC1 confirmed that the 7-kDa -synuclein fragment was recovered in fraction 9 (Fig. 4, B and C). MALDI-TOF mass analysis of fraction 9 gave signals corresponding to a molecular mass of 7873.5, which nearly matched to that of residues 31-109 of human -synuclein (predicted average mass: 7869) (Fig. 4D). These results strongly suggested that residues 31-109 of -synuclein represent the proteinase K-resistant core unit of -synuclein filaments.

View larger version (32K): [in this window] [in a new window]   Fig. 4.   Purification and mass spectrometric analysis of the proteinase K-resistant 7-kDa fragment of -synuclein filaments. A, in vitro assembled -synuclein filaments were treated with various concentrations of proteinase K (PK µg/ml, indicated below the lanes) and analyzed by immunoblotting with NAC1. B, separation profiles of in vitro assembled and unassembled -synuclein proteins treated with proteinase K (500 µg/ml, 30 min) by reverse phase-HPLC on an Aquapore RP300 column. The arrow in the lower panel indicates a peak unique to assembled -synuclein. C, immunoblot analysis of fractions separated in B with NAC1. D, MALDI-TOF mass spectrometry spectrogram of purified ~7-kDa proteinase K-resistant fragments of -synuclein filaments. E, schematic diagram of the location of the ~7-kDa core of the -synuclein filament.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The mechanisms by which -synuclein is assembled into highly ordered filaments and forms intracellular inclusions in the brains of patients with synucleinopathies including PD, DLB, and MSA are unknown. It has been shown that recombinant -synuclein can assemble into filaments that closely resemble the abnormal -synuclein filaments in synucleinopathy brains (12). Thus, in vitro modeling of -synuclein assembly is a useful strategy for the study of the molecular mechanisms of -synuclein fibril formation as well as for screening of small molecules that affect the formation of pathological -synuclein filaments.

In this study, we have shown that the in vitro assembled -synuclein filaments closely resemble the pathological filaments of synucleinopathy brains in their biochemical and structural characteristics. In vitro assembled -synuclein filaments and those from synucleinopathy brains shared very similar solubility profiles, i.e. insolubility in detergents (Triton X and Sarkosyl) and effective solubilization in high concentrations of urea. Furthermore, proteinase K treatment revealed that these two types of -synuclein filaments show very similar resistance profiles to protease digestion.

Biochemical analysis of the proteinase K-resistant -synuclein filament cores strongly suggested that the ~7-9-kDa fragments truncated at amino and carboxyl termini constitute the core portion of -synuclein filaments. Giasson et al. (24) have reported similar proteinase K-resistant -synuclein fragments, of which F1 and F2 fragments may correspond to our 8 and 7-kDa fragments, respectively. Conway et al. (20) have also reported a similar proteinase K-resistant -synuclein fragment that may correspond to our 7-kDa fragment. We have further extended the characterization of the proteinase K-resistant -synuclein core fragments by two complementary strategies, i.e. immunoelectron microscopy and protein chemical analysis.

Immunoelectron microscopic analysis showed that intact -synuclein filaments are labeled by antibodies that recognize the amino or carboxyl termini of -synuclein (no. 36 and Syn102, respectively), whereas an antibody that recognizes the central region of -synuclein (NAC1) failed to label them. In sharp contrast, proteinase K treatment abolished the immunoreactivities for the amino- and carboxyl-terminal portions, whereas NAC1 immunoreactivity was retrieved, probably because removal of the surface structures exposed the antigen buried at the filament cores. Taken together with the immunochemical data discussed above, it is strongly suggested that the central region of -synuclein (encompassing the NAC1 epitope) constitutes the proteinase K-resistant core of -synuclein filaments. Our observation that treatment of tissue sections with proteinase K or formic acid strongly enhanced the NAC1 immunoreactivities of synucleinopathy lesions on tissue sections (data not shown) may support this view.

Protein sequence and mass spectrometric analyses of the proteinase K-resistant fragments revealed that the central portion of -synuclein corresponding to amino acid residues 31-109, which is half the size of holoprotein, constitutes the core of -synuclein filaments. Recently, Giasson et al. (24) reported that the 12-amino acid stretch (⁷¹VTGVTAVAQKTV⁸²) within the central hydrophobic region of -synuclein is necessary and sufficient for its fibril formation, based on the sequence differences between - and -synucleins. However, the reason why -synuclein, which contains a hydrophobic stretch similar to that of -synuclein (see Fig. 1A), poorly assembles into filaments (14, 15) is unknown. It is tempting to speculate that the carboxyl-terminal region of our 7-kDa fragment (residues 98-109), which is unique to -synuclein but not found in - or -synucleins, may contribute to its fibril formation. It is also interesting to note that the amino acid residues substituted by missense mutations linked to familial PD are located within (A53T) or adjacent to (A30P) this 7-kDa core fragment. The pathogenic effects of these mutations to promote formation of -synuclein filaments or oligomers could be related to their effects on the conformational changes of this core portion. Further studies using variously modified or truncated recombinant -synuclein are needed to clarify these points.

There are a number of interesting similarities between -synuclein filaments deposited in synucleinopathy brains and tau filaments in Alzheimer's disease or tauopathy brains. Tau is a microtubule-associated protein harboring three or four tandem repeat sequences that serve as the microtubule binding domain. Tau can also be assembled into filaments in vitro from holoprotein or microtubule binding domain fragments (31-33). Using treatment by various proteases, it has also been shown that ~7-15-kDa fragments containing microtubule binding tandem repeats represent the protease-resistant core of the tau filaments that contributes to their stability (29, 34, 35). The amino- and carboxyl-terminal portions of tau are located peripheral to this core and constitute the superficial layer of filaments as a fuzzy coat (34, 35). It is tempting to speculate that there may be a common mechanism between -synuclein and tau filaments whereby basic charged, tandem repeat sequences of 80~95 amino acids in size form detergent-insoluble, protease-resistant cores of highly ordered filaments from natively unfolded neuronal cytosolic proteins. Further structural analyses of pathological -synuclein filaments will pave the way to unravel the mechanism whereby abnormal fibrous protein aggregates are formed and lead to neuronal dysfunction and eventually death in a wide variety of neurodegenerative disorders.

	ACKNOWLEDGEMENTS

We thank Dr. Michel Goedert for a cDNA encoding human -synuclein, Dr. Virginia M.-Y. Lee for Syn102, Dr. Pekka Jäkälä for NAC1, Dr. Yasuo Ihara for making MALDI-TOF mass available and Minami Baba, Akihiko Koyama, and Hideo Fujiwara for helpful discussions.

	FOOTNOTES

^* This work was supported by grants-in-aid from the Ministry of Education, Science, and Culture (to M. H. and T. I.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Both contributors served as senior authors.

^** To whom correspondence should be addressed: Dept. of Molecular Neurobiology, Tokyo Inst. of Psychiatry, Tokyo Metropolitan Organization for Medical Research, 2-1-8 Kamikitazawa, Setagaya-ku, Tokyo 156-8585, Japan. Tel.: 81-3-3304-5701; Fax: 81-3-3329-8035; E-mail: masato@prit.go.jp.

Published, JBC Papers in Press, March 13, 2002, DOI 10.1074/jbc.M110551200

² H. Miake, H. Mizusawa, T. Iwatsubo, and M. Hasegawa, unpublished observations.

	ABBREVIATIONS

The abbreviations used are: PD, Parkinson's disease; DLB, dementia with Lewy bodies; GCIs, glial cytoplasmic inclusions; MSA, multiple system atrophy; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; HPLC, high pressure liquid chromatography; MALDI-TOF, matrix-assisted laser desorption/ionization-time of flight.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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