A Hydrophobic Stretch of 12 Amino Acid Residues in the Middle of alpha -Synuclein Is Essential for Filament Assembly*

Benoit I. GiassonDagger, Ian V. J. Murray§, John Q. Trojanowski, and Virginia M.-Y. Lee

From the Center for Neurodegenerative Disease Research, Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

Received for publication, September 29, 2000, and in revised form, October 27, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Neuronal and oligodendrocytic aggregates of fibrillar alpha -synuclein define several diseases of the nervous system. It is likely that these inclusions impair vital metabolic processes and compromise vialibity of affected cells. Here, we report that a 12-amino acid stretch (71VTGVTAVAQKTV82) in the middle of the hydrophobic domain of human alpha -synuclein is necessary and sufficient for its fibrillization based on the following observations: 1) human beta -synuclein is highly homologous to alpha -synuclein but lacks these 12 residues, and it does not assemble into filaments in vitro; 2) the rate of alpha -synuclein polymerization in vitro decreases after the introduction of a single charged amino acid within these 12 residues, and a deletion within this region abrogates assembly; 3) this stretch of 12 amino acids appears to form the core of alpha -synuclein filaments, because it is resistant to proteolytic digestion in alpha -synuclein filaments; and 4) synthetic peptides corresponding to this 12-amino acid stretch self-polymerize to form filaments, and these peptides promote fibrillization of full-length human alpha -synuclein in vitro. Thus, we have identified key sequence elements necessary for the assembly of human alpha -synuclein into filaments, and these elements may be exploited as targets for the design of drugs that inhibit alpha -synuclein fibrillization and might arrest disease progression.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

alpha -synuclein (alpha -syn)1 is the major component of several pathological lesions diagnostic of specific neurological disorders, including Parkinson's disease, the Lewy body (LB) variant of Alzheimer's disease, dementia with LB, multiple system atrophy, and neurodegeneration with brain iron accumulation type 1 (formerly known as Hallervorden-Spatz disease; Refs. 1-11). In neurons, abnormal alpha -syn can form LBs, Lewy neurites, neuronal cell inclusions, and axonal spheroids. Moreover, oligodendrocytic aggregates of alpha -syn, known as glial cytoplasmic inclusions, are found abundantly in multiple system atrophy, but it is unknown how they cause disease (6, 8-10).

alpha -Syn cellular inclusions comprise fibrils formed by polymerized alpha -syn. This notion is supported by the intense labeling of these fibrils by antibodies specific to alpha -syn in situ (1, 4, 6, 9, 10) as well as by the partial purification of alpha -syn immunoreactive filaments from multiple system atrophy and dementia with LB brains (5, 8). Furthermore, in vitro, alpha -syn has an intrinsic propensity to polymerize into fibrils that resemble authentic filaments in pathological lesions (12-16).

alpha -Syn is a small, 14-kDa protein that has sequence homology to three other proteins termed beta -synuclein (beta -syn), gamma -synuclein (gamma -syn), and synoretin (17, 18). These proteins are more highly homologous within the amino-terminal half, which encompasses 5-6 degenerate KTKEGV repeats. Of these proteins, alpha -syn has the greatest sequence homology to beta -syn. Furthermore, the expression and intracellular localization of alpha - and beta -syn are very similar in that both proteins are predominantly expressed in neurons of the central nervous system and are concentrated at the presynaptic terminal (19-22). In contrast, gamma -syn is predominantly expressed in neurons of the peripheral nervous system, although it is also found at low levels in brain but diffusely distributed throughout the cytoplasm (23). Synoretin is similar to gamma -syn with respect to amino acid sequence and intracellular distribution, but synoretin is mainly expressed in the retina (17).

Intriguingly, although alpha - and beta -syn have sequence homology and very similar biological properties, alpha -syn, but not beta -syn, is found in fibrillar pathological lesions (1, 4, 5, 7-9, 24). To determine the reason for this difference, we compared the primary sequences of human alpha - and beta -syn, and we recognized that a hydrophobic stretch of amino acids within the middle hydrophobic region of alpha -syn is lacking in beta -syn and that there is a significant sequence divergence within the carboxyl-terminal region of these two proteins (Fig. 1). Deletion of amino acids 71-82 within the hydrophobic region abrogated the ability of human alpha -syn to polymerize, whereas the introduction of charged residues within the region significantly reduced the rate of filament formation. Conversely, substituting the carboxyl-terminal region of beta -syn for that of alpha -syn did not affect polymerization. Proteolytic digestion of assembled alpha -syn as well as coassembly experiments with a synthetic peptide corresponding to the hydrophobic region further confirmed the key role of this region in fibrillogenesis.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Expression and Purification of alpha -Synuclein-- All synuclein cDNAs were subcloned into the NdeI and HindIII restriction sites of the bacterial expression vector pRK172, and the respective proteins were expressed in Escherichia coli BL21 (DE3). Bacterial pellets were resuspended in high-salt buffer (0.75 M NaCl, 50 mM Tris, pH 7.4, 1 mM EDTA) containing a mixture of protease inhibitors, heated to 100 °C for 10 min, and centrifuged at 70,000 × g for 30 min. The supernatants were applied onto a Superdex 200 gel filtration column (Amersham Pharmacia Biotech) and separated by size exclusion using high-salt buffer. The fractions were assayed for the presence of the synuclein proteins by SDS-polyacrylamide gel electrophoresis (PAGE) followed by Coomassie Blue R-250 staining. The proteins were concentrated using Centriprep-10 (Millipore Corp., Bedford, MA), dialyzed against 10 mM Tris, pH 7.5, applied to a Mono Q column (Amersham Pharmacia Biotech), and eluted with a 0-0.5 M NaCl gradient. Protein concentration was determined using the bicinchoninic acid protein assay (Pierce) and bovine serum albumin as a standard.

Western Blotting-- Proteins were resolved on slab gels by SDS-PAGE (25) and electrophoretically transferred onto nitrocellulose membranes (Schleicher & Schuell, Keene, NH) in buffer containing 48 mM Tris, 39 mM glycine, and 10% methanol. Membranes were blocked with a 1% solution of powdered skimmed milk dissolved in Tris-buffered saline-Tween (50 mM Tris, pH 7.6, 150 mM NaCl, 0.1% Tween 20), incubated with antibodies to alpha -syn and beta -syn, followed by either goat anti-mouse or goat anti-rabbit antibodies conjugated to horseradish peroxidase, developed with Renaissance Enhanced Luminol Reagents (NEN Life Science Products, Inc.) and exposure onto X-Omat Blue XB-1 films (Eastman Kodak Co.).

Filament Assembly and Centrifugal Sedimentation-- Native and hybrid synuclein proteins were assembled into filaments by incubation at 37 °C in 100 mM sodium acetate, pH 7.0, with continuous shaking. Samples were centrifuged at 100,000 × g for 20 min, and SDS sample buffer (10 mM Tris, pH 6.8, 1 mM EDTA, 40 mM dithiothreitol, 1% SDS, 10% sucrose) was added to pellets and supernatants, which were heated to 100 °C for 15 min. Synuclein proteins were resolved by SDS-PAGE, stained with Coomassie Blue R-250, and quantified by densitometry.

Negative Staining Electron Microscopy-- Assembled synuclein filaments were absorbed to 300-mesh carbon-coated copper grids, stained with 1% uranyl acetate, and visualized with a Joel (Peabody, MA) 1010 transmission electron microscope. Images were captured with a Hamamatsu (Bridgewater, MA) digital camera using AMT (Danvers, MA) software. For diameter determination, the width of 100-120 filaments was measured using Image-Pro Plus software (Media Cybernetics, Del Mar, CA).

Protease Protection Analysis-- Sixty µg of synuclein proteins in 30 µl of 100 mM sodium acetate, pH 7.0, were digested with 4.2 µg of proteinase K at 37 °C. At different time points, the reaction was stopped by adding SDS-sample buffer and heating to 100 °C for 10 min. Samples from each time point were resolved on 15% SDS-polyacrylamide gels.

Circular Dichroism Spectrometry-- Synuclein proteins prepared at a concentration of 5 mg/ml in 100 mM phosphate buffer, pH 7.0, were analyzed by circular dichroism either immediately or after incubation at 37 °C for 2 days with constant shaking. Proteins were diluted 1:20 in distilled water and transferred to a 0.1-cm quartz cuvette, and the spectra between 190 and 260 nm were collected with a Aviv (Lakewood, NJ) 62DS spectrophotometer.

Antibodies-- The production and epitope mapping of anti-alpha -syn antibodies LB509, SNL-4, SNL-1, and Syn 208 and anti-beta -syn antibody Syn 207 were reported previously (26, 27). The rabbit anti-alpha -syn antibody NAC-1 was raised to a synthetic polypeptide (amino acids 75-91 in human alpha -syn) conjugated to maleimide activated keyhole limpet hemocyanin (Pierce).

Synthetic Peptide-- The peptide VTGVTAVAQKTA corresponding to amino acids 71-82 in human alpha -syn (Fig. 1) was synthesized and purified on reverse phase high performance liquid chromatography by the Biotechnology Resource Center at Yale University (New Haven, CT).



View larger version (27K):
[in this window]
[in a new window]
 
Fig. 1.   Schematic depicting the sequence alignment of human alpha - and beta -syn and mutants of alpha -syn. The amino acids identical between the two proteins are illustrated in a black background. The arrowhead indicates the position where the carboxyl-terminal domain of beta -syn was exchanged for the carboxyl-terminal domain of alpha -syn to generate the protein alpha /beta -syn. The bar between amino acids 71-82 depicts the residues deleted in the protein Delta 71-82 alpha -syn. The asterisk indicates the position of the Ala residue mutated to either an Arg or Glu.

Generation of Mutant and Hybrid Synuclein cDNAs-- The A76R and A76E alpha -syn DNAs were engineered by creating the respective nucleotide substitutions in the wild-type cDNA using complimentary sets of synthetic single-stranded DNA containing the mutant sequence (i.e. A76R primers, forward/reverse, ACGGGTGTGACAAGAGTAGCCCAGAAGACAGTG/CACTGTCTTCTGGGCTACTCTTGTCACACCCGT; and A76E primers, forward/reverse, ACGGGTGTGACAGAAGTAGCCCAGAAGACAGTG/CACTGTCTTCTGGGCTACTTCTGTCACACCCGT) and the QuikChange site-directed mutagenesis kit (Stratagene). The deletion of the nucleotide sequence coding for residues 71-82 in alpha -syn cDNA (Fig. 1) was created using the ExSite polymerase chain reaction site-directed mutagenesis kit (Stratagene) and oligonucleotides that specifically bind to the DNA sequence adjacent to the targeted deleted sequence (GAGGGAGCAGGGAGCATTGCAGC and CACTGCTCCTCCAACATTTGTCACTTG). The cDNA encoding the hybrid protein alpha /beta -syn, which consist of amino acid residues 1-97 of alpha -syn and 87-134 of beta -syn (Fig. 1), was created by polymerase chain reaction with oligonucleotides that amplified the two separate cDNA fragments of alpha - and beta -syn, respectively (alpha -syn, primer 1, CATATGGATGTATTCATGAAAGGAC; and primer 2, GAATTCCTCCTTTTTGACAAAGCCAGTGGCTG; beta -syn, primer 3, TGTCAAAAAGGAGGAATTCCCTACTGATC; and primer 4, AGGGACAGAAGCTTGCTGCTGGTG). The inner oligonucleotides (primers 2 and 3) contain extended complimentary sequences that were used to unite both initial polymerase chain reaction products by subsequent polymerase chain reaction reactions with primers 1 and 4. The sequences of the constructs were verified using T7 primer and an ABI Prism 377 DNA sequencer (PE Biosystems, Foster City, CA) as a service provided by the Nucleic Acid/Protein Research Core Facility at the Children's Hospital of Philadelphia.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Human beta -Syn Fails to Fibrilize in Vitro-- To assess whether human beta -syn is capable of self-assembly into filaments, we compared the ability of alpha - and beta -syn to form filaments using previously established conditions for the assembly of alpha -syn (12). Centrifugal sedimentation analysis and electron microscopy (EM) were used to determine the assembly of human alpha - and beta -syn. Both proteins were found in the soluble fraction before incubation for assembly (Fig. 2A), but the majority of alpha -syn was present in the pelletable fraction after 2 days of incubation, whereas beta -syn remained largely soluble (Figs. 2, B and C, and 3). EM analysis showed that human alpha -syn filaments were abundant within 2 days (Fig. 4A), but no fibrils could be detected with beta -syn after incubation to induce fibrilization (data not shown). beta -Syn failed to assemble even after 9 days in vitro, as demonstrated by quantitative sedimentation analysis of beta -syn (Fig. 3). Further extensive EM inspection of grids coated with beta -syn incubated under assembly conditions for up to 6 weeks did not reveal the presence of filaments. Finally, circular dichroism studies demonstrated that both proteins had random coiled conformations before assembly, but only alpha -syn acquired a beta -pleated sheet conformation during assembly conditions (Fig. 5, A and C).



View larger version (43K):
[in this window]
[in a new window]
 
Fig. 2.   Centrifugal sedimentation analysis of the assembly of recombinant synuclein proteins and derivatives thereof. Assembly of protein was monitored by sedimentation at 100,000 × g for 20 min as described under "Materials and Methods." Superntant (S) and pellet (P) fractions were loaded on 12% polyacrylamide gels. A, unassembled proteins at 5 mg/ml were predominantly in the superntant fractions. B and C, syn proteins (2.5 or 5 mg/ml) were incubated at 37 °C for 48 h with continuous shaking. D and E, alpha -syn incubated alone or with either beta -syn or Delta 71-82 at 37 °C for 48 h with shaking. F, A76E and A76R mutants of alpha -syn were incubated alone or with alpha -syn for 48 h at 37 °C with shaking. Proteins resolved by SDS-PAGE were visualized with Coomassie Blue R-250 (CB) or by Western blot analysis with antibodies specific to alpha -syn (LB 509) or beta -syn (Syn 207). A total of 5 µg or 50 ng of protein was loaded for each set of assembly experiments analyzed by Coomassie Blue staining or Western blot analysis, respectively. alpha , beta , and alpha /beta , alpha -syn, beta -syn, and alpha /beta -syn, respectively; A76E and A76R, respective mutant proteins.



View larger version (53K):
[in this window]
[in a new window]
 
Fig. 3.   Quantification of synuclein filament assembly as analyzed by centrifugal sedimentation. Proteins (5 mg/ml) in 100 mM acetate, pH 7.0 were incubated for 2, 4, and 9 days at 37 °C with continuous shaking followed by sedimentation at 100,000 × g for 20 min. Supernatants and pellets were resolved by SDS-PAGE and after Coomassie Blue R-250 staining quantified by densitometry. The percentage of protein in the pellet is expressed on the y axis. n = 4.



View larger version (128K):
[in this window]
[in a new window]
 
Fig. 4.   Transmission electron microscopy of negatively stained synuclein filaments. Wild-type human alpha -syn (A), alpha /beta -syn (B), and A76R human alpha -syn (C) were incubated for assembly as described under "Materials and Methods" at a concentration of 5 mg/ml for 2 days. D, under the same condition, filaments of A76E human alpha -syn were visualized after 9 days. The peptide corresponding to residues 71-82 in human alpha -syn (864 µM) also assembled into filaments (E), and at the same concentration it coassembled with 2.5 mg/ml human alpha -syn (F). The average diameter of each filament population is indicated below each respective micrograph. Scale bar, 125 nm.



View larger version (33K):
[in this window]
[in a new window]
 
Fig. 5.   Circular dichroism spectra of synuclein protein before (A and B) and after (C and D) assembly for 2 days at 37 °C with continuous shaking as described under "Materials and Methods."

The Requirement of Hydrophobic Residues 71VTGVTAVAQKTV82 in alpha -syn for Filament Assembly-- Alignment of the primary sequences of human alpha - and beta -syn showed two major regional differences between the proteins. A stretch of hydrophobic amino acids in the middle of alpha -syn is absent in beta -syn (Fig. 1). Also, the carboxyl termini of the two proteins are relatively divergent compared with the amino termini. To ascertain the importance of the hydrophobic region and the carboxyl-terminal region in driving filament assembly, two types of recombinant synuclein protein were generated. First, amino acid residues 71-82 in human alpha -syn were deleted to generate a protein termed Delta 71-82 (see Fig. 1). Second, a hybrid comprising the first 97 amino acids of human alpha -syn and the last 48 amino acids of human beta -syn was generated, termed alpha /beta -syn (Fig. 1). Before assembly, both recombinant proteins were recovered mainly in the supernatant after centrifugal sedimentation (Fig. 2A) and had a predominantly random coil conformation (Fig. 5, A and B). On incubation, the assembly rate of alpha /beta -syn paralleled that of alpha -syn (Fig. 3), and it formed filaments of similar diameter and appearance (Fig. 4, compare A with B). The fibrillization of alpha /beta -syn also was associated with a change into a beta -pleated sheet conformation (Fig. 5D). On the other hand, Delta 71-82 did not assemble under similar assembly conditions, as monitored by sedimentation analysis (Figs. 2, B and C, and 3), and transmission EM did not reveal the presence of filaments even after prolonged incubation for up to 6 weeks (data not shown). Finally, the lack of assembly was paralleled by a lack of conformational change, as detected by circular dichroism (Fig. 5D).

The possibility that beta -syn or Delta 71-82 could either coassemble with alpha -syn or inhibit the assembly of alpha -syn was determined by coassembly experiments. Neither beta -syn nor Delta 71-82 had any impact on the assembly of alpha -syn, as assayed by sedimentation (Fig. 2, D and E) and by EM (data not shown). Furthermore, neither protein coassembled with alpha -syn. Although alpha -syn can be separated from beta -syn by SDS-PAGE because of its greater mobility, the identity of the bands was also confirmed by Western blotting with an antibody specific to human alpha -syn (LB509) and an antibody specific to beta -syn (Syn 207).

Effects of Introducing Charged Residues within the Hydrophobic Residues 71VTGVTAVAQKTV82 of alpha -Syn-- To further confirm the importance of this hydrophobic region in fibrillogenesis, a single residue in the middle of this region, Ala-76, was substituted with either a positive (arginine) or a negative (glutamic acid) charge residue. Before assembly, the A76R and A76E mutations in recombinant alpha -syn proteins did not alter the normal random coil conformation of human alpha -syn in solution (Fig. 5B), and both recombinant proteins were in the soluble fraction when analyzed by centrifugal sedimentation (data not shown). The A76R mutation reduced the rate of filament formation when compared with wild-type alpha -syn at 2 days after assembly, but by 4 days the majority of the A76R protein was assembled (Figs. 2F and 3). Fibrils comprising A76R alpha -syn (Fig. 4C) were similar to those assembled of the wild-type protein (Fig. 4A). By contrast, the A76E mutation had a much greater impact on the rate of filament assembly. Even after 4 days of incubation, the mutant protein was predominantly found in the soluble fraction after sedimentation analysis (Figs. 2F and 3), and only very rare and short fibrils were observed by EM (data not shown). However, after 9 days of assembly, the A76E alpha -syn protein eventually polymerized into fibrils that were morphologically indistinguishable from those generated from the wild-type protein (Fig. 4, compare A with D). The presence of either the A76R or A76E mutant in coassembly experiments did not seem to inhibit the assembly of wild-type protein, as assayed by sedimentation (Fig. 2F), and observation by EM of grids coated with samples of both proteins coincubated for assembly (data not shown).

The Hydrophobic Region Containing Residues 71VTGVTAVAQKTV82 of alpha -Syn Is Resistant to Protease Digestion of alpha -Syn Filaments-- Digestion of alpha -syn fibrils with proteinase K was used to identify regions that are probably buried within and therefore are core elements of these filaments. Digestion of unassembled human alpha -syn resulted in the rapid proteolysis of the protein within 30 min (Fig. 6A). Conversely, several lower Mr fragments, resistant to proteolysis, were detected when assembled alpha -syn filaments were treated with proteinase K for 0.5 h (Fig. 6B). The acquired resistance to proteinase K was specific to assembled alpha -syn filaments, because two other proteins that are assembly incompetent, beta -syn and Delta  71-82, were completely digested even after assembly incubation (Fig. 6C). To identify the proteolytic resistant polypeptides, Western blot analysis was performed using antibodies with defined epitopes throughout the alpha -syn molecule (Fig. 6, D and E), and the approximate sequences of the three major polypeptides, designated F1-F3, were deduced on the basis of their immunoreactivity with the different antibodies and their apparent molecular mass on SDS-PAGE gels (Fig. 6, D and E). All three peptides contain amino acid residues 71-82 within the middle hydrophobic region of alpha -syn.



View larger version (46K):
[in this window]
[in a new window]
 
Fig. 6.   Within alpha -syn filaments, the central hydrophobic region is protected against proteolytic digestion. Unassembled (A) or polymerized (B) human alpha -syn was untreated (U) or digested with proteinase K for the time indicated above each lane. C, alpha -syn, beta -syn, and Delta 71-82 were incubated under assembly conditions (100 mM sodium acetate, pH 7.0, at 37 °C for 2 days with shaking) and undigested (-) or digested (+) with proteinase K for 30 min. Note that proteolytic resistant fragments are only observed with alpha -syn. In A-C, proteins were resolved on 15% SDS-polyacrylamide gels and visualized by staining with Coomassie Blue R-250. D, the protected peptides were identified by using antibodies mapped to specific epitopes (26). Western blot analysis of assembled alpha -syn undigested (-) or digested (+) with proteinase K was performed using the antibodies indicated above each panel. The epitope of each antibody is also indicated in parentheses above each panel. E, Schematic summarizing the approximate location of the three major fragments (F1-F3) resistant to proteolytic digestion as determined by antibody recognition and apparent molecular mass on SDS-PAGE. The black region defines amino acids 61-95, also known as NAC (see "Discussion"). The mobility of molecular mass markers are indicated on the left.

The Synthetic Peptide 71VTGVTAVAQKTV82 Can Self-assemble and Induce the Assembly of Human alpha -Syn-- To further assess the importance of amino acids 71-82 in human alpha -syn for fibrillization, we asked whether synthetic peptides corresponding to this sequence self-assemble into fibrils. Fig. 4E shows that peptide 71-82 polymerizes into filaments, but these filaments are much straighter than those assembled from the full-length alpha -syn (Fig. 4, compare A with E). However, when full-length alpha -syn was assembled in the presence of peptide 71-82, only the more serginous fibrils, typical of full-length alpha -syn, were observed, suggesting that this peptide was incorporated into the same fibrils as full-length alpha -syn (Fig. 4F). On a molar basis, peptide 71-82 is less efficiently assembled than full-length alpha -syn; nevertheless, its ability to self-assemble can also be observed by light scattering (Fig. 7A). Finally, the addition of peptide 71-82 was found to enhance the assembly of full-length alpha -syn. After a 48-h incubation period, with less vigorous agitation to reduce the rate of assembly, the majority of alpha -syn when assembled alone was recovered in the supernatant, whereas in the presence of peptide 71-82, alpha -syn was found primarily in the pellet after sedimentation analysis (Fig. 7).



View larger version (26K):
[in this window]
[in a new window]
 
Fig. 7.   The synthetic peptide 71-82 in human alpha -syn can self-assemble and promote the assembly of the full-length alpha -syn. Fibrillization was assayed by measuring light scattering at a wavelength of 400 nm (A) or sedimentation at 100,000 × g for 20 min followed by Coomassie Blue R-250 staining of a 12% SDS-PAGE gel in which supernatants (S) and pellets (P) were loaded on separate lanes after incubation for 24 h (B). Unassembled peptide 71-82 (864 µM) and alpha -syn (2.5 mg/ml) had an A400 of <0.02. Assembly was conducted with less vigorous continuous shaking to reduce the rate of assembly. alpha -syn was at a concentration of 2.5 mg/ml, whereas peptide 71-82 was used at either a 2.5-fold higher molar concentration (432 µM; low) or a 5-fold higher molar concentration (864 µM; high).



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Our study used a number of different assays to show that amino acid residues 71-82 within the middle of the hydrophobic domain of alpha -syn are necessary and sufficient for alpha -syn to polymerize into filaments. The importance of residues 71-82 for filament formation was realized when we found that human alpha -syn demonstrates an intrinsic ability to assemble into filaments, whereas human beta -syn cannot polymerize into filaments even after prolonged (6 weeks) exposure to assembly conditions. These results are consistent with a recent report (28). However, it cannot be completely excluded that beta -syn would never form filaments under any circumstances, because another study suggested that a small number of beta -syn filaments were observed after 5 weeks of incubation (29). Although we did observe a slight increase in the amount of pelletable beta -syn after prolonged incubation (Fig. 3), we attributed this to nonspecific aggregation because of the lack of visible filaments by EM. Taken together, these findings indicate that beta -syn is far less prone to polymerize into fibrils relative to alpha -syn.

We sought to explain the dramatic difference in assembly properties of human alpha - and beta -syn by comparing their sequences, which revealed two major differences: 1) beta -syn lacks a hydrophobic amino acid stretch found near the middle region of alpha -syn; and 2) the carboxyl termini of alpha - and beta -syn are significantly divergent. The ability of a hybrid protein containing the amino terminus of alpha -syn and the carboxyl terminus of beta -syn to assemble into filaments demonstrates that differences within the carboxyl-terminal region of alpha - and beta -syn do not account for the paucity of filament formation by beta -syn. Conversely, a 12-amino acid deletion of the middle hydrophobic region within alpha -syn (Delta 71-82) obliterated polymerization. The importance of the hydrophobic region is further supported by the inhibitory influences of the substitution of a single uncharged amino acid to a charged residue (either Glu or Arg). The introduction of either of these single substitutions did not completely inhibit assembly, but it did slow down the rate of filament assembly, especially the A76E mutation. Thus, these results demonstrate that the introduction of a single charged residue is not sufficient to disrupt the hydrophobic intermolecular interactions driving filament formation.

Protection of unassembled and assembled alpha -syn from proteinase K digestion was used to screen for segments of the protein buried in the core, because they are likely to be involved in filament assembly. Three predominant proteolytic resistant fragments (F1-F3) were observed. These fragments mapped to the middle hydrophobic region and contained amino acids 61-95, which has also been termed the nonamyloid component (NAC) of Abeta amyloid plaques (30). The NAC peptide was initially purified from SDS-insoluble fractions enriched in amyloid cores from Alzheimer's disease brains, and it was thought to be the second most abundant component of plaques after beta -amyloid. Whether this peptide is actually an intrinsic component of amyloid plaques is still a matter of contention (30-33). It may have been purified as a contaminant of amyloid cores, since almost all brains from Alzheimer's disease patients contain variable amounts of aggregated alpha -syn in LBs and in Lewy neurites. The proteolytic resistance of this region within fibrillar alpha -syn shown in our study may explain why the NAC peptide was selectively recovered with amyloid cores, since both NAC and Abeta may be resilient to proteolysis by endogenous proteases during purification or to the protease that was used in the final stage of purification.

To further confirm the role of the hydrophobic region in the polymerization of alpha -syn, a synthetic peptide corresponding to amino acids 71-82 in human alpha -syn was assayed for its ability to generate filaments and to coassemble with full-length alpha -syn. Peptide 71-82 readily self-assembled under the same conditions used for alpha -syn, and it further promoted the assembly of alpha -syn. Consistent with these results and the notion that the hydrophobic region is essential for assembly, it was reported that a peptide corresponding to amino acids 1-18 in NAC (i.e. 61-78 in alpha -syn) can also form filaments, but not a peptide corresponding to amino acids 19-35 in NAC (i.e. 79-95 in alpha -syn; Ref. 34). Furthermore, a carboxyl-terminal alpha -syn truncation mutant (1-89) not only formed filaments, it actually assembled faster than full-length alpha -syn (29). Collectively, these studies raise the possibility that aberrant proteolysis of alpha -syn and the accumulation of a polypeptide constituting the hydrophobic region may act together as a nidus for the formation of large inclusions. Indeed, LBs isolated from dementia with LB brains contain truncated alpha -syn fragments (1).

The inefficiency of beta -syn to form filaments likely explains the paucity of beta -syn within pathological lesions defined by alpha -syn immunoreactivity. The only exception is a recent study (35) in which an accumulation of both alpha - and beta -syn was observed in mossy fibers terminals that synapse on hilar neurons in patients with Parkinson's disease or dementia with LB. However, this pathology is different from other alpha -syn lesions, because it is not defined by the presence of abundant fibrils as seen in LBs and glial cytoplasmic inclusions. The accumulation of alpha - and beta -syn in synaptic terminals is likely attributable to the degeneration of presynaptic terminals independent of alpha -syn fibrillogenesis, a notion that is consistent with the accumulation of other synaptic proteins such as synaptophysin, synapsin, and synaptobrevin (35).

A detailed understanding of the protein motifs required in the fibrillogenesis and subsequent formation of pathological inclusions is critical to plan potentially effective therapeutic strategies targeted at reducing the formation of pathological alpha -syn aggregation. Candidates for such therapy may include small molecules that could interfere with the assembly process by binding to the hydrophobic region. However, as demonstrated by the peptide corresponding to amino acids 71-82 in human alpha -syn, such treatments could produce an effect opposite to that desired by increasing aggregation. Pharmacological agents that could prevent alpha -syn from converting into a beta -pleated sheet conformation may be effective. Indeed, this structural alteration is associated with filament formation in vitro, and pathologically aggregated alpha -syn is also likely to be in a beta -sheet conformation, as suggested by the staining of pathological lesions with thioflavin S (36). Further refinement of in vitro assembly systems and the development of in vivo models are key to developing and testing putative therapies for diseases characterized by filamentous alpha -syn inclusions.


    ACKNOWLEDGEMENTS

We thank Drs. K. S. Reddy and C. Moser for assistance with circular dichroism studies and the Biochemical Imaging Core Facility of the University of Pennsylvania for assistance with the EM studies.


    FOOTNOTES

* This work was supported in part by grants from the NIA, National Institutes of Health, and a Pioneer Award from the Alzheimer's Association. B. I. G. and I. V. J. M. contributed equally to this study.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.

Dagger Recipient of a fellowship from the Human Frontier Science Program Organization.

§ Recipient of a fellowship from the Medical Research Council of Canada.

To whom correspondence should be addressed: Center for Neurodegenerative Disease Research, Dept. of Pathology and Laboratory Medicine, Maloney 3, Hospital of the University of Pennsylvania, Philadelphia, PA 19104-4283. Tel.: 215-662-6427; Fax: 215-349-5909; E-mail: vmylee@mail.med.upenn.edu.

Published, JBC Papers in Press, November 1, 2000, DOI 10.1074/jbc.M008919200


    ABBREVIATIONS

The abbreviations used are: syn, synuclein; EM, electron microscopy; LB, Lewy body; NAC, nonamyloid component of amyloid plaques; PAGE, polyacrylamide gel electrophoresis.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES


1. Baba, M., Nakajo, S., Tu, P., Tomita, T., Nakaya, K., Lee, V. M.-Y., Trojanowski, J. Q., and Iwatsubo, T. (1998) Am. J. Pathol. 152, 879-884[Abstract]
2. Wakabayashi, K., Matsumoto, K., Takayama, K., Yoshimoto, M., and Takahashi, H. (1997) Neurosci. Lett. 239, 45-48[CrossRef][Medline] [Order article via Infotrieve]
3. Takeda, A., Mallory, M., Sundsumo, M., Honer, W., Hansen, L., and Masliah, E. (1998) Am. J. Pathol. 152, 367-372[Abstract]
4. Arima, K., Uéda, K., Sunohara, N., Hirai, S., Izumiyama, Y., Tonozuka-Uehara, H., and Kawai, M. (1998) Brain Res. 808, 93-100[CrossRef][Medline] [Order article via Infotrieve]
5. Spillantini, M. G., Crowther, R. A., Jakes, R., Hasegawa, M., and Goedert, M. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 6469-6473[Abstract/Free Full Text]
6. Arima, K., Uéda, K., Sunohara, N., Arakawa, K., Hirai, S., Nakamura, M., Tonozuka-Uehara, H., and Kawai, M. (1998) Acta Neuropathol. (Berl.) 96, 439-444[CrossRef][Medline] [Order article via Infotrieve]
7. Spillantini, M. G., Schmidt, M. L., Lee, V. M.-Y., Trojanowski, J. Q., Jakes, R., and Goedert, M. (1997) Nature 388, 839-840[CrossRef][Medline] [Order article via Infotrieve]
8. Spillantini, M. G., Crowther, R. A., Jakes, R., Cairns, N. J., Lantos, P. L., and Goedert, M. (1998) Neurosci. Lett. 251, 205-208[CrossRef][Medline] [Order article via Infotrieve]
9. Tu, P., Galvin, J. E., Baba, M., Giasson, B., Tomita, T., Leigth, S., Nakajo, S., Iwatsubo, T., Trojanowski, J. Q., and Lee, V. M.-Y. (1998) Ann. Neurol. 44, 415-422[Medline] [Order article via Infotrieve]
10. Wakabayashi, K., Hayashi, S., Kakita, A., Yamada, M., Toyoshima, Y., Yoshimoto, M., and Takahashi, H. (1998) Acta Neuropathol. (Berl.) 96, 445-452[CrossRef][Medline] [Order article via Infotrieve]
11. Wakabayashi, K., Yoshimoto, M., Fukushima, H., Koide, R., Horikawa, Y., Morita, T., and Takahashi, H. (1999) Neuropathol. Appl. Neurobiol. 25, 363-368[CrossRef][Medline] [Order article via Infotrieve]
12. Giasson, B. I., Uryu, K., Trojanowski, J. Q., and Lee, V. M.-Y. (1999) J. Biol. Chem. 274, 7619-7622[Abstract/Free Full Text]
13. Conway, K. A., Harper, J. D., and Lansbury, P. T. (1998) Nat. Med. 11, 1318-1320[CrossRef]
14. El-Agnaf, O. M. A., Jakes, R., Curran, M. D., and Wallace, A. (1998) FEBS Lett. 440, 67-70[CrossRef][Medline] [Order article via Infotrieve]
15. Hashimoto, M., Hsu, L. J., Sisk, A., Xia, Y., Takeda, A., Sundsmo, M., and Masliah, E. (1998) Brain Res. 799, 301-306[CrossRef][Medline] [Order article via Infotrieve]
16. Narhi, L., Wood, S. J., Steavenson, S., Jiang, Y., Wu, G. M., Anafi, D., Kaufman, S. A., Martin, F., Sitney, K., Denis, P., Louis, J.-C., Wypych, J., Biere, A. L., and Citron, M. (1999) J. Biol. Chem. 274, 9843-9846[Abstract/Free Full Text]
17. Surguchov, A., Surgucheva, I., Solessio, E., and Baehr, W. (1999) Mol. Cell. Neurosci. 13, 95-103[CrossRef][Medline] [Order article via Infotrieve]
18. Clayton, D. F., and George, J. M. (1998) Trends Neurosci. 21, 249-254[CrossRef][Medline] [Order article via Infotrieve]
19. Jakes, R., Spillantini, M. G., and Goedert, M. (1994) FEBS Lett. 345, 27-32[CrossRef][Medline] [Order article via Infotrieve]
20. Shibayama-Imazu, T., Okahashi, I., Omata, K., Nakajo, S., Ochiai, H., Nakai, Y., Hama, T., Nakamura, Y., and Nakaya, K. (1993) Brain Res. 622, 17-25[CrossRef][Medline] [Order article via Infotrieve]
21. George, J. M., Jin, H., Woods, W. S., and Clayton, D. F. (1995) Neuron 15, 361-372[Medline] [Order article via Infotrieve]
22. Iwai, A., Masliah, E., Yoshimoto, M., Ge, N., Flanagan, L., Rohan de Silva, H. A., Kittel, A., and Saitoh, T. (1995) Neuron 14, 467-475[Medline] [Order article via Infotrieve]
23. Buchman, V. L., Hunter, H. J. A., Pinõn, L. G. P., Thompson, J., Privalova, E. M., Ninkina, N. N., and Davies, A. M. (1998) J. Neurosci. 18, 9335-9341[Abstract/Free Full Text]
24. Lippa, C. F., Schmidt, M. L., Lee, V.-M.-Y., and Trojanowski, J. Q. (1999) Ann. Neurol. 45, 353-357[CrossRef][Medline] [Order article via Infotrieve]
25. Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve]
26. Giasson, B. I., Jakes, R., Goedert, M., Duda, J. E., Leight, S., Trojanowski, J. Q., and Lee, V. M.-Y. (2000) J. Neurosci. Res. 274, 528-533[CrossRef]
27. Jakes, R., Crowther, R. A., Lee, V. M.-Y., Trojanowski, J. Q., Iwatsubo, T., and Goedert, M. (1999) Neurosci. Lett. 269, 13-16[CrossRef][Medline] [Order article via Infotrieve]
28. Biere, A. L., Wood, S. J., Wypych, J., Steavenson, S., Jiang, Y., Anafi, D., Jacobsen, F. W., Jarosinski, M. A., Wu, G. M., Louis, J.-C., Martin, F., Narhi, L. O., and Citron, M. J. Biol. Chem. 275, 34574-34579
29. Serpell, L. C., Berriman, J., Jakes, R., Goedert, M., and Crowther, R. A. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 4897-4902[Abstract/Free Full Text]
30. Uéda, K., Fukushima, H., Masliah, E., Xia, Y., Iwai, A., Yoshimoto, M., Otero, D. A. C., Kondo, J., Ihara, Y., and Saitoh, T. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 11282-11286[Abstract]
31. Masliah, E., Iwai, A., Mallory, M., Uéda, K., and Saitoh, T. (1996) Am. J. Pathol. 148, 201-210[Abstract]
32. Culvenor, J. G., McLean, C. A., Cutt, S., Campbell, B. C. V., Maher, F., Hartmann, T., Beyreuther, K., Masters, C. L., and Li, Q.-X. (1999) Am. J. Pathol. 155, 1173-1181[Abstract/Free Full Text]
33. Bayer, T. A., Jakala, P., Hartmann, T., Havas, L., McLean, C., Culvenor, J. G., Li, Q. X., Masters, C. L., Falkai, P., and Beyreuther, K. (1999) Neurosci. Lett. 266, 213-216[CrossRef][Medline] [Order article via Infotrieve]
34. El-Agnaf, O. M. A., Jakes, R., Curran, M. D., Middleton, D., Ingenito, R., Bianchi, E., Pessi, A., Neill, D., and Wallace, A. (1998) FEBS Lett. 440, 71-75[CrossRef][Medline] [Order article via Infotrieve]
35. Galvin, J. E., Uryu, K., Lee, V. M.-Y., and Trojanowski, J. Q. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 13450-13455[Abstract/Free Full Text]
36. Duda, J. E., Lee, V. M.-Y., and Trojanowski, J. Q. (2000) J. Neurosci. Res. 61, 121-127[CrossRef][Medline] [Order article via Infotrieve]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.